Conditions: Anal Cancer High-grade Squamous Intraepithelial Lesion HIV Infection Human Papilloma Virus Infection
Keywords: anal cancer high-grade squamous intraepithelial lesion anal HSIL HIV HIV/AIDS human papillomavirus HPV
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 4446
Healthy volunteers: no

Brief summary:
The randomized phase of the trial compared topical or ablative treatment with active monitoring in preventing anal cancer in patients with human immunodeficiency virus (HIV) and high-grade squamous intraepithelial lesions (HSIL). Anal HSIL is tissue in the anal canal that has been damaged by infection with human papillomavirus (HPV) and is at risk for turning into anal cancer.

The ANCHOR Data Safety Monitoring Board (DSMB) determined that the primary study endpoint was completed, based on the data and statistical analysis presented to them on 07SEP2021.

In the post-randomization phase of this trial, all enrolled participants are offered treatment for HSIL and/or follow-up, at the participant's choice.

Detailed description:
PRIMARY OBJECTIVES: The primary objective of this study has been completed for efficacy.

I. To determine the effectiveness of treating anal HSIL to reduce the incidence of anal cancer in human immunodeficiency virus (HIV)-infected men and women.

SECONDARY OBJECTIVES:

I. To determine the safety of infrared coagulation (IRC), electrocautery, imiquimod, laser and 5- fluorouracil treatments for anal HSIL.

II. To assess the responsiveness (sensitivity to change) and clinical significance of the ANCHOR Health-Related Symptom Index (A-HRSI) subscales by comparing change scores within groups of participants as defined by participant responses to the participant global impression of change (PGIC) item. (completed FEB2020)

TERTIARY OBJECTIVES:

Collect clinical specimens and data to create a bank of well-annotated specimens that will enable correlative science:

I. Identification of viral factors in HSIL progression to cancer; II. Identification of host factors in HSIL progression to cancer; III. Identify host and viral biomarkers of progression from HSIL to cancer; IV. Identify medical history and behavioral risk factors for HSIL progression to cancer.

QUALITY OF LIFE OBJECTIVES (completed FEB2022) I. Primary QOL Objective: To compare arms in terms of changes in physical symptoms and impacts from T2 to T3, adjusting for T1.

ANCILLARY (COVID SUPPLEMENT) SUBSTUDY OBJECTIVES:

I. Determine the prevalence of SARS-CoV-2 detection in anal and oropharyngeal swabs among people living with HIV (PLWH) being screened for and enrolled in the ANCHOR study.

II. Determine the relationship between prevalent anal SARS-CoV-2 positivity, anal HPV infection, and anal high-grade squamous intraepithelial lesions (HSIL).

III. Determine the 6-month incidence of SARS-CoV-2 detection in anal and oropharyngeal swabs among participants in the active monitoring arm being assessed for the first time for treatment and individuals already enrolled in the COVID substudy under protocol version 15.0.

IV. Determine the relationship between prevalent or incident SARS-CoV-2 detection and regression of anal HPV infection or HSIL among active monitoring arm participants already enrolled in the COVID substudy under protocol version 15.0, and those who continue the protocol and who choose not to be treated at visit 101.

OUTLINE: The randomized strategy to study the efficacy of HSIL treatment to reduce the risk of progression to anal cancer, as compared to active monitoring, was discontinued for all participants.

Patients are randomized to 1 of 2 treatment arms. (accrual closed SEP2021)

ARM I: Patients are directed to receive either topical or ablative treatment at the discretion of the clinician. Patients receiving topical treatment apply topical imiquimod intra-anally, peri-anally or both thrice weekly for up to 16 weeks, or topical 5-fluorouracil twice daily for 5 days every 2 weeks for up to 16 weeks. Patients receiving ablative treatment using infrared coagulation, hyfrecation/electrocautery, or laser. Patients may undergo excision under anesthesia if the clinician believes none of the other treatment approaches will be effective. The number and timing of such treatments will be at the discretion of the investigator. Patients with persistent HSIL should continue a protocol-approved treatment or a new protocol treatment should be considered.

ARM II: Patients undergo active monitoring with HRA examinations and anal cytology every 6 months. Every 12 months, patients undergo biopsies of visible lesions.

Participants on both arms are to be followed for up to 5 years after randomization of the last participant.

Post-randomization phase: Individuals in the treatment arm may continue treatment, and participants in the active monitoring arm are offered treatment. If upon assessment participants continue to have HSIL but do not intend to get treatment, they will be monitored for the potential for disease progression to anal cancer.

Arms:
arm 1: Patients are directed to receive either topical or ablative treatment at the discretion of the clinician. Patients receiving topical treatment apply imiquimod intra-anally, peri-anally or both thrice weekly for up to 16 weeks, fluorouracil twice daily for 5 days every 2 weeks for up to 16 weeks, or trichloroacetic acid every 3 weeks up to 12 weeks. Patients receiving ablative treatment using infrared photocoagulation therapy, hyfrecation/electrocautery (thermal ablation therapy), or laser therapy. Patients may undergo excision under anesthesia if the clinician believes none of the other treatment approaches will be effective. The number and timing of such treatments will be at the discretion of the investigator. Patients with persistent HSIL should continue a protocol-approved treatment or a new protocol treatment should be considered. All participants will have samples collected for laboratory biomarker analysis. arm 2: Patients undergo active monitoring with examinations for clinical observation every 6 months. Every 12 months, patients undergo biopsies of visible lesions. Patients have cytology sampling performed at every visit. All participants will have samples collected for laboratory biomarker analysis.

Interventions: intervention 1: imiquimod intervention 2: fluorouracil intervention 3: infrared photocoagulation therapy intervention 4: thermal ablation therapy intervention 5: laser therapy intervention 6: clinical observation intervention 7: laboratory biomarker analysis
Intervention descriptions: intervention 1: Applied topically intervention 2: Applied topically intervention 3: Undergo infrared coagulation intervention 4: Undergo hyfrecation/electrocautery therapy intervention 5: Undergo laser therapy intervention 6: Undergo active monitoring (High Resolution Anoscopy \[HRA\]) with biopsies intervention 7: Correlative studies

Protocol document:
Prot_SAP_000.pdf: 
ANCHOR Protocol (AMC-A01 Version 15.0) 22JAN2021 
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SUMMARY OF CHANGES 
 
ANCHOR Study: Anal Cancer Prevention Study 
Anal Cancer/HSIL Outcomes Research Study 
Version 15.0 
 
NCI Protocol #: AMC-A01 
Local Protocol #: ANCHOR 
 
NCI Version Date: 22JAN2021 
Protocol Date: 22JAN2021 
 
I. 
Comments Requiring a Response – Major issues from the 22 December 2020 
disapproval letter for protocol version 14: 
# 
Section 
Comments 
1. 
8.5.11, 
8.6.13 
Response to comment 2 is unclear: the protocol should make it clear whether 
missed follow-up evaluation visits are considered a protocol violation. 
 
Please note:  CTEP does not approve open-ended missed follow up and 
endpoint re-evaluation visits that can affect the primary endpoint analysis.  
Guidelines and time limits must be made clear, including when to consider 
successive missed visits a protocol deviation. 
 
It would appear that the investigators wish to follow patients regardless of  
potential impact on the primary endpoint.  If this is the case, then a definition 
of when a patient can no longer viably contribute to the primary endpoint must 
be included, along with a sensitivity and feasibility analysis for when the 
protocol itself can no longer accomplish it’s primary goal. 
 
PI Response: There are no stated exemptions from protocol deviations 
from missed 6-month visits. We stated in our prior response that these are 
unavoidable deviations. If a site performs a telehealth visit because the 6-
month visit cannot be performed, it will still require reporting as a missed 
visit and is still considered a protocol deviation. This has been 
communicated previously to sites per CTEP guidance regarding 
management of the effects COVID-19 on research visits. At CTEP’s 
request, this is now repeated in the protocol. 
 
Participants are evaluable for the primary endpoint until they develop a 
confirmed anal cancer diagnosis or expire, or withdraw informed consent 
for participation. This is described in section 4.5 and the investigators find 
no other reason to omit a participant from the primary analysis (ITT). As 
stated in the prior response, telehealth visits also do not supplant in-office 
visits in determining person-years of follow-up completed. The approach 


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to determining the amount of follow-up for all cases involves calculating 
the duration between the latest date HRA was performed and the 
randomization date, and to compare visit completion rates to the expected 
visit completion rates based on the participant’s visit schedule. The DSMB 
reviews this information during its regular reviews of the protocol, in 
conjunction with the interim analyses and the dropout rate. While visits 
have been missed due to necessary COVID-19 -related restrictions, the 
protocol is not at risk of failing to accomplish its primary analysis (last 
reviewed by the DSMB November 2020, and to be reviewed at least 
annually for the remainder of trial duration). 
 
II. 
Recommendations from the 22 December 2020 disapproval letter for protocol 
version 14: 
# 
Section 
Comments 
2. 
8.1.12 
The protocol indicates that collection tubes containing oropharyngeal swabs 
from ARAS-CoV-2 testing must be stored at -70C or colder until shipment to 
the biorepository (e.g., see page 89).  The investigators should confirm that all 
sites that will participate in the ancillary SARS-CoV-2 study have the facilities 
to store the collection tubes at this temperature. 
 
PI Response: The study team qualified the freezers available at each site 
before site initiation. If the clinical site does not have a -70C (or colder) 
refrigerator, the manual of procedures specifies appropriate alternate 
procedures (shipping specimens on the same day as collection, or, if 
collected on a Friday or before a holiday, storing specimens frozen at -20C 
and then shipping on dry ice on the next available business day). The study 
specimens may also be stored at -20C and the protocol has been revised 
accordingly. This level of detail for these special cases is not required for the 
protocol document and is appropriately managed in a manual of 
procedures. 
3. 
10.6 
Under Aim 1 in section 10.6 the protocol states: “With a sample size of 400 for 
each swab type, the 95% confidence interval for the SARS-CoV-2 prevalence 
will be no greater than +/- 0.3%.”  The 0.3% figure appears incorrect. Under the 
worst-case scenario of a prevalence of 0.5 (50%), the 95% CI width would be 
5.0%.  Possibly the investigators are computing the width under the hypothesized 
prevalence of 10%, but in that case the width would be 3.0% and not 0.03%.  
Assumptions should be clarified, and corrections made as needed. 
 
PI Response: A key sentence was omitted in this section.  We postulated that 
the prevalence of SARS-CoV-2 would be 10%; thus, the width of the 95% 
confidence interval would be ± 3.0%. 


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III. 
Comments Requiring a Response – Major issues from the 24 November 2020 
disapproval letter for protocol version 13: 
# 
Section 
Comments 
1. 
2.3.11 
This section states, “These results provide evidence of A-HRSI clinical 
responsiveness that will be further explored as part of the ANCHOR trial 
through refined assessment windows”. Please clarify what additional 
psychometric work on the tool is needed.  
PI Response: Please refer to the subsequent sentences that describe how 
the windows for these evaluations were refined for the QoL objective. This 
sentence introduces this subsequent description for the QoL objective. 
Minor phrasing changes were applied so that the reader appropriately 
identifies the context of this paragraph. 
2. 
8.5.11, 
8.6.13 
Amended sections 8.5.11 and 8.6.13 allow replacing follow-up evaluation 
visits with a virtual visit. This may compromise evaluation of the primary 
endpoint and hurt study ability to address its primary goal.  Every effort should 
be made to maintain the follow up visit schedule (missed visits should be 
rescheduled, and if a visit cannot be rescheduled it should be considered 
protocol non-compliance).  
PI Response: As stated in our prior response, the study team’s intent is 
that every effort must be made to maintain the follow-up visit scheduled. 
No waiver for the 6-month visit is provided with these changes. Missed 
visits due to COVID-19 may be unavoidable deviations due to local site 
closures or restrictions. The intent of this provision is to provide a 
provision for telehealth when in-person visits are not possible or the 
participant is unwilling to attend a visit, for the purposes of collecting 
follow up information for safety, study arm adherence, supporting 
participant retention, and determining whether the participant has any 
anal symptoms that may warrant an immediate visit for the participant’s 
care. Clinic staff report that this process supports retention by 
maintaining communication with the participant when the 6-month visit 
cannot be scheduled. Sites are still instructed to conduct a makeup 6-
month visit once this is feasible again. This statement has been added to 
the protocol to emphasize this intent. 
The study team believes this process minimizes the effect of COVID-19 on 
the primary endpoint by allowing clinicians to remain in contact with 
participants when visits are not feasible and to identify participants who 
require immediate care due to concern for potential cancer, on the basis 
of reported symptoms. The study team recognizes this information cannot 
supplant evaluation for the primary endpoint by HRA with biopsy, but in 
the presence of pandemic-related restrictions preventing visit conduct, this 
is the best available approach to weigh when the potential benefit of 
conducting a visit or providing care outweighs the risk of SARS-CoV-2 


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transmission in the clinic. The information from these follow-up calls are 
collected in a separate case report form in the database that will not affect 
the follow-up calculation for the primary analysis; this will be based on the 
time elapsed between the participant’s randomization date and the date of 
the last HRA performed. 
3.  
10.1.2 
It is not clear why this is an secondary objective: To assess the responsiveness 
(sensitivity to change) and clinical significance of the A-HRSI subscales by 
comparing change scores within groups of participants as defined by 
participant responses to the PGIC item (n=500). Hasn’t this work been 
completed in February with a sample size of 100 patients? 
PI Response: This was a copy/paste error that has been deleted. See section 
10.1.4 for the QoL objectives, which are also stated in section 1.5. 
4.  
10.5 
There is no explanation of how missing data will be handled. 
PI Response: The sample sizes for the QoL substudy and QoL objective 
account for rates of non-response and dropout, based on the substudy. In 
addition to the specified analyses that assume data are missing at random, 
multiple imputation methods will be applied as a sensitivity analysis to 
assess the impact of missing data that may not be missing at random. 
 
IV. 
Comments Requiring a Response – Major Issues from the 25 June 2020 Disapproval 
Letter for protocol version 12: 
# 
Section 
Comments 
5.  
2.3.11 
Aims of the A-HRSI substudy were to validate the HR-QoL questionnaire-final 
data collection was completed in February 2020 and final results are still being 
analyzed. Based on the interim results, the questionnaire is now being 
implemented in the ANCHOR study. However, the results of the psychometric 
work are not reported in the protocol. Please provide the results, scoring, 
responsiveness over time and the change in score that is considered clinically 
meaningful. There is no information regarding clinical significance of 
calculated non-normalized ES for the A-HRSI subscales.  
PI Response: Results from the A-HRSI substudy have been added to the 
background section (section 2.3.11). In section 10.5, we comment on the 
minimum differences we can detect based on our sample size calculations 
for the final implementation of A-HRSI as compared to those seen in the 
responsiveness substudy when comparing those with worse ECOG PS 
scores versus those with no change. The A-HRSI substudy provided a 
preliminary indication of change that would be considered clinically 
meaningful. 


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Section 
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6.  
4.1 
8.5.10, 
8.6.13 
Section 4.1 states patients will undergo HRA every 6 months, and biopsy of 
HSIL every 12 months. New sections 8.5.1 and 8.6.13 provide for visit deferrals 
with the possibility of telemedicine consultation instead. There seems to me 
limit on length of deferrals on these follow-up exams, or provisions for 
scheduling a deferred follow-up visit. 
Please reconcile section 4.1 with the appropriate sections in 8, and provide 
more direction for how much deferral can occur before requiring patients to 
come off study. 
PI Response: Per the guidance of the ANCHOR DSMB during its second 
visit in December 2015, participants should not be taken off study unless 
they expire or their disease satisfies the primary endpoint (progression to 
cancer). As this is a time to event study, if a participant remains willing 
and able to return to the trial, a visit should be scheduled to perform HRA 
with biopsy regardless of whether prior visits were missed or performed 
as a telemedicine follow-up visit. Telemedicine visits during the COVID 
pandemic are a means to continue to collect data on participant safety and 
study arm compliance if an in-person visit is not feasible due to COVID-
related restrictions only.  
7.  
8.2, 8.7 
2.3.11 
There is not justification provided for the 9 timepoints of measurement.  
PI Response: As discussed during a teleconference with representatives 
from CTEP on 25AUG2020, time 2 (T2) will be revised to occur 2-7 days 
after randomization, to correspond with the period during which most 
participants are symptomatic after the initial treatment or monitoring arm 
HRA, and time 3 (T3) has been revised to occur at 4 weeks after 
randomization, at which point the investigators would not expect 
participants to be symptomatic. Subsequent time points 4-8 are now 
scheduled to occur around annual visits through the fifth year of study 
participation, to evaluate any long-term psychological effects of each study 
arm’s strategy. Time 1 (baseline) remains within 2 weeks before 
randomization. Time points 4-8 will be used in an exploratory analysis in 
order to characterize prospective changes in HR-QoL. 
8.  
8.4.7 
The value of completing the HR-QoL questionnaire 2 weeks post-
randomization is unclear. Is there an expected change from the baseline 
measurement in such a short period of time?  
PI Response: Please see our responses to items #3 and 6 for the rationale 
for time point 2 and its role as the expected timeframe in which physical 
effects of the monitoring and treatment strategies will be measured. 
9.  
8.6.10.1 
Please explain the new provision to allow treatment of HSIL if it was biopsied 
6 months prior.   


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PI Response: This provision permits empirical treatment of clinically 
suspected HSIL lesions in the treatment only arm only if the biopsies are 
collected at the same visit as they are treated and not the prior six month 
visit. The new provision allows clinicians to treat these lesions to avoid 
having to biopsy the lesion, wait for the result and bring the participant 
back at a separate visit for treatment, as long as their suspicion of cancer 
is low. To confirm they that did not unintentionally treat a cancer, we do 
however require that they biopsy the lesion before treating it. 
10.  
10.5.2 
There is no stated hypothesis for the HR-QoL sub-study.  
PI Response: The primary endpoint for the QoL objective is changes in 
physical symptoms and impacts from T2 to T3, adjusting for T1. It is 
anticipated that participants assigned to the treatment arm will experience 
significant reduction in physical symptoms and impacts from T2 to T3 
versus those in the active monitoring arm. The secondary endpoint for the 
QoL objective is changes in psychological symptoms from T3 to T4, 
adjusting for T1. There is uncertainty of the timing of psychological 
symptoms related to lack of treatment among those in the active 
monitoring arm, but it is anticipated that participants assigned to the 
active monitoring arm will experience significant increases in 
psychological symptoms from T3 to T4, a later time point compared with  
those in the treatment arm. The exploratory endpoint is HR-QoL changes 
in physical symptoms/impacts and psychological symptoms from T4 
through the subsequent T5-T8 follow-ups. 
11.  
10.5.2 
The HR-QoL study endpoint is not clear. The investigators state they will 
perform mixed model repeated measures ANOVA with change at baseline 
calculated at each follow up visit for each subscale. Do the investigators intend 
to do a longitudinal analysis? What comparison and timepoint(s) are of interest?  
PI Response: Please see our response to item #6. The analyses for these 
endpoints are detailed in section 10.5.2. 
12.  
10.5.2 
It is not appropriate to use 5% two-sided alpha per sub-scale, because of 
multiplicity of analyses. The overall type one error should be controlled for at 
5% level. 
PI Response: For the primary endpoint, we are testing two subscales and 
anticipate that participants assigned to the treatment arm will experience 
significant reduction in physical symptoms and impacts from T2 to T3 versus 
those in the active monitoring arm. Therefore, we will conduct two tests at the 
one-sided alpha level of 0.025, based on applying a Bonferroni correction to 
maintain an overall one-sided alpha level of 0.05. 
13.  
10.5.2 
For responsiveness, the investigators consider the possibility of a scenario 
leading to type one error +0.31. If this scenario is realistic, analysis does not 


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Section 
Comments 
make sense. 
PI Response: This language was not a part of the current amendment for 
the main QoL objective but was related to the responsiveness substudy. It 
was  intended to show increased Type I error if effect sizes were smaller 
than what the substudy was designed around. 
 
V. 
Scientific and Substantive Changes 
# 
Section 
Description of Change 
14.  
Synopsis 
1.6 
2.6 
3.5 
3.6 
3.7 
8.1.12 
8.2 
8.5 
10.1 
10.6 
13.0 
Appendix I 
The protocol has been revised throughout to add a supplemental study 
to describe detection of SARS-CoV-2 in anal swab samples from 
PLWH being screened for the ANCHOR study; examine its 
relationship to prevalent anal HPV infection and HSIL in the 
screening population; and determine its effect on the natural history of 
anal HPV infection and HSIL by examining its relationship to 
regression of HSIL and clearance of HPV infection in the subset of 
enrolled participants randomized to the active monitoring arm. 
Accordingly, our specific aims are outlined in Section 1.6.  
15.  
Synopsis 
Schema 
1.5 
2.3.11 
2.4 
2.4.1 
2.4.2 
3.3 
3.4 
3.6.1 
3.7 
8.3 
8.4 
8.5 
8.6 
8.7 
8.8 
8.9 
10.1 
10.2 
The protocol was revised to report completion of the A-HRSI scale 
responsiveness substudy and to implement the A-HRSI instrument in 
the protocol as a formal QoL aim. The objectives, eligibility criteria, 
study procedures, statistical section, and appendices were revised 
accordingly. The QoL aim will also include collection of data using 
the Spanish language version of the A-HRSI as validated in the AMC-
A04 protocol, Development of a Health-Related Symptom Index for 
Spanish-Speaking Persons Diagnosed with and either Treated or 
Monitored for Anal High-Grade Squamous Intraepithelial Lesions 
(HSIL). 


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10.4 
Appendix I 
Appendix X 
Appendix XI 
Appendix XII 
Appendix XIII 
16.  
2.3 
A definition for an anal cancer case that qualifies for the primary 
endpoint was added to the protocol, based on current National 
Comprehensive Cancer Network (NCCN) guidelines for the extent of 
disease that is considered anal squamous cell carcinoma. 
17.  
4.3 
The exclusion criterion prohibiting recent receipt of other 
investigational drugs (excepting investigational ART agents, 
treatments for Hepatitis C, and treatments/vaccinations for SARS-
CoV-2) has been extended as prohibited therapy during study 
participation. 
18.  
6.4 
Data on COVID-19 diagnoses (suspected and confirmed) will now be 
collected as routine adverse events, for the purpose of identifying 
cases in the future as needed for ancillary research proposals in 
development. 
19.  
8.5.11 
8.6.13 
Telephone follow-up procedures were added to the protocol for 6-
month visits unable to be conducted due to COVID-19, to formalize 
the study’s procedures for maintaining contact with participants and 
evaluating patient safety and adherence to the assigned study arm to 
the extent feasible during the SARS-CoV-2 pandemic. 
20.  
Appendix III 
Guidance regarding insufficient or inconclusive cytology at screening 
was added, requiring repeat testing if it occurs. Valid results must be 
available prior to randomization (may occur up to 12 weeks prior to 
randomization). 
 
VI. 
Administrative and Editorial Changes 
# 
Section 
Description of Change 
21.  
Global 
The protocol version and version date were updated to version 15.0, 
dated 22JAN2021. 
22.  
Global 
Spelling, grammar, and editorial changes (abbreviations, company 
names) were applied throughout the document. 
23.  
Global 
The manufacturer name for topical medications was updated from 
Valeant Pharmaceuticals to Bausch Health. 


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Section 
Description of Change 
24.  
List of Tables 
List of Figures 
Lists of tables and figures were added to the protocol, and each table 
and figure renumbered accordingly. 
25.  
2.1.1 
The background section was updated with current anal HSIL 
incidence estimates. 
26.  
8.5.9.1 
8.6.10.1 
Statements regarding empiric treatment of HSIL in the treatment arm 
following biopsy at a given visit were repeated in the evaluations 
section at visits after randomization. 
27.  
Appendix VI 
The AMC Data and Safety Monitoring Plan, which applies to all AMC 
protocols, was updated from version 6.0 to version 9.0. Key revisions 
include: the addition of an introduction to address the variety of 
systems the AMC uses for individual trials; changes to the data entry 
systems used by domestic AMC trials (OPEN/Rave) activated 
September 1, 2020 and later; participation with a single IRB for new 
domestic AMC protocols; updated procedures for data reporting, 
determination of requirements for DSMB review, and safety 
review/pharmacovigilance; and administrative changes (updates to 
document organization, external links, and group terminology). 
 


ANCHOR Protocol (AMC-A01 Version 15.0) 22JAN2021 
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ANCHOR Study: Anal Cancer/HSIL Outcomes Research Study 
(AMC Protocol #A01) 
 
A Collaboration of the 
AIDS Malignancy Consortium 
Human Papillomavirus Working Group 
and the University of California, San Francisco 
Sponsored by: 
National Cancer Institute 
Office of HIV and AIDS Malignancy (OHAM) 
NCT Registration Number: 
NCT02135419 
Regulatory Status: 
 
IND exempt drugs 
IDE exempt study devices 
Commercially Available 
Agents:  
5-Fluorouracil (5-FU) (NSC 19893) 
Topical Imiquimod Cream (5%) (NSC 741062) 
Protocol Chair: 
Joel Palefsky, MD, CM, F.R.C.P.(C) 
Protocol Co-Chair: 
J. Michael Berry, MD 
AMC-A01 Version 15.0, 22 January 2021 
NCI Version Date 22 January 2021 


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PROTOCOL SIGNATURE PAGE 
 
I,          
 
 
 
, Principal Investigator at site _______________, agree to 
conduct and follow this protocol: ANCHOR Study (AMC-A01, Version 15.0, 22JAN2021), as 
written according to NCI, OHRP, and FDA guidelines, and group policies. I understand that no 
deviations from the protocol eligibility criteria or waivers for protocol deviations will be permitted. 
 
 
_____________________________________ 
____________________________________ 
PI Signature  
 
 
 
 
Date (mm/dd/yyyy) 
 


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TABLE OF CONTENTS 
 
SUMMARY OF CHANGES ................................................................................................................ i 
I. 
Comments Requiring a Response – Major issues from the 22 December 2020 
disapproval letter for protocol version 14: ..................................................................... i 
II. 
Recommendations from the 22 December 2020 disapproval letter for protocol 
version 14: ..................................................................................................................... ii 
III. 
Comments Requiring a Response – Major issues from the 24 November 2020 
disapproval letter for protocol version 13: ................................................................... iii 
IV. 
Comments Requiring a Response – Major Issues from the 25 June 2020 
Disapproval Letter for protocol version 12: ................................................................. iv 
V. 
Scientific and Substantive Changes ............................................................................ vii 
VI. 
Administrative and Editorial Changes ....................................................................... viii 
PROTOCOL SIGNATURE PAGE .................................................................................................... 2 
TABLE OF CONTENTS .................................................................................................................... 3 
LIST OF TABLES ............................................................................................................................... 7 
LIST OF FIGURES ............................................................................................................................. 8 
PROTOCOL ROSTER ....................................................................................................................... 9 
PROTOCOL SYNOPSIS .................................................................................................................. 10 
PROTOCOL SCHEMA .................................................................................................................... 13 
1.0 
OBJECTIVES ........................................................................................................................ 19 
1.1 
Hypothesis ................................................................................................................... 19 
1.2 
Primary Objective ....................................................................................................... 19 
1.3 
Secondary Objectives .................................................................................................. 19 
1.4 
Exploratory Objectives ............................................................................................... 19 
1.5 
Quality of Life Objectives .......................................................................................... 20 
1.6 
Ancillary Study Objectives ......................................................................................... 20 
2.0 
BACKGROUND .................................................................................................................... 22 
2.1 
Anal Cancer ................................................................................................................ 22 
2.2 
Study Agents and Treatments ..................................................................................... 27 
2.3 
Study Design and Rationale ........................................................................................ 31 
2.4 
Overview of Study Design .......................................................................................... 44 
2.5 
Correlative Studies ...................................................................................................... 49 
2.6 
Ancillary Study: SARS-CoV-2 and Anal HPV Infection ........................................... 54 
3.0 
PATIENT SELECTION ....................................................................................................... 60 


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3.1 
Eligibility Criteria ....................................................................................................... 60 
3.2 
Exclusion Criteria ....................................................................................................... 62 
3.3 
Inclusion Criteria for the HRQoL Substudy ............................................................... 62 
3.4 
Inclusion Criteria for the QoL Objective .................................................................... 63 
3.5 
Inclusion Criteria for the SARS-CoV-2 Ancillary Study ........................................... 63 
3.6 
Number of Participants to be Enrolled ........................................................................ 63 
3.7 
Participant Enrollment Procedures ............................................................................. 63 
4.0 
TREATMENT PLAN ........................................................................................................... 65 
4.1 
Group 1: Active Monitoring Arm ............................................................................... 65 
4.2 
Group 2: Treatment Arm ............................................................................................ 65 
4.3 
General Concomitant Medication and Supportive Care Guidelines ........................... 71 
4.4 
Concurrent HPV-related Disease Treatment ............................................................... 72 
4.5 
Duration of Follow-Up ............................................................................................... 73 
4.6 
Criteria for Removal from the Study (Drop-Out) ....................................................... 74 
5.0 
DOSING DELAYS/DOSE MODIFICATIONS .................................................................. 75 
5.1 
Dose Modifications Guidelines ................................................................................... 75 
6.0 
ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS ............................. 76 
6.1 
Potential Risks Lists .................................................................................................... 76 
6.2 
Adverse Event Characteristics and Definitions .......................................................... 77 
6.3 
Expedited Adverse Event Reporting ........................................................................... 79 
6.4 
Routine Adverse Event Reporting .............................................................................. 81 
6.5 
Secondary Malignancy ................................................................................................ 82 
6.6 
Second Malignancy ..................................................................................................... 82 
7.0 
PHARMACEUTICAL AND STUDY DEVICE INFORMATION ................................... 83 
7.1 
5-Fluorouracil 5% Cream ........................................................................................... 83 
7.2 
Topical Imiquimod Cream (5%) ................................................................................. 84 
7.3 
Study Devices ............................................................................................................. 85 
7.4 
Drug Supply and Accountability ................................................................................ 85 
8.0 
CLINICAL AND LABORATORY EVALUATIONS ....................................................... 87 
8.1 
Special Instructions and Definitions of Evaluations ................................................... 87 
8.2 
Screening Evaluations (Visit 0) .................................................................................. 90 
8.3 
Baseline Visit with Randomization Evaluations (Visit 1) .......................................... 91 
8.4 
Evaluations between Six Month Visits (Interim Visits) ............................................. 93 
8.5 
Evaluations at Visit 2 (6 Months) for All Participants ................................................ 93 


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8.6 
Evaluations at Follow-Up (Visits 3-10+), Every 6 Months until Study 
Completion .................................................................................................................. 94 
8.7 
Final Evaluations, Off Study ....................................................................................... 96 
8.8 
Early Study Discontinuation Evaluations ................................................................... 97 
8.9 
QoL Objective ............................................................................................................. 98 
9.0 
BIOMARKER STUDIES ..................................................................................................... 99 
9.1 
Viral Factors in HSIL Progression to Cancer ............................................................. 99 
9.2 
Identification of Host Factors in HSIL Progression to Cancer ................................. 101 
9.3 
Viral and Host Biomarker Predictors of Risk of Progression from HSIL to 
Cancer ....................................................................................................................... 104 
9.4 
Medical History and Behavioral Risk Factors for Progression from HSIL to 
Cancer among Those Who Are Enrolled .................................................................. 110 
10.0 
STATISTICAL CONSIDERATIONS ............................................................................... 112 
10.1 
Study Design/Endpoints ............................................................................................ 112 
10.2 
Sample Size/Accrual Rate ......................................................................................... 112 
10.3 
Stratification Factors ................................................................................................. 115 
10.4 
Statistical Analysis .................................................................................................... 115 
10.5 
Quality of Life Analyses ........................................................................................... 116 
10.6 
SARS-CoV-2 Ancillary Study Analyses .................................................................. 118 
10.7 
Data and Safety Monitoring ...................................................................................... 119 
11.0 
ROLE OF DATA MANAGEMENT .................................................................................. 122 
11.1 
CRF Instructions ....................................................................................................... 122 
11.2 
Data Quality .............................................................................................................. 122 
11.3 
Data Monitoring ........................................................................................................ 122 
12.0 
ETHICAL AND REGULATORY CONSIDERATIONS ................................................ 123 
12.1 
IRB Approval and Informed Consent ....................................................................... 123 
12.2 
Changes to the Protocol ............................................................................................ 123 
12.3 
Women and Minorities ............................................................................................. 123 
13.0 
REFERENCES .................................................................................................................... 125 
APPENDIX I: SCHEDULE OF EVALUATIONS ....................................................................... 137 
Participants Randomized to Active Monitoring Arm ............................................................ 137 
Participants Randomized to Treatment Arm, Treatment with Imiquimod Or 5-
Fluorouracil Cream ................................................................................................... 139 
Participants Randomized to Treatment Arm, Treatment with Infrared Coagulation, 
Hyfrecation/Electrocautery, or Laser ........................................................................ 141 


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Participants Randomized to Treatment Arm, Surgery........................................................... 143 
Health-Related Quality of Life (QoL) Objective Schedule of Evaluations ........................... 145 
APPENDIX II: PERFORMANCE STATUS SCALES ................................................................ 146 
APPENDIX III: ANAL CYTOLOGY AND HPV SAMPLING .................................................. 147 
APPENDIX IV: HIGH RESOLUTION ANOSCOPY (HRA) AND ANAL BIOPSIES ........... 148 
APPENDIX V: RISK FACTOR QUESTIONNAIRE .................................................................. 151 
APPENDIX VI: AMC DATA AND SAFETY MONITORING PLAN ...................................... 156 
APPENDIX VII: PARTICIPANT INSTRUCTIONS FOR APPLYING IMIQUIMOD 5% 
CREAM 
161 
APPENDIX VIII: PARTICIPANT INSTRUCTIONS FOR APPLYING EFUDEX (5-
FLUOROURACIL) 5% CREAM .................................................................................................. 162 
APPENDIX IX: REGULATORY STATUS OF INVESTIGATIONAL AGENTS ................... 163 
APPENDIX X: ANCHOR HEALTH-RELATED SYMPTOM INDEX..................................... 141 
APPENDIX XI: ANCHOR HEALTH-RELATED SYMPTOM INDEX – SPANISH 
LANGUAGE VERSION ................................................................................................................. 143 
APPENDIX XII: PATIENT VERSION OF THE EASTERN COOPERATIVE GROUP 
PERFORMANCE STATUS............................................................................................................ 145 
APPENDIX XIII: PARTICIPANT GLOBAL IMPRESSION OF CHANGE (PGIC) ............. 146 
 
 
 


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LIST OF TABLES 
 
Table 2-A: Descriptive statistics for change in subscales (T3 minus T2) according to change in 
PGIC and ECOG PS, mean (SD) ...................................................................................................... 43 
Table 2-B: Responsiveness comparison of changes (T3 minus T2), mean difference (95% 
confidence interval) ............................................................................................................................ 43 
Table 2-C: A-HRSI completion time points for QoL objective ..................................................... 48 
Table 5-A:1. Dose modifications ....................................................................................................... 75 
Table 6-A: Mapping adverse event attribution assignments to causal relationship designations .. 
 
 
..................................................................................................................................... 79 
Table 10-A: Power associated with varying drop-out rates and drop-in rates from the active 
monitoring arm with fixed sample size (N=5058), two-sided significance level of 0.05, incidence 
rate for the active monitoring arm (200/100,000) and the treatment arm (50/100,000) ............ 113 
Table 10-B: Sample size = 5058, two-sided significance level = 0.05, 3 year accrual, 5 year 
follow-up 
................................................................................................................................... 114 
Table 10-C: Power associated with varying drop-out rates and drop-in rates from the active 
monitoring arm with fixed sample size (N=5058), two-sided significance level of 0.05, incidence 
rate for the active monitoring arm (200/100,000) and the treatment arm (50/100,000) ............ 115 
Table 10-D: Smallest differences that can be detected for comparing change for specified time 
points according to treatment group for various scenarios based on pilot data with 90% power 
using a one-sided alpha of 0.025 ..................................................................................................... 117 
Table 10-E: Statistical test boundaries and associated operating characteristics. ..................... 120 
Table 12-A: Accrual targets ............................................................................................................ 124 
 
 


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LIST OF FIGURES 
 
Figure 2-D Paired Lesion Comparisons ........................................................................................... 51 
Figure 4-A: Participant initiates imiquimod/5-FU at visit 1 (randomization visit) ..................... 70 
Figure 4-B: IRC, hyfrecation, or electrocautery performed at visit 1 (randomization visit)...... 70 
Figure 6-B: Timeframes and Exceptions for Expedited SAE Reporting ...................................... 80 
 
 


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PROTOCOL ROSTER 
 
ANCHOR: Anal Cancer Prevention Study 
Anal Cancer/HSIL Outcomes Research Study 
 
Protocol Chair: 
Protocol Statistician: 
Joel Palefsky, MD, CM, F.R.C.P.(C) 
513 Parnassus Ave Box 0654 
University of California, San Francisco 
San Francisco, CA 94143 
Tel: 415-476-1574 
Fax: 415-476-9364 
Email: jpalefsky@medicine.ucsf.edu 
Jeannette Lee, PhD 
4301 West Markham, #781 
Ed III, Room 3212 
University of Arkansas for Medical Sciences 
Little Rock, AR 72205 
Tel: 501-526-6712 
Fax: 501-526-6729 
Email: jylee@uams.edu 
 
 
Protocol Co-Chair: 
Data Management/Operations: 
J. Michael Berry, MD 
ANCHOR Data Management Center 
1600 Divisadero Street Room A740 
The Emmes Company, LLC 
University of California, San Francisco 
401 N. Washington Street, Suite 700 
San Francisco, CA 94115 
Rockville, MD 20850 
Tel: 415-353-7443 
Tel: 301-251-1161 
Fax: 415-353-7464 
Fax: 301-830-6799 
Email: JMichael.Berry@ucsf.edu 
Email: anchordmc@emmes.com 
 
 
National Coordinator: 
Arizona Regional ACSR Biorepository: 
Abigail Arons, MPH 
Monika Schmelz, PhD 
University of California, San Francisco 
Assistant Professor of Pathology 
Mission Hall: Global Health & Clinical Sciences 
Building 
Department of Pathology 
550 16th Street, 3rd Floor, UCSF Box 0886 
1501 N. Campbell Ave. Room 5103 
San Francisco, CA 94158-2549 
P.O. Box 245043 
Tel: (415) 514-8155 
Tucson, AZ 85724 
Fax: (415) 476-5348 
Tel: 520-626-8864 
Email: abigail.arons@ucsf.edu 
Fax: 520-626-6081 
 
Email: schmelz@pathology.arizona.edu 
 
 


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PROTOCOL SYNOPSIS 
 
Title: 
ANCHOR Anal Cancer Prevention Study 
 
Phase of Study: 
Phase III 
 
Participating Institutions: 
This protocol will be open to sites that have been approved for 
participation by the ANCHOR Coordinating Committee and have 
at least 2 practitioners certified in high resolution anoscopy (HRA) 
and 1 practitioner certified in HSIL treatment (infrared coagulation 
(IRC), hyfrecation/electrocautery, or laser ablation) by the 
ANCHOR HRA Training and Certification Committee. At least 
fifteen sites will be targeted for participation. 
 
Accrual Target: 
5,058 participants 
 
Population: 
 
Men and women age 35 and older with HIV infection and biopsy-
proven high-grade squamous intraepithelial lesions (HSIL) at 
baseline. Eligible participants will have no history of anal cancer, 
or treatment or removal of HSIL. Approximately 17,385 
participants who provide informed consent for study participation 
will be screened to identify 5,058 eligible participants with 
previously untreated HSIL. 
 
Regimen: 
Eligible participants will be randomized to treatment or active 
monitoring at baseline. Participants will be followed every six 
months for HSIL outcomes for up to five years after the last 
participant’s date of randomization. Throughout the study, the 
incidence of invasive cancer in both arms will be monitored, and 
biospecimens and associated participant data will be collected for 
correlative science studies. Effects of the treatment and monitoring 
strategies on participants’ quality of life will be assessed using the 
ANCHOR Health Related Symptom Index (A-HRSI) in a subset of 
consenting participants (n=500) before randomization and at 7 time 
points through 5 years after randomization). 
 
Ancillary Studies: 
 
Anal shedding of SARS-CoV-2: relationship to anal HPV infection 
and high-grade squamous intraepithelial lesions in people living 
with HIV (Availability of Urgent Competitive Revision and 
Administrative Supplements on Coronavirus Disease 2019, PA-18-
59), Section 2.6 
 
Duration: 
Minimum of 5 years of follow-up for each participant 
 


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Primary Objective: 
To determine whether treating anal high-grade squamous 
intraepithelial lesions (HSIL) is effective in reducing the incidence 
of anal cancer in HIV-infected men and women. 
 
Secondary Objectives: 
 
 
• To determine the safety of infrared coagulation (IRC), 
electrocautery, imiquimod, laser and 5- fluorouracil treatments 
for anal HSIL. 
• To assess the responsiveness (sensitivity to change) and clinical 
significance of the ANCHOR Health-Related Symptom Index 
(A-HRSI) subscales by comparing change scores within groups 
of participants as defined by participant responses to the 
participant global impression of change (PGIC) item. 
 
Exploratory Objectives: 
 
Collect clinical specimens and data to create a bank of well-
annotated specimens that will enable correlative science: 
• Identification of viral factors in HSIL progression to cancer; 
• Identification of host factors in HSIL progression to cancer; 
• Identify host and viral biomarkers of progression from HSIL to 
cancer; 
• Identify medical history and behavioral risk factors for HSIL 
progression to cancer. 
 
Quality of Life Objectives: 
Primary QOL Objective: To compare arms in terms of changes in 
physical symptoms and impacts from T2 to T3, adjusting for T1. 
Secondary QOL Objective: To compare arms in terms of changes 
in psychological symptoms from T3 to T4, adjusting for T1. 
Exploratory QOL Objective: To assess of long-term HR-QoL 
changes in physical symptoms/impacts and psychological 
symptoms from T4 through the subsequent T5-T8 follow-ups 
overall and by arm. 
 
Ancillary Study 
Objectives: 
1. Determine the prevalence of SARS-CoV-2 detection in anal and 
oropharyngeal swabs among people living with HIV (PLWH) 
being screened for the ANCHOR study 
2. Determine the relationship in the ANCHOR screening 
population between prevalent anal SARSCoV-2 positivity, anal 
HPV infection, and anal high-grade squamous intraepithelial 
lesions (HSIL) 
3. Determine the 6-month incidence of SARS-CoV-2 detection in 
anal and oropharyngeal swabs among participants with anal 
HSIL newly enrolled into the ANCHOR study 


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4. Determine the relationship between prevalent or incident 
SARS-CoV-2 detection and regression of anal HPV infection or 
HSIL among participants newly enrolled into the ANCHOR 
study and who are randomized to the monitoring arm 
 
 


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Visit 0 / Screening
Up to 17,385 HIV+ volunteers
Visit 1 / Randomization
5,058 participants with biopsy-proven anal 
HSIL
Active Monitoring
Observation of HSIL with HRA and biopsies
Visits 2-11+ (every 6 months)
Anal cytology, HRA, research specimens at 
each visit. Biopsies of visible HSIL only every 
12 months or anytime if cancer suspected.
Final visit
Determine anal cancer incidence in 
monitoring arm
Anal cytology, HRA with minimum 4-quadrant 
biopsy, research specimens
Treatment
HRA and biopsies, with ablation or topical 
treatment for HSIL
Visits 2-11+ (every 6 months)
Anal cytology, HRA, biopsies of visible HSIL, 
treatment as needed, research specimens at 
each visit; biopsy anytime if cancer suspected.
Final visit
Determine anal cancer incidence in 
treatment arm
Anal cytology, HRA with minimum 4-quadrant 
biopsy, research specimens
PROTOCOL SCHEMA 
 
 
All participants followed with 6-month visits until 5 years after the last participant is randomized. 
 
During the study, interim visits are permitted between 6-month visits at the provider’s discretion for treatment 
(treatment arm only), safety assessments, and/or closer monitoring for progression to cancer. 


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ANCHOR Health-Related Symptom Index (A-HRSI) Validation and Implementation 
Schema 
Group C/Validation:
100 participants
Goal: Demonstrate A-HRSI scale responsiveness
Consent for optional A-HRSI validation substudy during 
consenting process for main ANCHOR trial
Initiated July 2019
Time 1: Pre-randomization
Administer A-HRSI and ECOG PS
Time 2: 2-10 weeks post-randomization
Administer A-HRSI, ECOG PS, PGIC
Time 3: 10-16 weeks post-randomization
Administer A-HRSI, ECOG PS, PGIC
Statistical analysis  to complete validation, determination of 
sample size, time points for ANCHOR protocol 
implementation - Completed February 2020
ANCHOR protocol modification (v13) to administer 
validated A-HRSI in a sample (n=500) of randomized 
participants at 8 time points: baseline, 2-7 days post 
randomization, 4 weeks post randomization, and at 12, 
24, 36, 48, and 60 months after randomization


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LIST OF ABBREVIATIONS 
5-FU 
5-fluorouracil 
A-HRSI 
ANCHOR Health-Related Symptom Index 
ACSR 
AIDS and Cancer Specimen Resource 
ADL 
Activities of daily living 
AE 
Adverse event 
AIDS 
Acquired immunodeficiency syndrome 
AIN 
Anal intraepithelial neoplasia 
AM 
Active monitoring 
AMC 
AIDS Malignancy Consortium 
AML 
Acute myelocytic leukemia 
AMC 
AIDS Malignancy Consortium 
ANC 
Absolute neutrophil count 
ANCHOR 
Anal Cancer/HSIL Outcomes Research Study 
APOT 
Amplification of Papillomavirus Oncogene Transcript 
ART 
Antiretroviral therapy 
ASCCP 
American Society for Colposcopy and Cervical Pathology 
ASIL 
Anal squamous intraepithelial lesions 
AWE 
Acetowhite epithelium 
BID 
Bis in die (twice a day) 
cART 
Combination antiretroviral therapy 
CBC 
Complete blood count 
CDC 
Centers for Disease Control and Prevention 
CDUS 
Clinical Data Update System 
CLIA 
Clinical Laboratory Improvement Amendments 
CR 
Complete response 
CRT 
Chemoradiation therapy 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
CTEP-AERS 
Cancer Therapy Evaluation Program Adverse Event Reporting System 
CTMS 
Clinical Trials Monitoring Service 


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DAB 
Diaminobenzidine 
DARE 
Digital anorectal examination 
DARF 
Drug accountability record form 
DE 
Differentially expressed 
DHHS 
Department of Health and Human Services 
DMC 
Data management center 
DMR 
Differentially methylated region 
DNA 
Deoxyribonucleic acid 
DSMB 
Data and Safety Monitoring Board 
ECOG 
Eastern Cooperative Oncology Group 
ELISA 
enzyme-linked immunosorbent assay 
EMT 
Epithelial-mesenchymal transition 
ePRO 
Electronic patient-reported outcomes 
FCBP 
Female of childbearing potential 
FDA 
Food and Drug Administration 
FDR 
False discovery rate 
FFPE 
Formalin-fixed paraffin-embedded 
FU 
Follow-up 
GO 
Gene Ontology 
HIER 
Heat-induced epitope retrieval 
HIV 
Human immunodeficiency virus 
HPV 
Human papillomavirus 
HRA 
High resolution anoscopy 
HRQoL 
Health-related quality of life 
HSIL 
High grade squamous intraepithelial lesions 
IDB 
Investigational Drug Branch 
IDE 
Investigational device exemption 
IHC 
Immunohistochemistry 
IND 
Investigational new drug 
IRB 
Institutional review board 
IRC 
Infrared coagulation 


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ITT 
Intent-to-treat 
LAST 
Lower anogenital squamous terminology 
LCM 
Laser capture microdissection 
LSIL 
Low grade squamous intraepithelial lesions 
KPS 
Karnofsky Performance Score 
MDS 
Myelodysplastic syndrome 
MOP 
Manual of Procedures 
MSM 
Men who have sex with men 
NCBI 
National Centre for Biotechnology Information 
NCI 
National Cancer Institute 
NCTN 
NCI Clinical Trials Network 
ODMC 
Operations and Data Management Center 
OHAM 
Office of HIV AIDS Malignancy 
PCR 
Polymerase chain reaction 
PGIC 
Participant global impression of change 
PIO 
Protocol Information Office 
PR 
Partial response 
PS 
Performance status 
RACE 
Rapid amplification of cDNA ends 
RCT 
Randomized controlled trial 
RLU 
Relative light units 
RMA 
Robust Multi-Array 
RNA 
Ribonucleic acid 
SAE 
Serious adverse event 
SCC 
Squamous cell carcinoma 
SCJ 
Squamocolumnar junction 
SIL 
Squamous intraepithelial lesion 
SIR 
Standardized incidence ratio 
SISCCA 
Superficially invasive squamous cell carcinoma 
SOC 
System organ class 
SOP 
Standard Operating Procedure 


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STD 
Sexually transmitted disease 
TNF 
Tumor necrosis factor 
TUA 
Treatment under anesthesia 
TZ 
Transformation zone 
UADE 
Unanticipated Adverse Device Effect 
UCSC 
University of California, Santa Cruz 
UCSF 
University of California, San Francisco 
 
 
 


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1.0 
OBJECTIVES 
1.1 
Hypothesis 
Treatment of anal high-grade squamous intraepithelial lesions (HSIL) will lead to a 
reduction of 75% of incident anal cancer compared with a population with anal HSIL that 
is observed without treatment. 
1.2 
Primary Objective 
To determine whether treating anal HSIL is effective in reducing the incidence of anal 
cancer in HIV-infected men and women. 
1.3 
Secondary Objectives 
1.3.1 To determine the safety of infrared coagulation, electrocautery, imiquimod, laser, 
and 5-fluorouracil treatments for anal HSIL. 
1.3.2 To assess the responsiveness (sensitivity to change) and clinical significance of the 
A-HRSI subscales by comparing change scores within groups of participants as 
defined by participant responses to the PGIC item. 
1.4 
Exploratory Objectives 
1.4.1 Collect clinical specimens and data to create a bank of well-annotated specimens 
that will enable: 
a) Identification of viral factors in HSIL progression to cancer 
1.4.1.a.1 
Determine the HPV type in cancer and compare to that of overlying 
HSIL and HSIL biopsies collected concurrently that did not progress 
to cancer. 
1.4.1.a.2 
Determine the strain variant of HPV 16 in participants who 
progressed to anal cancer and compare to participants with HSIL 
biopsies who did not progress to cancer. 
1.4.1.a.3 
Determine the HPV integration site in overlying anal cancer to that 
of HSIL overlying the cancer and HSIL biopsies collected 
concurrently that did not progress to cancer. 
b) Identification of host factors in HSIL progression to cancer 
1.4.1.b.1 Perform gene expression array analysis comparing expression in 
anal cancer with HSIL overlying the cancer. Perform gene 
expression array analysis comparing expression in HSIL biopsies 
that progressed to cancer with non-progressing HSIL biopsies at 
other locations. Perform similar analyses comparing expression in 
HSIL biopsies that progressed to cancer with the same lesion at 
earlier time points prior to progression. 
1.4.1.b.2 
Characterize genetic changes in anal cancers compared with HSIL 
overlying the cancer. Characterize genetic changes in HSIL biopsies 
that progressed to cancer compared with non-progressing HSIL 
biopsies at other locations. Characterize genetic changes in HSIL 


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biopsies that progressed to cancer with the same lesion at earlier 
time points prior to progression. 
c) Identify host and viral biomarkers of progression from HSIL to cancer 
1.4.2 Evaluate medical history and behavioral risk factors for HSIL progression to 
cancer. 
1.5 
Quality of Life Objectives 
1.5.1 Primary QOL Objective: To compare arms in terms of changes in physical 
symptoms and impacts from T2 to T3, adjusting for T1. 
Hypothesis: Participants assigned to the treatment arm will experience significant 
reductions in physical symptoms and impacts from T2 to T3 as compared to those 
in the active monitoring arm. 
1.5.2 Secondary QOL Objective: To compare arms in terms of changes in psychological 
symptoms from T3 to T4, adjusting for T1.  
Hypothesis: Due to potential uncertainty related to lack of treatment for those in 
the active monitoring arm, participants assigned to the active monitoring arm will 
experience significant increases in psychological symptoms from T3 to T4 as 
compared to those in the treatment arm. 
1.5.3 Exploratory QOL Objective: To assess of long-term HR-QoL changes in physical 
symptoms/impacts and psychological symptoms from T4 through the subsequent 
T5-T8 follow-ups overall and by arm. 
1.6 
Ancillary Study Objectives 
1.6.1 Determine the prevalence of SARS-CoV-2 detection in anal and oropharyngeal 
swabs among people living with HIV (PLWH) being screened for the ANCHOR 
study. 
Hypothesis: 10% of PLWH will have prevalent anal swab SARS-CoV-2 positivity, 
and will have concurrent oropharyngeal positivity. 
1.6.2 Determine the relationship in the ANCHOR screening population between 
prevalent anal SARSCoV-2 positivity, anal HPV infection, and anal high-grade 
squamous intraepithelial lesions (HSIL). 
Hypothesis: Anal SARS-CoV-2 positivity will be associated with higher quantities 
of anal HPV DNA and higher prevalence of anal HSIL among those who are high-
risk HPV-positive. 
1.6.3 Determine the 6-month incidence of SARS-CoV-2 detection in anal and 
oropharyngeal swabs among participants with anal HSIL newly enrolled into the 
ANCHOR study. 
Hypothesis: Among PLWH enrolled into the ANCHOR study who were SARS-
CoV-2-negative at screening, 10% will have incident anal and oropharyngeal 
positivity at 6 months. 
1.6.4 Determine the relationship between prevalent or incident SARS-CoV-2 detection and 
regression of anal HPV infection or HSIL among participants newly enrolled into the 


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ANCHOR study and who are randomized to the monitoring arm.Hypothesis: In the 
monitoring arm of the ANCHOR Study, prevalent or incident anal SARS-CoV-2 detection 
will be associated with a lower rate of HSIL regression and clearance of anal HPV DNA than 
those who remain SARS-CoV-2-negative for 6 months.


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2.0 
BACKGROUND 
2.1 
Anal Cancer 
2.1.1 Incidence of anal cancer in the general population and in high-risk groups 
Anal cancer is a growing problem in the United States. In the U.S. general 
population, the incidence of anal cancer from 2006-2010 was 1.5/100,000 among 
men and 1.9/100,000 among women (1). Updated estimates for the period 2011-
2015 report the incidence at 1.15/100,000 among men and 1.93/100,000 among 
women (131). Compared with the general population, the standardized incidence 
ratio (SIR) of anal cancer is increased more than 100-fold among some risk groups 
of HIV-infected persons who are successfully treated with combination 
antiretroviral therapy (cART) (2). Among men in the general population, the 
incidence is highest among Black men, at 1.9/100,000 (3). The American Cancer 
Society projected that there would be 7090 cases and 880 deaths from anal cancer 
in 2013 (4). At the time the protocol was initiated, the most recent data with actual 
number of cases were available from 2009. There were 2,210 cases of cancer of the 
anus, anal canal, and anorectum among males and 3,624 cases among females. 
There were 302 deaths among males and 516 among females from these cancers. 
As of 2019, the rates of anal cancer were reported to be increasing at a rate of 2.7% 
per year from the period 2001-2015, with the number of cases from the period 2011-
2015 at 9,598 among males and 18,683 among females; for the period from 2001-
2016, there were 4,720 deaths among males and 7,391 among females (131). 
The incidence of anal cancer is largely concentrated among several groups well-
known to be at increased risk: men who have sex with men (MSM) (5), HIV-
infected men and women (6), men and women immunosuppressed for reasons other 
than HIV infection, including solid organ transplant (7)(8), women with a history 
of HPV-related cancer or high-grade squamous intraepithelial lesions (HSIL) 
elsewhere in the anogenital tract (9), and those with a history of genital warts (10). 
Of all the groups listed above the highest incidence of anal cancer is among HIV-
infected men and women, particularly MSM (2)(6). 
Overall it is estimated that the proportion of individuals with anal cancer who are 
also infected with HIV increased from 1980-1984 to 2001-2005, rising from 1.1% 
to 28.4% among males, and from 0% to 1.2% among females (11). From 1980-
2005, the HIV epidemic had little impact on the trends in anal cancer among 
females, among whom the incidence has been increasing annually by 3.3%. 
However, the HIV epidemic has had a strong impact on the trends in anal cancer 
incidence among males, with an annual increase of 1.7% among men without HIV 
infection and 3.4% among men with HIV infection. The risk of anal cancer was 
elevated 52-fold in HIV-infected men who have sex with men (MSM), 32-fold in 
HIV-infected men, and 24-fold in HIV-infected women compared with the general 
population (11). 
Recent studies indicate an incidence of 131/100,000 among HIV-infected MSM in 
the U.S. from NA-ACCORD (6). The incidence of anal cancer was 46/100,000 
among HIV-infected men other than MSM, and 30/100,000 among HIV-infected 
women. The incidence of anal cancer among HIV-infected individuals had also 


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been reported outside the U.S. In the period from 2005-2008, compared with the 
general population, HIV-infected MSM from the French Hospital Database on HIV 
had a SIR for anal cancer of 109.8 (2). Other HIV-infected men had an SIR of 49.2 
and HIV-infected women had an SIR of 13.1 (2). Among the four most common 
non-AIDS-defining malignancies, the largest increase has been in anal cancer (12). 
Several studies show an increase in the incidence of anal cancer since cART 
became available (11)(13)(14)(15). The relationship between risk of anal cancer, 
CD4 level, and complete versus incomplete HIV suppression is unclear. In the 
French study cited above, among patients who maintained a CD4 count above 
500/mm3 for at least 2 years, a CD4 nadir below 200/mm3 was associated with a 
higher SIR for anal cancer than a CD4 nadir above 200/mm3 (2). Another report 
showed that being on cART with an undetectable HIV viral load 60% or more of 
the time was associated with reduced incidence of anal cancer compared with those 
who had an undetectable HIV viral load for less than 60% of the time (13). 
However, in that study the incidence of anal cancer was high, even among those 
receiving cART, and similar to the data reported in NA-ACCORD. Overall a 
cART-associated reduction in the incidence of anal cancer has not materialized (2). 
While it is possible that better control of HIV infection with earlier initiation of 
cART may moderate the increased risk of anal cancer, the long-term effect of cART 
on anal cancer risk has not yet been studied. Moreover, among HIV-infected 
individuals, the great majority of people currently living with HIV were begun on 
cART later in the course of their HIV infection than is currently recommended. A 
recent meta-analysis estimated that 1 in 377 HIV-infected MSM with anal HSIL 
progress every year to anal cancer since the introduction of cART (16), and since 
current cART has extended the life spans of HIV-infected individuals, this may 
result in a 10% lifetime risk of anal cancer or higher among HIV-infected MSM if 
nothing is done (17). 
Taken together, these data highlight two important points: HIV-infected men and 
women are disproportionately affected by anal cancer, and the impact of HIV 
infection on anal cancer will likely continue to increase. Second, most of the cases 
of anal cancer in terms of absolute numbers occur among HIV-uninfected men and 
women, and thus the results of the ANCHOR study will impact on both HIV-
infected and HIV-uninfected populations. In the absence of active intervention, the 
number of cases of anal cancer will likely continue to grow in the general 
population in the U.S. with varying degrees of contribution by the at-risk groups 
described above. Data on the global incidence of anal cancer are not as reliable but 
it is certain that there are many thousands more cases and deaths from anal cancer 
each year world-wide. Combined with the possibility that anal cancer is 
preventable, the incidence of anal cancer is unacceptably high and calls for urgent 
intervention. 
2.1.2 Anal cancer may be preventable 
There are two approaches that may be used to prevent anal cancer. Primary 
prevention, in the form of vaccination against HPV infection may be useful to 
reduce infection with HPV, the underlying causative agent of anal cancer. Although 


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there is great potential for HPV vaccination to reduce anal cancer in the long term, 
there are several limitations to this approach as described below. Secondary 
prevention, which consists of screening for, and treating HSIL prior to progression 
to cancer, is the approach needed for the many individuals who have already been 
exposed to HPV and who have developed HPV-associated precancerous lesions. 
Current cervical cancer prevention programs rely on both approaches, and in the 
long-term, it is likely that both will be needed for anal cancer prevention. 
2.1.3 A study of anal HSIL treatment in prevention of anal cancer is needed despite the 
availability of an HPV vaccine 
Like cervical cancer, most cases of anal cancer are caused by HPV 16 and HPV 18 
(18). Vaccination with the quadrivalent and bivalent vaccine has been shown to 
reduce anal infection with HPV 16 and HPV 18 in both males and females naïve to 
those types (19)(20). Vaccination has also been shown to reduce the risk of anal 
HSIL in males (20). In the long-term, HPV vaccination may represent an excellent 
tool for reduction of anal cancer. Vaccination is currently routinely recommended 
for males (quadrivalent) and females (bivalent or quadrivalent) with a target of 11-
12 years of age, and up to 26 years of age for all women, and for men who are 
MSM, or HIV-infected/immunocompromised (21). Routine vaccination is 
recommended for other men up to age 21 years. However, the great majority of 
HIV-infected individuals currently at risk for anal cancer are older than 26 years 
and do not qualify for HPV vaccination. Even if the vaccine is made available to 
them, a high proportion will already have been exposed to HPV 16 and 18. 
Even among those who might benefit from vaccination, the impact of the vaccine 
has been reduced by poor uptake. Uptake among young women is limited at this 
time (22) with only 32% of eligible women receiving all three doses of the vaccine 
in 2010, and uptake among young men is even more limited. While herd immunity 
due to vaccination of females may contribute to protection against HPV even 
among those who have not been vaccinated, it is likely to be very limited among 
MSM. There are several reasons for the poor rates of HPV vaccination in the U.S. 
including varying levels of access, fear of HPV vaccine side effects, limited 
understanding of the benefits of HPV vaccination, and fear of vaccination in 
general. Globally the cost of the vaccine and lack of the necessary infrastructure to 
administer the vaccine are also major reasons. Finally, given the long period of time 
required for progression from cervical or anal HSIL to invasive cancer, it is 
expected that it will be decades before any reduction in cancer incidence is 
realized., even among those who have been vaccinated, it will likely be several 
decades before a vaccine-related reduction in anal cancer is observed and the full 
potential of the vaccine to reduce cancer incidence will most likely not be realized. 
In summary, the combination of low vaccine uptake among vaccine-eligible men 
and women means that the full potential of the vaccine to reduce cancer incidence 
will likely not be realized unless rates of uptake improve. Combined with the fact 
that most HIV-infected men and women are too old for vaccination or were exposed 
to HPV 16 and 18 before vaccination became available, millions of men and women 
remain susceptible to HPV 16- and HPV 18-related HSIL and cancer. For these 


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individuals, secondary prevention in the form of identifying and treating HSIL may 
be the only option to reduce the risk of anal cancer. Determination of the efficacy 
of HSIL treatment to prevent anal cancer is therefore a current and public health 
concern for the foreseeable future for this target population. 
2.1.4 Similarities between anal and cervical cancer inform secondary prevention 
approaches to anal cancer prevention 
Cervical and anal cancer share many biological features. As described above, like 
cervical cancer, most cases of anal cancer are associated with HPV 16 or 18 
infection (18). For both the cervix and the anus, the transformation zone is the 
primary target area for HPV due to the conjunction of two types of epithelium. In 
the cervix, these are the squamous epithelium of the exocervix and columnar 
epithelium of the endocervical canal. In the anus, the anorectal junction contains a 
transformation zone where the squamous epithelium of the anus is juxtaposed to 
the columnar epithelium of the rectum. HPV-associated changes in the cervix may 
be manifest as a spectrum of changes ranging from cervical low-grade squamous 
intraepithelial lesions (LSIL) to cervical HSIL. It is now well known that the 
precursor lesion to cervical cancer is cervical HSIL and the identification and 
removal of cervical HSIL before progression to cancer occurs is the basis for the 
success of the cervical cytology screening program. Similarly, anal squamous 
intraepithelial lesions (ASIL) span the same spectrum of changes as cervical 
lesions, and there is evidence that anal HSIL is the precursor to invasive anal cancer 
(23)(24) (25). In recognition of the similarity of HPV-associated anal and cervical 
disease, the American Society for Colposcopy and Cervical Pathology (ASCCP) 
and College of American Pathologists recently recommended standardization of 
terminology across all anogenital sites where HPV-associated cancers occur 
(LAST) (26). 
Prior to the introduction of cervical cytology screening programs, the incidence of 
cervical cancer in the U.S. was 40-50/100,000. Largely due to the success of 
cervical cytology screening, in which the cervical cancer precursor lesion, cervical 
HSIL, is sought and treated before progression to cancer occurs, the incidence of 
cervical cancer has declined to approximately 8/100,000 (27). Typically, an 
abnormal cervical cytology leads to referral for colposcopy, in which the lesions 
are visualized directly under magnification and with the aid of 5% acetic acid. The 
lesions are biopsied to establish the grade of the lesion, and HSIL is ablated using 
one or more different methods, most often loop electroexcision procedure in the 
U.S. 
Due to its success in reducing the incidence of cervical cancer, the cervical cytology 
screening program is considered to be one of the most successful cancer prevention 
programs ever implemented. Notably, however, the program was introduced prior 
to the era of evidence-based medicine, and its effect on reduction of cervical cancer 
only became apparent after following millions of women for several decades. While 
there are similarities between cervical and anal cancer, evidence must be developed 
as to whether anal cancer prevention is effective, to inform public health policy. 


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2.1.5 A study of anal HSIL treatment in prevention of anal cancer is needed even though 
it is known that treatment of cervical HSIL is effective in prevention of cervical 
cancer 
Although anal cancer and cervical cancer are very similar, it cannot be assumed 
that treatment of anal HSIL will be effective in preventing anal cancer like 
treatment of cervical HSIL is effective in preventing cervical cancer. Compared 
with the cervix, a high proportion of anal lesions are large and multifocal, especially 
in HIV-infected individuals. The lesion recurrence rate and incidence of new 
lesions may be higher than is seen in the cervix in these groups. Compared with 
removal of cervical lesions using loop electroexcision procedure, complete removal 
of anal lesions can be comparatively difficult and may lead to more post-treatment 
discomfort given the need for regular bowel movements. Complete eradication of 
HSIL may therefore be more difficult in the anus than in the cervix. In addition, 
data on the efficacy of individual treatments for anal HSIL are far more limited than 
for treatment of cervical HSIL. While cervical cancer screening was adopted as 
standard of care prior to the era of evidence-based medicine, adoption of new 
medical practices such as screening and treatment of anal HSIL is increasingly 
requiring rigorous demonstration of the value of these practices. 
Although a growing number of groups around the U.S. have begun anal screening 
programs, but at this time only a small fraction of individuals at risk for anal cancer 
have had access to any form of anal screening and treatment for anal HSIL. With 
the exception of the State of New York, there are no formal public health guidelines 
recommending anal screening and treatment of anal HSIL. This is because no 
studies have yet been done to demonstrate that treatment of HSIL reduces the 
incidence of anal cancer. The American Society of Colorectal Surgery recently 
issued guidelines recommending that HSIL be treated, but based on low-quality 
evidence (28). The United States Public Health Service has indicated that evidence 
of the efficacy of HSIL treatment is needed before treatment of anal HSIL can be 
recommended (29). CDC STD guidelines for 2014 are being updated, and will state 
that “Some centers perform anal cytology to screen for anal cancer among high-
risk populations (e.g. HIV-infected persons, MSM), followed by high-resolution 
anoscopy for abnormal cytologic results (e.g. ASC-US or worse). More evidence is 
needed concerning the natural history of anal intraepithelial neoplasia, the best 
screening methods and target populations, the safety and response to treatments, 
and other programmatic considerations before screening can be routinely 
recommended” (Park I, personal communication). Many insurance companies will 
not cover the cost of screening for anal HSIL or for treating it in the absence of 
guidelines establishing these services as standard of care. At the same time, 
standard of care guidelines require evidence that treatment of HSIL reduces the 
incidence of anal cancer. Conversely, if efficacy is not demonstrated, it is important 
that individuals with HSIL not be subjected to ineffective treatments that can confer 
medical harm and unnecessary cost to the healthcare system. 


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2.2 
Study Agents and Treatments 
2.2.1 Overview of treatment of anal HSIL 
As noted previously, treatment of anal HSIL in HIV-infected men and women is 
more challenging than in HIV-uninfected individuals. This is because HSIL tends 
to be larger in size and number in HIV-infected individuals. They may also recur 
more often at the site of prior treatment, and incident lesions may develop more 
often at sites that were not previously shown to have HSIL (metachronous lesions).  
All of the treatments described below are used routinely by clinicians involved in 
treating HSIL. With the exception of treatment under anesthesia (surgical excision), 
which we expect to be necessary for only a very small percentage of patients with 
HSIL, each is readily available for use in the office setting by a wide variety of 
medical professionals, including MDs, nurse practitioners, and physician assistants. 
Thus, if the ANCHOR study shows that treatment of HSIL reduces the incidence 
of anal cancer, it will be possible to immediately implement the study findings. 
2.2.2 Infrared coagulation 
Infrared coagulation (IRC) is an office-based procedure that uses the Redfield IRC 
1900 or 2100, a therapeutic device to treat anal HSIL. It delivers short pulses of a 
narrow beam of visible and infrared light through a small contact tip applicator that 
is applied directly to the target tissue, transmitted down the rigid quartz glass of the 
light guide. The tungsten-halogen lamp (150 watts of power) is the light source. 
This light causes thermal coagulation that results in tissue necrosis. The depth of 
coagulation is determined by the total amount of energy delivered, which is 
adjustable via pulse duration setting (0.5 to 3.0 seconds in 0.1-second intervals) in 
repeated applications. The depth of tissue destruction is directly proportional to the 
duration of infrared impulses. The device is cleared by the Food and Drug 
Administration for use in the treatment of hemorrhoids, tattoo removal, chronic 
rhinitis, and anal condyloma. 
The procedure typically takes about 30 minutes to perform. It is generally well 
tolerated, and it is not uncommon for patients to go to work after the procedure. 
Overall the procedure is safe and can be performed by a wide variety of non-
surgeon medical professionals. It requires only local anesthetic injected directly 
into or around the lesions to be treated, and does not require smoke evacuation 
apparatus or protection. 
There may be occasional mild intra- and post-procedural pain, and bleeding for up 
to 2 weeks. There may also be mild textural changes of anal canal mucosa for 
several weeks post-procedure. The sequence of events is similar to that seen with 
cryosurgery: hemorrhagic blistering and necrosis of the treatment site followed by 
a shallow erosion and ulcer, and then healing over several weeks. There is a small 
risk of infection from the procedure. The risk of serious bleeding prompting 
emergency room evaluation is <1%. The PI of this protocol and his colleagues have 
performed thousands of IRC procedures, and most patients easily tolerate multiple 
procedures. 


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Most published data show that on an individual lesion basis, the “cure” rate is high, 
defined as absence of HSIL in the treated area (30)(31)(32)(33) as well as when 
combined with excision under anesthesia for the patients with the most extensive 
disease (34). In these studies a therapeutic response is defined as absence of HSIL, 
and the remaining tissue may include normal tissue or LSIL. We expect that even 
if LSIL is present after IRC (or any treatment), that the main goal of treatment will 
have been attained, since there is no evidence that LSIL is a cancer precursor. 
In the largest study reported to date, the probability of curing an individual lesion 
after the first IRC ablation was 67% in HIV-infected men (30) and increased with 
additional ablations. Most recurrence was due to the development of metachronous 
lesions occurring in 82% of HIV-infected men after their first infrared coagulator 
treatment. At their last visit, 82% of HIV-infected MSM were free of HSIL. A 
similar result was obtained in AMC, in which the safety of IRC was studied in a 
multisite prospective cohort study performed by the AMC HPV Working Group 
(32). 
2.2.3 Electrocautery and surgical lasers 
Electrocautery and laser ablation are ablative techniques with a long history of use 
in the operating room and the office for the treatment of HPV-associated anal 
lesions. A recent publication showed no statistically significant differences between 
electrocautery and IRC in their efficacy and safety profile (33). In the ANCHOR 
study, clinicians may use IRC, hyfrecator/electrocautery, and surgical lasers 
interchangeably for in office removal of anal HSIL. Legally marketed 
electrosurgical units and carbon dioxide lasers that are indicated for excision, 
coagulation, and/or ablation of tissue, and that are appropriate for use in 
dermatologic, general, and/or gastrointestinal surgical procedures may be used for 
in-office anal HSIL treatment on this protocol. The choice of therapy typically 
depends on the prior training of the clinician and the technique with which they 
have the most experience. Non-surgeons in the U.S. who treat anal HSIL were 
primarily trained by Dr. Palefsky and his group to use IRC, but those with a surgical 
background may prefer electrocautery. The main advantages of electrocautery are 
that it is faster than IRC to perform and the electrocautery probe tip can be more 
precisely applied to the lesion than the IRC probe tip, which is larger in diameter. 
The main disadvantage of electrocautery is that it generates a smoke plume, 
requiring protection from smoke for providers and patients in the room. 
2.2.4 Treatment under anesthesia 
On this protocol, the term treatment under anesthesia (TUA) will be used to 
designate excision of HSIL lesions, performed by a surgeon on participants who 
are given spinal or general anesthesia. Procedures may be performed in an operating 
room as a surgical referral or in an office setting on participants with conscious 
sedation. The procedure will generally be performed using electrocautery devices 
or surgical lasers indicated for the excision, ablation, or coagulation of tissues, as 
described above. For TUA, selection of the medical devices to be used will be left 
to the discretion of the treating clinician. Combined with post-procedural IRC, it 
has been shown to be effective in treatment of HSIL for patients with disease too 


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widespread to treat with targeted ablation therapy (34). TUA is usually required for 
only a small percentage of patients being treated for anal HSIL. These include 1) 
disease that is so extensive that it cannot be treated by any of the other methods 
described in this section, or 2) if a lesion is so large that it cannot be sufficiently 
biopsied in the office to establish or exclude a diagnosis of cancer to the satisfaction 
of the clinician. The disadvantage of TUA is that it is expensive compared with the 
other methods. In addition, by definition, TUA is only done when more extensive  
excision is required and patients typically experience more post-procedure side 
effects, including pain, bleeding, and infection after the procedure when compared 
with less aggressive treatment modalities. The costs of TUA will only be covered 
by the study with the QA committee’s approval. 
2.2.5 Topical 5% fluorouracil cream (5-FU) 
Topical treatment with 5-FU was first reported in 1962 for treatment of skin cancers 
following a report that systemic 5-FU induced regression of keratosis (35). It 
inhibits DNA synthesis by blocking the conversion of uracil deoxyribonucleotide 
to thymine deoxyribonucleotide, resulting in thymine deficiency and inhibition of 
cell division. Its most marked effect is on rapidly proliferating tissues where it can 
cause erythema and edema followed by erosion, ulceration, and necrosis. It has 
been used since the 1970s to treat HPV-related lower genital tract disease in women 
(36)(37). In one study of HIV-infected women, topical 5-FU was shown to be 
effective in preventing recurrence of cervical HSIL following loop electroexcision 
procedure (36). Most patients who use 5-FU exhibit local side effects in the form 
of pain or discomfort associated with epithelial ulceration. Moderate to severe side 
effects were reported by 48% of patients (38). In a subsequent randomized 
controlled trial, 27% of patients treated with 5-FU had grade 3 or 4 side effects (39). 
Topical 5-FU is typically used when disease is too extensive for IRC or 
electrocautery. It is given with the intent to clear as much of the lesion as possible, 
and while it is unusual for a large lesion to clear completely, it may be particularly 
useful to “debulk” a lesion to the point where it can them be treated with a targeted 
ablative approach such as IRC or electrocautery. Among 20 patients with extensive 
HSIL who were treated with topical 5-FU at UCSF, complete response was seen in 
only 3 and no response was seen in one, but 16 had a significant decrease in volume 
of disease to 25-50% that allowed for in-office IRC. At UCSF the protocol for use 
of topical 5-FU is for the patient to apply intra-anal or perianal 5-FU twice daily 
for 5 days, followed by a 9-day rest period (Jay N and Palefsky J, personal 
communication). This cycle can be repeated eight times. 
There are limited reports of use of topical 5-FU specifically for treatment of anal 
HSIL. 7 of 8 patients treated with topical 5-FU cream for perianal Bowen’s disease 
applied twice weekly for 16 weeks had no evidence of Bowen’s disease on biopsy 
at 12 months post-treatment (40). In another study using intra-anal 5-FU complete 
responses were seen in 12 of 34 patients with anal HSIL and partial responses with 
regression to LSIL in 8 of 34 patients (38). 
In a recently reported randomized controlled trial of 5-FU, imiquimod or 
electrocautery among 148 HIV-infected MSM with AIN (57% with HSIL) the 


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response rate was 29, 39, and 48% respectively (39). Severe side effects were 
reported as 27, 43, and 18% respectively, and recurrence rates were 57, 25, and 
17% at 6 months post-treatment. Of note the treatment regimens in this study were 
different from what we typically do and what is proposed for the ANCHOR study. 
The investigators used lower doses of 5-FU than we typically use, but their study 
participants also appeared to have more side effects than we have experienced with 
our patients. 
2.2.6 Topical 5% imiquimod cream 
Like 5-FU, imiquimod is a topical agent that has typically been used when the 
extent of disease has been too large to allow for use of a targeted ablative modality 
such as IRC or electrocautery. Imiquimod has been used for treatment of external 
genital condyloma since 1997. It is a synthetic compound that exhibits antiviral 
activity by up-regulating the immune response, at least in part through toll-like 
receptors. It may lead to a Th1 cytokine response that activates HPV-specific cell-
mediated immunity and clearance of lesions. Imiquimod has been used for 
treatment of HPV-associated mucosal disease including vulvar (41) (42), penile 
(43)(44) and anal SIL (45). 
The advantage of imiquimod is that, like topical 5-FU cream it can be used to treat 
extensive disease that is too large for targeted ablation. It has a long safety record 
for treatment of condyloma, including in the setting of HIV infection. The 
disadvantages of imiquimod are side effects may be severe, particularly when 
patients are experiencing a robust clinical response. These may be local irritation 
or pain, or may be systemic flu-like symptoms. Another disadvantage is that there 
are relatively few randomized study data on the efficacy of imiquimod in treatment 
of perianal or anal disease. A randomized, controlled trial of 100 patients with 
anogenital warts assigned to 5% imiquimod cream or placebo for 16 weeks showed 
a low rate of complete response (11% drug vs. 6% placebo). 47% of patients in the 
imiquimod group reported greater than 50% reduction in total wart area vs. 20% in 
the placebo arm. Response rates were higher in HIV-uninfected patients compared 
with HIV-infected patients (62% and 31% respectively) (46). 
There are limited reports that demonstrate efficacy using imiquimod specifically 
for treatment of anal HSIL in cohort studies and case reports (47). In one study, 14 
of 19 (74%) HIV-infected MSM had complete regressions after treatment with 
imiquimod (48). In a randomized, double-blind placebo controlled study of 
imiquimod for intra-anal HSIL, 4 of 28 participants on active drug resolved and 8 
downgraded to LSIL, compared with only 1 of 25 in the placebo arm who had a 
spontaneous regression (49). Participants were treated with open-label imiquimod 
after completing the study, and overall 29 of 47 (61%) had sustained regression of 
anal HSIL. 
Our clinical impression over the years is that imiquimod has relatively limited value 
for treatment of anal HSIL, particularly in the setting of HIV infection where 
induction of immune response may be attenuated. However, we have not studied it 
formally in the U.S., and in light of the RCT data described above, the AMC HPV 
Working Group was recently approved by CTEP to perform a randomized trial of 


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imiquimod vs. 5-FU vs. observation for extensive HSIL in HIV-infected men and 
women. We have also elected to leave imiquimod on the list of treatment modalities 
available to ANCHOR study clinicians but may change that pending the outcome 
of the AMC study described above or other study data as they become available. 
2.3 
Study Design and Rationale 
In this trial we will screen HIV-infected men and women for HSIL and if they meet the 
enrollment criteria for randomization, we will randomize them to treatment vs. active 
monitoring (AM) and follow them for up to five years after the last participant’s date 
of randomization. Throughout the study we will be monitoring the incidence of 
invasive cancer in both arms and reporting regularly to a Data and Safety Monitoring 
Board (DSMB). 
We will also be collecting samples and clinical data that will allow us to perform 
the correlative science studies described in Section 9.0, which are focused on 
exploiting a unique opportunity to understand progression from HSIL to cancer. 
Definition of anal cancer 
The anus is composed of the anal canal and the perianus. Proximally, the anal canal 
is bordered by the squamocolumnar junction (SCJ). This border is not static and 
anal squamous cell carcinoma (SCC) may occasionally be diagnosed proximal to 
the SCJ. These SCCs, diagnosed in the distal rectum, proximal to the SCJ, will be 
classified as anal cancers. 
Distally, the anus includes the perianus, defined as a circumferential area with a 5 
cm radius from the anal verge, (after the buttocks have been retracted gently), and 
includes the squamous mucocutaneous junction (151). For a perianal SCC to be 
classified as an anal cancer, it must be part of a lesion (LSIL/HSIL/cancer) that is 
located wholly or partially within the perianus. If located outside the perianus, it 
must be contiguous with a LSIL or HSIL lesion that resides as least partially within 
the perianal region. An exception may be in the situation where an anal cancer is 
diagnosed outside the perianus, that was previously documented to be part of a 
lesion that was at least partially within the perianus, but at the time of cancer 
diagnosis a lesion within the boundaries of the perianus is no longer present due to 
prior treatment. Among women, the anterior border of the perianus is defined as the 
posterior half of the perineum. SCC located on the posterior half of the perineum 
are considered anal cancers even if they extend to the anterior, vulvar region. 
2.3.1 Rationale for selected approach and trial design 
To have an impact on standard of care guidelines, it is critical that a study of the 
value of anal HSIL treatment in prevention of anal cancer be designed to provide 
the highest quality of evidence. The highest quality evidence will be obtained in a 
randomized controlled trial (RCT). Such a trial would require HIV-infected men 
and women with biopsy-proven HSIL, randomized to a treatment or active 
monitoring arm, and followed for up to five years. 
Careful consideration has been given to alternative, less costly options for study 
designs that will yield the data needed to definitively define standard of care for 
anal cancer prevention. Least informative are small, non-randomized treatment 


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studies of anal HSIL. There are few of these published to date. Two such studies 
showed that anal cancers occurred in patients whose anal HSIL was not treated, in 
contrast to patients whose HSIL was treated and who did not develop cancer (50) 
(52). Although encouraging, these small studies are subject to many potential biases 
and do not constitute definitive evidence of the value of anal HSIL treatment. 
A more robust study design would be an ecologic study, in which the rates of anal 
cancer at locations where HSIL is sought and treated are compared with locations 
where there is no such screening and treatment. 
There are two recent reports comparing the incidence of anal cancer in the San 
Francisco region, where the UCSF Anal Neoplasia Clinic has been treating patients 
since the early 1990s, with incidence reported from registries outside California. 
Individuals with anal cancer in the San Francisco region had a 39% lower mortality 
risk than those reported from registries outside California but there was no decrease 
in the incidence of invasive cancer in the San Francisco region (53). There are also 
data showing that while rates of anal cancer may not have decreased in the San 
Francisco area during this time, they may have increased more outside of California 
(54). 
Unfortunately, there are several limitations to these kinds of analyses with regard 
to inferences about the efficacy of treating anal HSIL in prevention of anal cancer. 
Methodologically-sound ecologic studies of anal HSIL treatment are not truly 
feasible for several reasons. First, screening and treatment for anal HSIL have 
begun on a small scale on an ad hoc basis in many centers with large populations 
of at-risk men and women. There is little standardization of clinical screening and 
treatment practices, and it is not possible to collect reliable data on treatment 
outcomes. Second, even in those locations where screening and treatment are 
performed, we estimate that only a small percentage of at-risk individuals have been 
screened to date and the effect of screening on reducing anal cancer incidence at 
these locations will be limited at best. This is true even in San Francisco, where we 
estimate that fewer than 10% of individuals with anal HSIL will have been treated, 
despite having one of the most active treatment programs in the country. Taken 
together, the data described above hint at the potential benefits of treating HSIL, 
but do not obviate the need for a RCT. 
2.3.2. Prevention of anal cancer is highly desirable 
One option to reduce mortality from anal cancer would be to focus efforts on 
finding cancers as early as possible, instead of trying to prevent them by treating 
HSIL. In the absence of screening programs to identify anal cancer or anal HSIL in 
at-risk populations, most anal cancers are currently diagnosed when individuals 
present with anal pain, bleeding, or a mass. Occasionally asymptomatic cancers are 
detected on digital anorectal examination (DARE) or incidentally at procedures 
such as hemorrhoidectomy. Treating cancers when they occur, which is the current 
approach, has the advantage of not over-treating many individuals whose HSIL 
would never have progressed to cancer if left untreated. 
However, there are several reasons why focusing on diagnosis after cancer develops 
is a poor strategy. Many cancers found incidentally or because of symptoms will 


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be diagnosed at more advanced stages. The survival rate declines substantially 
when the cancers are diagnosed at a more advanced stage (55). Even at stage 1, the 
most common stage at which anal cancer is diagnosed, nearly 30% of individuals 
die within 5 years. At more advanced stages, the mortality increases further, and 
when metastatic cancer is diagnosed (Stage IV) the 5-year survival rate is only 21%. 
Like cervical cancer, anal cancer may be preventable. An important reason to focus 
on cancer prevention rather than treatment of existing cancer is that while the 
standard anal cancer treatment, chemoradiation therapy (CRT), is often successful 
at the earlier stages of disease, it also leads to substantial morbidity (56). Acute 
toxicity is a major issue, particularly in HIV-infected patients, with one study 
reporting Grade 3 or 4 toxicity in nearly half of the patients (57). Chronic toxicity 
is also a major issue. Radiation therapy leads to radiation proctitis in a substantial 
proportion of patients, with anal pain and bleeding for years after successful 
therapy. Some require a colostomy for adverse late effects of CRT. The late effects 
likely result more from the radiation therapy rather than the chemotherapy. The 
symptoms related to treatment generally peak within the first 2 years, but radiation 
injury can develop after an interval of 5 to 10 years. Anal ulcers, fistulae, severe 
fibrosis, skin ulceration, and rectal stenosis may occur. There may be major issues 
with sexual function after CRT and there may be adverse psychosocial effects 
including depression. Radiation-induced pelvic and hip insufficiency fractures are 
also increasingly being recognized and reported, and are more common in women. 
Although HIV-infected individuals on cART are now commonly able to complete 
a full course of CRT, a study from the United Kingdom showed that CRT was 
associated with a reduction in CD4 level. The reduced CD4 level persisted after 
completion of therapy and may have contributed to the deaths of patients in 
remission (58). 
Currently we have no way to distinguish HSIL that will progress to cancer from 
HSIL that will never progress. This is a problem, since treatment of HSIL, while 
well tolerated, does have consequences in terms of acute morbidity and cost to the 
healthcare system. Further, HSIL is common among HIV-infected individuals, and 
a high proportion of HIV-infected men and women will be diagnosed with anal 
HSIL at some point if screening programs are implemented. Of note, however, even 
with the need to “over-treat” anal HSIL, screening for and treating anal HSIL is 
likely to be cost-effective compared with treating anal cancer. Several years ago 
Drs. Sue Goldie and Palefsky published formal cost-effectiveness studies of 
screening for and treating ASIL in HIV-infected and HIV-uninfected MSM 
(59)(60). Both of these studies showed these procedures to be highly cost-effective. 
Since that time, we believe that our treatment approaches have improved further, 
and that computation of cost-effectiveness today would yield even better numbers. 
Although the treatment of anal HSIL is not inexpensive, and may require multiple 
attempts and visits, anal screening and treatment is highly cost-effective because 
the risk of anal cancer is not evenly distributed throughout the population and we 
know who the high-risk groups are. A more recent study from the United Kingdom 
concluded that anal screening was not cost-effective for MSM, but the assumptions 
that went into their model were very different from those used in the Goldie papers, 
stemming from paucity of data and perhaps the very different cost structures of the 


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U.K. and U.S. health care systems (61). Unlike cervical screening, which is 
recommended for all sexually active women, we only need to screen a subset of the 
population to have a substantial impact on the incidence of anal cancer. It is also 
possible that biomarkers of progression from HSIL to cancer can be identified, and 
this might reduce the number of HSIL lesions that need to be treated. This is one of 
the major aims of the correlative science component of the ANCHOR study and if 
successful, it will allow clinicians to target their therapy to only those HSIL at 
highest risk of progression. This would reduce unnecessary treatment and likely 
improve cost-effectiveness even further. 
2.3.3 Impact of preventing anal cancer through secondary prevention 
In considering the value of preventing anal cancer, it is critical to consider both 
prevention of deaths and impact on quality of life among those who survive 
treatment of anal cancer. Simple treatments such as those that will be evaluated in 
the ANCHOR study to prevent development of cancer are far less morbid than 
treating invasive cancer (56). Clearly prevention of anal cancer through 
identification and treatment of HSIL would be preferable to waiting until cancers 
develop. If we can prevent most anal cancers, we will reduce the need for CRT in 
thousands of patients per year in the U.S., and prevent about 800 deaths per year. 
Worldwide we will reduce the need for CRT in many more thousands of patients, 
and likely prevent several thousand deaths per year. To address the issue of 
unnecessary treatment of anal HSILs that would not have progressed if left 
untreated, ANCHOR study has a robust correlative science component designed to 
identify biomarkers that have the potential to distinguish anal HSIL at high risk of 
imminent progression to cancer from anal HSIL that can be safely observed. Given 
the biological similarity to other HPV-related cancers, these data may be of value 
in prevention of cervical and HPV-related oral cancer, and may contribute to 
prevention of many hundreds of thousands, if not millions, of cases of cancer and 
deaths. The ANCHOR study correlative science program also has the potential to 
lead to new therapies for anal HSIL and cancer that would simplify treatment, and 
these too may potentially of value in prevention and treatment of cervical and oral 
cancer. 
2.3. 
Implementation of the ANCHOR study findings if the data support treating anal 
HSIL to reduce the risk of anal cancer 
If the ANCHOR study shows that treatment of anal HSIL significantly reduces the 
incidence of anal cancer, it is likely that these results will quickly lead to standard 
of care guidelines. The ANCHOR study is a “strategy” trial, in which the strategy 
of treating HSIL is being tested for its ability to reduce the incidence of anal cancer. 
It is not a treatment trial of any one specific treatment modality. Thus, if the 
ANCHOR study shows that treatment of anal HSIL is effective in reducing anal 
cancer, standard of care guidelines will be adopted that use the most current 
information on how to treat anal HSIL, including the methods used in this study. If 
better methods become available in the future, the demonstration that treatment of 
anal HSIL reduces the risk of cancer will likely allow those newer treatments to be 
adopted as standard of care by showing their efficacy in treating HSIL. This is much 
easier and less expensive than needing to show efficacy in reducing the incidence 


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of cancer, as is proposed for the ANCHOR study. 
Conversely, if the data from the ANCHOR study show that treating HSIL is not 
effective in reducing the incidence of cancer, we expect that dissemination of these 
findings will lead to a reduction and probably cessation of anal HSIL screening and 
treatment activities. If so, data from the ANCHOR study may still lead to an overall 
improvement in patient outcomes. Here it is possible that new progression 
biomarkers and therapeutic approaches identified in the ANCHOR study 
correlative science program may allow us to identify and treat anal cancers much 
earlier than is the case currently. This in turn may lead to successful treatment of 
these cancers with much less morbidity than CRT. It is very possible that the data 
will also be of value for prevention and treatment of cervical and oral cancer. 
If treating HSIL is effective in reducing the incidence of anal cancer, then methods 
to identify those with anal HSIL through screening of at-risk individuals will 
become standard of care as well. As with treatment, there are several approaches 
that are currently available, but newer screening methods are being tested and 
others will be tested in the future. Correlative science studies enabled by the 
ANCHOR study as well as several others currently in progress will likely lead to 
optimization of screening strategies for anal HSIL, to the identification of those 
patients with HSIL who are at increased risk of progression to anal cancer, and 
potentially to new treatment approaches to HSIL and cancer.  
2.3.5 The impact of the ANCHOR study for setting public health policy extends well 
beyond HIV-infected men and women 
In addition to patients with HIV infection, those with a history of genital warts, or 
signs of perianal HPV-related lesions, whether warts or HSIL, may also potentially 
benefit from screening. With the growing incidence of anal cancer among the 
general population of women, we may reach a point where screening larger 
numbers of women who do not fit into any of these groups (e.g., women reporting 
a history of anal intercourse), may also become useful, but this will require further 
study. Although the non-HIV-infected groups listed above are not eligible for the 
ANCHOR study for several reasons outlined in Section 2.3.7, we also expect that 
the standard of care guidelines adopted for HIV-infected men and women will 
apply to the non-HIV-infected groups as well. Anal HSIL in HIV-infected men and 
women is harder to treat than in HIV-uninfected men and women for a number of 
reasons, including multifocal lesions, large lesion area, and high risk of HSIL 
recurrence after treatment. We believe that if the ANCHOR study shows that 
treatment reduces the risk of anal cancer in HIV-infected individuals, then the 
results will be generalizable to other groups in whom treatment of HSIL will be less 
challenging. Since most cases of anal cancer in the general population are occurring 
in HIV-uninfected individuals, it is clear that the data from ANCHOR study will 
have an impact on populations at risk for anal cancer that extend well beyond those 
with HIV infection. 
2.3.6 The impact of the ANCHOR study extends beyond anal cancer 
The ANCHOR study is also designed to have wide impact through its correlative 
science component. Multiple samples will be collected from patients during the 


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trial, and we plan to analyze them through a series of separately funded correlative 
studies. The ANCHOR study is unique because it focuses on prevention of 
progression from HSIL to cancer. A study such as this is not currently possible at 
any other anatomic site at which HPV-associated cancer develops. A study of 
progression to cervical cancer is not possible because it is currently standard of care 
to treat cervical HSIL. A study of progression to oral cancer is not possible because 
it is difficult to reliably identify oral cancer precursor lesions for treatment. The 
ANCHOR study not only offers the opportunity to determine the efficacy of HSIL 
treatment to prevent cancer, but careful follow-up and sample collection from those 
in the active monitoring arm will allow us to better understand the molecular 
pathogenesis of progression to cancer. This may lead to new approaches to treating 
HSIL to prevent progression to cancer and potentially cancer itself. The study also 
provides the opportunity to identify biomarkers of progression to cancer. Given the 
strong biological similarities between anal, cervical and oral cancer it is very 
possible that biomarkers and treatments identified in the ANCHOR study will be 
applicable to cervical and HPV-associated oral cancer. Biomarkers of progression 
from cervical HSIL to cervical cancer would be of great value in resource-limited 
settings where the cost of treatment could be reduced if there were ways to identify 
those at highest need of intervention. This is often the case in developing countries 
that lack organized cervical cancer screening programs. In developed countries, this 
information could be valuable to identify those at highest need of close follow-up 
after treatment of cervical HSIL. Recurrence after treatment of cervical HSIL is 
particularly common in HIV-infected women (62) and this is just one group that 
might benefit from use of these biomarkers. Development of better treatments to 
reduce the incidence of HPV-related cancers regardless of the anatomic site at 
which they occur, or better biomarkers to identify those at risk, and who most need 
treatment of HSIL, has the potential to save hundreds of thousands, if not millions 
of lives. 
In summary, the ANCHOR study will have a major impact in several ways: 1) If 
treatment of HSIL is shown to be effective in reducing the incidence of anal cancer, 
it will establish standard of care guidelines for treatment of HSIL in at-risk 
populations. The results will be applicable to both HIV-infected and HIV-
uninfected at-risk populations; 2) Information obtained on molecular pathogenesis 
of progression from anal HSIL to anal cancer, and development of biomarkers of 
progression may lead to an ability to target those with anal HSIL most in need of 
treatment, and better methods treating anal HSIL to prevent anal cancer, and 3) the 
results of ANCHOR will likely extend to cervical and HPV-associated oral cancer. 
2.3.7 Rationale for eligibility criteria 
The ANCHOR study includes only HIV-infected men and women for several 
reasons. First, this is the group with the highest incidence of anal cancer. Second, 
since the incidence of cancer is highest in this group, we expect to obtain the 
necessary number of cases of anal cancer in the active monitoring arm with the 
smallest number of participants possible, and over the shortest amount of time 
possible. Third, given the challenges associated with treating HSIL in this group 
(multifocal lesions, risk of HSIL recurrence after treatment) we believe that if the 


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study shows that treatment reduces the risk of anal cancer in HIV-infected 
individuals, then the results will be generalizable to other groups in whom treatment 
of HSIL will be less challenging. In addition, performing a RCT like ANCHOR 
study in HIV-uninfected individuals would not be practical since such a study 
would require many more participants who would need to be followed for a 
substantially longer period of time. It would also be difficult to generalize the 
results of any study in HIV-uninfected individuals to HIV-infected individuals. 
We also have elected to study only HIV-infected men and women 35 years or older. 
This is meant to enrich the study population at risk for cancer since anal cancer 
occurs only rarely under this age even among HIV-infected individuals. Fewer than 
1% of anal cancers occur under the age of 35 years (1). 
Finally, our goal is to recruit to this study at locations around the U.S. that will 
allow us to enroll participants whose racial/ethnic background mirror the current 
demographics of the HIV epidemic, to ensure the generalizability of the results to 
the HIV-infected population into the future. 
We have considered other potential methods to enrich the study population for risk 
of cancer such as HPV 16 or 18 positivity as an enrollment criterion but other HPV 
types may also play a role in anal cancer, particularly in the HIV setting, and we 
want to ensure that our results are as generalizable as possible. Although HPV 16 
is the predominant HPV type in anal cancers overall (18), little is known about the 
distribution of HPV in anal cancers among HIV-infected men and women. Recent 
data suggest that HPV 16 may play a less prominent role in cervical cancers of 
HIV-infected women compared with HIV-uninfected women (63)(64), and it will 
be important to know whether this is also true of anal cancers. In one study from 
Germany, all five squamous cell carcinomas (SCCs) of the anal canal contained 
HPV 16 compared with only one of four anal margin SCCs. HPV 31, HPV 33 and 
HPV 68 were found in the other three anal margin SCCs) (52). 
We do not plan to use CD4 level, HIV viral load, history of cART, and nadir CD4 
level as entrance criteria, since our own data show that individuals on cART have 
a wide range of CD4 levels at the time of anal cancer diagnosis (25). A high 
proportion of HIV-infected individuals are on ART and thus use or lack of use of 
cART also would not be a useful entrance criterion. Other than the recent 
publication using VA data suggesting that having more time with an undetectable 
HIV viral load was protective against developing anal cancer (13), there are 
insufficient data to use HIV viral load as an entrance criterion. It would be difficult 
to reliably quantify a prospective participant’s past viral load control, and 
regardless of what it was at the time of study entry, we would feel ethically obliged 
to work with the participant and their HIV care provider to maximize the likelihood 
that they have the best viral load control possible. 
2.3.8 Equipoise in the treatment and active monitoring arms 
We have had many discussions with specialists in medical ethics and with many 
members of the HIV community. We believe that there is equipoise in the treatment 
and active monitoring arms of the ANCHOR study. Participants in the active 
monitoring arm are at risk of progressing to anal cancer. However, we do not know 


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that treatment of anal HSIL is effective to reduce anal cancer, and individuals in the 
treatment arm may be at similar risk, hence the need for the ANCHOR study. If 
HSIL treatment is shown to be ineffective in prevention of incident HSIL, then 
participants in the active monitoring arm are being spared treatments that may be 
causing unnecessary morbidity. 
We have designed the study to minimize the risk of harmful outcomes associated 
with developing invasive cancer for ethical reasons and to maximize the likelihood 
of study retention. For this reason, we are following participants in both arms very 
closely, i.e., at 6-month intervals or shorter. With close follow-up, it is possible (if 
not probable) that if a cancer does develop, it will be an early cancer. Detecting 
cancers early is likely to be advantageous since treatment is usually successful at 
the early stages, and is less successful at later stages. Cancers that occur while still 
defined as superficially invasive squamous cell carcinoma (SISCCA) (26) may be 
treatable with local excision. Local excision is not standard of care for intra-anal 
SISCCA but this is a research question that will be addressed in a study under 
investigation by the AMC HPV Working Group (participants in the ANCHOR 
study who are diagnosed with SISCCA may be referred to that study if it is still 
recruiting). Local removal could potentially spare participants the side effects 
associated with CRT. However, in the informed consent process, we will be explicit 
with the participants that despite the frequent monitoring in the study, those who 
develop cancer may need CRT with its associated morbidity, and they may also die 
of their anal cancer. 
It is very possible that in the absence of the ANCHOR study, many individuals who 
agree to participate and who are randomized to the active monitoring arm would 
not have undergone any form of screening- screening is not currently standard of 
care and is not available to most at-risk individuals. If they were going to develop 
cancer, they would have done so anyway, outside the context of any study and these 
individuals would eventually present with symptoms from their cancer. The cancers 
may be diagnosed at a later stage than if they were being monitored in the 
ANCHOR study, would more likely require CRT and they may have increased risk 
of death. Being in the active monitoring arm therefore offers the possibility of 
earlier diagnosis of cancer than if they were not in the study at all, a better chance 
of survival and very possibly fewer side effects of cancer treatment. 
Being in the treatment arm offers the possibility of reduced risk of developing anal 
cancer compared with being in the active monitoring arm. However, as noted 
above, we do not yet know if this is the case, and it is possible that individuals in 
the treatment arm will be undergoing a series of procedures with little or no clinical 
benefit. Many of the treatments described above lead to a high clearance rate per 
lesion, but patients may still have a focus of anal HSIL somewhere even after 
successful treatment of a given lesion because of metachronous disease. We will 
therefore allow ongoing treatment of HSIL throughout the course of the study. 
Although the HSIL treatment procedures that we will use in the ANCHOR study 
are generally well tolerated, participants in the treatment arm may experience 
treatment-associated side effects, including post-procedure pain or discomfort, 
bleeding and although rare, infection or stenosis. 


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The DSMB will review data including progression rates to cancer in both arms on 
a regular basis (e.g., 6 months, and not less than once per year) and will discontinue 
the study if the data indicate that it is no longer ethical to continue. The DSMB will 
also monitor study data relevant to the assumptions so that necessary changes can 
be made to accrual strategies, eligibility criteria, sample size, or other study 
parameters in a timely manner. Interim analyses are described in Section 10.5.2. 
In summary, participants in both arms who develop anal cancer may need CRT, 
and may be at risk of death due to anal cancer. It is not known if this risk is different 
between the two arms. Participants in both arms may also benefit from early 
diagnosis of anal cancer and may be spared the side effects of CRT. Some might 
benefit from being in the study compared with not being followed at all, which is 
currently the case for most at-risk individuals. Finally, if the study hypothesis is 
rejected, i.e., that treatment of HSIL is not effective to reduce the incidence of anal 
cancer, then the participants in the active monitoring arm will have been spared the 
morbidity associated with treatments of those participating in the treatment arm. 
2.3.9 Feasibility of recruitment to the ANCHOR study 
In preparation for the ANCHOR study, we performed a series of studies examining 
willingness to enroll in the study. Funded by NIH, we performed focus groups in 
ten different U.S. cities comprised of HIV-infected men and women. In each city 
we obtained data from several groups, individually composed of HIV-infected 
White, Black and Hispanic men or women. Our goal was to assess the feasibility 
of performing the ANCHOR study among the demographic groups that currently 
comprise the HIV epidemic. Given that we expect to recruit to the ANCHOR study 
in 10 sites spread around the United States where the HIV epidemic is prominent, 
we sought to assess willingness to participate across a wide geographic and 
demographic spectrum of potential participants. We assessed willingness to 
participate in a trial with the same design as ANCHOR in an ethnically diverse 
sample of HIV-infected men (n=202) and women (n=39) in 10 US cities. Focus 
group participants were screened for eligibility (35 or more years of age and HIV-
infected) by telephone and assigned to one of four focus group types based on 
ethnicity and gender (Black, Hispanic or Latino, and White men, and a multi-ethnic 
group of women). Two moderators traveled to each of the ten cities to conduct the 
focus groups following a semi-structured guide. The Hispanic groups were 
conducted primarily in Spanish, primarily in English or both, depending on 
participant preference. 
We also performed an internet-based survey of 257 HIV-infected men and women 
in 20 cities. The online survey targeted the same population living in the same 10 
cities used for the focus group portion of the study, plus 9 additional US cities 
(Boston MA, Minneapolis MN, Omaha NE, Philadelphia PA, Sacramento CA, San 
Antonio TX, San Diego CA, Tampa FL, and Washington DC) and one Canadian 
city (Vancouver BC). The study was conducted using SurveyGizmo and 
participants were recruited through flyers mailed to AIDS service organizations and 
HIV clinics. We also directly surveyed HIV-infected men to determine their 
willingness to participate in study with the ANCHOR study design after being 
given a diagnosis of anal HSIL. 


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In summary, most HIV-infected individuals surveyed expressed an interest in 
participating in the ANCHOR study even with a 50% chance of being randomized 
to either arm. In so doing they would be supported by their HIV primary care 
provider. Survey respondents mostly preferred being in the treatment arm, but with 
appropriate support, would likely stay in the active monitoring arm. Among the 20 
participants who were surveyed after being given a diagnosis of HSIL, most 
expressed a willingness to be randomized even after having undergone procedures 
very similar to the ANCHOR study screening protocol and after having come to 
the clinic fully expecting to be treated for their HSIL. In the worst-case scenario, 
individuals may choose not to continue in the study after their screening visit, and 
if that is the case, we will screen additional individuals to reach our recruitment 
target of 5058 randomized men and women. Taken together, these data give us 
confidence that we will be successful in recruiting to the ANCHOR study as 
described in the schema, and that we will be able to successfully retain participants 
in both arms with sufficient ongoing education and support. 
2.3.10 Rationale for health-related quality of life assessment 
The ANCHOR study presents a unique opportunity for the assessment of health-
related quality of life (HRQoL) among those diagnosed with anal HSIL who are 
either treated or observed. At the time of protocol implementation, no measure 
existed that captures the specific symptoms and related experiences of living with 
or being treated or monitored for anal HSIL. Although definitions of HRQoL vary, 
there is general agreement that it is a “multi-domain concept that represents the 
patient’s general perception of the effect of illness and treatment on physical, 
psychological, and social aspects of life.” (125). The original funding application 
for the ANCHOR trial included a HRQoL measurement component using existing 
symptom measures not specific to anal HSIL. During the NCI’s Cancer Therapy 
Evaluation Program (CTEP) review of the initial protocol draft, CTEP reviewers 
strongly recommended that the PI work to develop a HRQoL measure that was 
specific to anal HSIL with the goal of implementing such a measure within the 
ANCHOR trial so as to provide important secondary outcomes. To this effect, the 
AMC began development (protocols AMC-A02, AMC-A03, and AMC-A04, 
described further below), with the ultimate aim of validating and implementing the 
new measure within the ANCHOR trial. 
There is a paucity of data on symptoms and concerns of persons diagnosed and 
treated for anal HSIL. Studies examining aspects of HRQoL in persons being 
screened for and treated for anal HSIL used diverse measures to assess HRQoL, 
ranging from possibly study-specific ad hoc items (126), symptom measures 
designed for HIV-positive persons but not specific to anal HSIL (127), and generic 
HRQoL measures (128), supplemented with validated sexual functioning items 
(128) or psychological distress measures (128). Although many of these measures 
showed sensitivity to the impact of anal cancer screening or treatment, the variety 
of measures used and lack of specificity for anal HSIL diagnosis, monitoring, and 
treatment support the need for a rigorously developed and validated measure to 
provide reliable, comparable data across anal HSIL populations and treatments. In 
addition, within the ANCHOR trial, different treatment modalities are permitted for 


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those participants assigned to the treatment study arm: Infrared coagulation, 
electrocautery, laser, treatment under anesthesia (vs. local anesthesia), topical 5% 
fluorouracil cream (5-FU), and topical 5% imiquimod cream. Thus, it is important 
to develop a HRQoL measure that can capture the array and severity of symptoms. 
2.3.11 Development of the ANCHOR Study Health-Related Symptom Index and study 
implementation for QoL assessment 
Initial development of the ANCHOR Study Health-Related Symptom Index 
(ANCHOR HRSI, or A-HRSI) was conducted under protocol AMC-A02, and 
included a four-phase process to establish content validity. This measure was 
specifically created for use in the ANCHOR trial using state-of-the-art instrument 
development methodology. Expert consultation was used to inform concept 
elicitation interviews with 41 participants who were eligible for ANCHOR. Based 
on these interviews, a 23-item measure was drafted and then tested in a second 
cohort of 45 participants eligible for ANCHOR in a process of cognitive 
interviewing. This resulted in a 25-item content-valid measure of physical 
symptoms, physical impacts and psychological symptoms (129). Reliability was 
then assessed in 100 ANCHOR participants across a 7-10 day timeframe, with 
Cronbach’s α in the fair to good range across the three domains (i.e., 0.79 – 0.82), 
indicating adequate internal consistency for the measure (130). The test-retest 
reliability was good across each of the three domains (intraclass correlation 
coefficients = 0.80 – 0.84). Construct validity of the measure was then established 
in a fourth cohort of individuals enrolled in ANCHOR (n=303) who completed A-
HRSI at a single time point, with the three domain model (i.e., physical symptoms, 
physical impacts, psychological symptoms) confirmed via confirmatory factor 
analysis, with these three domains strongly associated (Pearson’s r) with 
corresponding domains from the well-established MD Anderson Symptom 
Inventory and Functional Assessment of Cancer Therapy – General tools.  
The final version of the A-HRSI (both English and Spanish) includes 25-items that 
asks participants to rate the degree of prevalence or impact of their physical 
symptoms (10 items), physical impacts (6 items), or psychological symptoms (9 
items) on a 0 (not at all) to 4 (very much) scale. Items within each domain are 
averaged, with higher scores indicative of worse HRQoL. The measure takes 
approximately 6-10 minutes to complete either via telephone-facilitated interview 
or electronic patient-reported outcomes (ePRO) platform in the data entry system 
for the trial, AdvantageEDC.  
The ePRO system is a separate module of AdvantageEDC, the data entry system 
for this trial, that allows the participant to complete questionnaires directly in the 
data entry system. This eliminates any need to administer a paper questionnaire and 
to transcribe the data into the system. The ePRO data capture system is user-
friendly and compatible with major internet browsers and a variety of electronic 
devices. In an effort to evaluate participant behavior in using the ePRO, the amount 
of time spent on each ePRO item screen will be captured.  
The completed substudy also made use of the patient version of the Eastern 
Cooperative Oncology Group Performance Status (Patient ECOG) and at time of 


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follow-up assessment, the Patient Global Impression of Change (PGIC) scale. The 
Patient ECOG was adapted from clinician to patient language through focus groups, 
interviews, and comparisons of clinician and patient responses. This measure 
consists of a single item that asks participants to rate their current performance 
status from 0-4. This item takes approximately 1 minute to complete. The PGIC 
allows participants to consider, using a 7-point scale ranging from “very much 
worse” to “very much better,” whether their overall quality of life has changed since 
the last time they were assessed. This item takes approximately 1 minute to 
complete. 
To adapt A-HRSI for ANCHOR participants who prefer Spanish as their primary 
language for healthcare delivery, content validation of A-HRSI was completed in 
Spanish under AMC-A04. A-HRSI was translated into Spanish by the MSKCC 
Patient-Reported Outcomes, Community-Engagement, and Language (PRO-CEL) 
Core Facility. Using similar qualitative methodology as AMC-A02, cognitive 
interviews were completed across two rounds with a total of 17 participants who 
were eligible for ANCHOR. Through this process, changes to item and response 
translations were made based on suggestions from participants, but no substantive 
changes were implemented to alter item content. As such, the Spanish version of 
A-HRSI has been adequately content validated and is appropriate for use in the 
ANCHOR trial. 
Final stage of A-HRSI development and substudy results 
The aim of the A-HRSI substudy wass to assess the responsiveness (sensitivity to 
change) and clinical significance of the A-HRSI subscales by comparing change 
scores within groups of participants as defined by participant responses to the PGIC 
item. Responsiveness posits that a psychometric scale is able to detect the 
difference between patients who experience changes in symptoms and patients who 
experience no change. Thus, we used the PGIC item to document change between 
follow-up 1 (time 2) and FU 2 (time 3) time points (“Since the last time you 
completed a questionnaire, how would you rate your OVERALL QUALITY OF 
LIFE?”, marked on a 7-point scale from “Very much worse” to “Very much better” 
with a mid-point of “No Change”). Each participant was be categorized into one of 
3 groups: change for the worse (a response of “Very much worse”, “Moderately 
worse”, or “A little worse”), no change (“About the Same”), and change for the 
better (“A little better”, “Moderately better”, and “Very much better”). Changes in 
the A-HRSI scores were also be calculated (FU1 scores minus baseline scores). 
Responsiveness is supported if changes in the A-HRSI scores correspond reliably 
with the anticipated changes between the three groups. This can be addressed by a 
one-way ANOVA on the changes in the A-HRSI scores across the 3 groups 
(“worse”, “no change”, and “better”). 
The PGIC item has been used with recall periods up to 6 weeks. It may or may not 
work for recall periods exceeding 6 weeks. We collected the dates of assessments 
and thus can assess for trends in PGIC item. 
For this final stage of the A-HRSI development, up to 100 eligible participants who 
consented to participate to the ANCHOR study and to the optional A-HRSI 


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substudy to demonstrate measurement scale responsiveness (Section 1.3.2) were 
consented and enrolled between July- and October 2019, and were administered the 
A-HRSI and several legacy measures before randomization and at two time points 
post-randomization and treatment. Final data collection occurred in February 2020. 
This fifth cohort of individuals enrolled in ANCHOR (n=103) completed A-HRSI 
at three time points in order to establish clinical responsiveness (i.e., sensitivity to 
change) of the tool. A-HRSI and self-reported Patient Global Impression of Change 
(PGIC) scale and ECOG Performance Status (ECOG PS) item were administered 
either via ePRO tool or via telephone facilitated interview at time of enrollment up 
until time of trial randomization (T1), 14-70 days post-randomization (T2), and 71-
112 days post-randomization (T3). Participants at follow-up timepoints were 
categorized into two sets of three groups based on PGIC and ECOG PS responses 
(“worse,” “no change,” “better”), with the primary responsiveness analysis using 
these three groups in a one-way analysis of variance (ANOVA). Below we show 
the results for change at T3 vs. T2. The change from T3 to T2 in subscales showed 
those who had changed for the worse on the PGIC or ECOG PS had worsening A-
HRSI subscales as compared to those who changed for the better or had no change.  
Table 2-A: Descriptive statistics for change in subscales (T3 minus T2) according to change 
in PGIC and ECOG PS, mean (SD) 
 
PGIC 
ECOG PS 
A-HRSI 
Subscale 
Change for 
better 
N=34 
No Change 
 
N=38 
Change for 
the worse 
N=12 
Change for 
better 
N=18 
No Change 
 
N=43 
Change for 
Worse 
N=24 
Physical 
Symptoms 
-0.12 (0.86) 
0.05 (0.74) 
0.27 (0.65) 
-0.23 (0.61) 
-0.06 (0.80) 
0.37 (0.78) 
Physical 
Impacts 
0.01 (1.14) 
-0.06 (0.99) 
0.65 (1.25) 
-0.76 (1.18) 
0.10 (0.74) 
0.63 (1.22) 
Psychological 
Symptoms 
-0.22 (1.39) 
0.05 (1.19) 
0.90 (1.50) 
-0.34 (1.42) 
-0.08 (1.11) 
0.64 (1.55) 
 
Table 2-B: Responsiveness comparison of changes (T3 minus T2), mean difference (95% 
confidence interval) 
 
PGIC 
ECOG PS 
A-HRSI 
Subscale 
Change for worse vs. 
no change 
Change for worse 
vs. better 
Change for worse vs. 
no change 
Change for worse vs. 
better 
Physical 
Symptoms 
0.22 (-0.30 to 0.73) 
P=0.401 
0.39 (-0.13 to 0.90) 
P=0.144 
0.43 (0.05 to 0.82) 
P=0.027 
0.60 (0.13 to 1.07) 
P=0.013 
Physical 
Impacts 
0.71 (-0.01 to 1.43) 
P=0.052 
0.65 (-0.08 to 1.38) 
P=0.081 
0.53 (0.02 to 1.03) 
P=0.041 
1.39 (0.77 to 2.01) 
P<0.001 
Psychological 
Symptoms 
0.86 (-0.02 to 1.73) 
P=0.055 
1.12 (0.23 to 2.01) 
P=0.014 
0.72 (0.04 to 1.39) 
P=0.037 
0.98 (0.14 to 1.82) 
P=0.022 
Note: P-values are two-sided and from post-hoc pairwise comparison of means in an analysis of variance. The overall 
F-statistic was significant for PGIC group for psychological symptoms (p=0.047) and for ECOG PS group for physical 
symptoms (p=0.026), physical impacts (p<0.001), and psychological symptoms (p=0.042). 
There was a significant moderate relationship (i.e., standardized response means = 
0.52 and 0.60 for physical impacts and psychological symptoms, respectively) 
between changes in A-HRSI subscales and changes in ECOG PS from T2 to T3. 
These results provide evidence of A-HRSI clinical responsiveness that will be 


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further explored as part of the ANCHOR trial through the refined assessment 
windows for the formal QoL objective: T1 will remain the same (enrollment up 
until time of trial randomization); however, T2 will now be revised to occur 2-7 
days post-randomization to correspond with the period during which most 
participants are symptomatic after their initial treatment or assignment to active 
monitoring. T3 will be revised to occur 4-weeks after randomization, at which time 
it is expected that participant symptoms or impacts due to treatment or assignment 
to active monitoring will be lessened. Additional assessment time points T4-T8 will 
then occur at annual visits through the fifth year of study participation in order to 
evaluate any long-term health-related symptom impacts related to treatment or 
assignment to active monitoring. 
A-HRSI implementation for QoL aim 
After validating the A-HRSI, the A-HRSI is being implemented with an 
amendment (protocol version 13.0), and administered to a sample of 500 ANCHOR 
participants who consent to participation for these additional surveys. The 
questionnaire will be administered before randomization and at seven time points 
thereafter (2-7 days, 4 weeks and 12, 24, 36, 48, and 60 months),  
2.4 
Overview of Study Design 
2.4.1 Guiding principles to the study design 
1) To address our primary objective, clinicians will make their best attempt to treat 
HSIL throughout the course of the study for those in the treatment arm. The 
approaches used in the ANCHOR study to treat anal HSIL are essentially 
identical to those used in current clinical practice in the U.S. The proposed 
treatment approach parallels real-world clinical practice, in which patients may 
respond well to a given treatment, but experience a recurrence or development 
of a new lesion. The treating clinician may then embark upon another course of 
treatment with the same modality or a new one depending on a number of 
factors. Our goal in this study is to ensure that the results are as generalizable 
as possible while also ensuring that clinical standard operating procedures are 
rigorously followed. Standardized protocols will be used to treat HSIL in which 
the choice of therapy will depend on the size, number and location of lesions 
but clinicians will be allowed some latitude in their choice of therapy within 
these well-defined parameters. 
Participant safety is our first priority and if a clinician or a participant feels 
uncomfortable about continuing in either arm, they will be withdrawn from the 
study. Within those parameters, we will emphasize ongoing participant support 
and education to maximize study retention within each arm. Participants who 
are shown to have anal cancer during screening will be immediately referred 
for appropriate therapy. For all participants diagnosed with cancer during the 
study, stage of cancer at diagnosis will be recorded at the time of study 
discontinuation. 
2) We have designed the study to make the procedures, including biopsies as 
similar as possible between the observation and treatment arms, with of course, 


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the exception of the treatment procedures in the treatment arm. We have 
included annual biopsies in both arms as a means of ongoing monitoring of 
disease status and to collect samples for correlative studies. However, biopsies 
may occur in either arm at additional visits. In both arms, this may occur if the 
clinician suspects progression to cancer at any visit. In the treatment arm, this 
may occur if the protocol for the treatment modality being used calls for 
documentation of disease status to enable a decision about the need for 
additional treatment. 
3) We will have regular meetings among all study investigators (every 6 months) 
and with study investigators at their sites that include ongoing and rigorous 
quality assurance monitoring. 
4) We will continuously monitor developments in the HPV/SIL/cancer field and 
if a new therapy emerges that should be implemented, we will modify the study 
design to allow it; this will not compromise the integrity of the study, since our 
goal is to do our best to eradicate HSIL. For example, a study will soon be 
initiated in the AMC comparing 5-fluorouracil cream to imiquimod for 
treatment of anal HSIL. If one of these is shown to be superior, we will modify 
the protocol to recommend prioritizing the use of the superior medication. 
5) Although we believe that most HSIL lesions can be treated successfully, there 
is still the possibility that some will progress to cancer after treatment, or that 
cancer may develop from a focus of HSIL that was clinically unrecognized and 
therefore not treated. For this reason, we have powered the study to detect a 
75% reduction, not 100% reduction, in the incidence of anal cancer in the 
treatment arm compared with the active monitoring arm. We have shown that a 
75% reduction in incident anal cancer in the treatment arm would be cost-
effective (56). It would likely be viewed in the community as a worthwhile 
clinical outcome and would likely result in implementation of screening for 
HSIL and treatment in at-risk populations. 
6) Management of HIV infection will not be part of the ANCHOR study per se. 
Rather, the ANCHOR study staff will work closely with the participant and 
their HIV care provider to ensure that the participant is receiving optimal HIV 
care. 
 
 


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2.4.2 Schema 
Information Visit: This is a recommended information session and may be done 
with potential participants individually or in groups. The study will be explained in 
detail, and knowledge will be assessed. Participants will be given a copy of the 
informed consent to take with them and encouraged to discuss participation in the 
study with their health care provider, or may give consent for participation at the 
information visit. Potential participants should be asked to return for a screening 
visit within 4 weeks; this is done to ensure that the he or she is truly committed to 
being in the study if qualified, and to answer additional questions that may have 
arisen since the information visit. By explaining the study in detail in advance, it is 
also expected that some potential participants may inform the study staff of issues 
that may preclude them from enrolling, such as prior history of HSIL or known 
abnormalities in blood tests such as platelets, etc. In so doing, we will maximize 
the efficiency of the screening visit that follows. If the participant gives informed 
consent at the information visit, screening procedures may be provided at that visit 
at the investigator’s discretion. 
Visit 0 (targeted for within 4 weeks of the information visit): This is the screening 
visit. Prior to performing any of the study procedures, the participants will be asked 
to sign the informed consent (if not signed previously) that gives us permission to 
screen them for HSIL and other eligibility criteria. The informed consent also gives 
us permission to randomize them if they meet all eligibility criteria after the 
screening visit. Participants who do not return for their screening visit (Visit 0) or 
baseline visit (Visit 1), or who are determined to be ineligible based on the results 
of the screening visit, will not be enrolled in the study. 
We will collect three anal swabs, the first for cytology and correlative research 
studies, the second for DNA-based research testing, and the third for RNA/protein 
analyses (please see Section 9.0). The clinician will perform a digital anorectal 
exam (DARE), HRA, and HRA-guided biopsies. Blood will be collected for 
complete blood count and serum storage. 
Anal biopsy and cytology samples will be read by the local pathologist with these 
results used to guide enrollment and ongoing decisions in the course of follow-up. 
Slides will be sent to a central laboratory for interpretation by a central study 
pathologist. Interpretations generated by the central pathologist will be used for the 
purposes of study data analysis. Readings that differ between the local and central 
pathologist with respect to presence or absence of HSIL or cancer will be 
adjudicated by a third pathologist. For scheduling/planning purposes, once the 
cytology and biopsy results are available as assessed by the local pathologist, the 
HRA clinician will indicate the treatment that would be done if one or more 
biopsies are positive for HSIL and the participant is randomized to the treatment 
arm. 
If the participant is determined to be eligible based on the results of testing done at 
the screening visit, he or she will be contacted and asked to come in for Visit 1. 
Eligible ANCHOR participants were also made aware of the optional health-related 
quality of life (HRQoL) questionnaire (substudy to validate the A-HRSI completed 


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enrollment in October 2019) to this protocol and provided informed consent to this 
study if they are interested in participating. Participants who were eligible and 
enrolled for the HRQoL objective (or the preceding substudy to validate the 
instrument) completed their initial HRQoL questionnaires via telephone-based or 
electronic administration up until the time of Visit 1 (within 2 weeks before 
randomization).  
Participants who are not eligible for randomization to the ANCHOR study will not 
be followed any further. At that time, they may potentially be eligible for other 
studies funded and performed outside the context of the ANCHOR study, e.g., a 
prospective study could be done (funded separately) of people who had LSIL but 
not HSIL to determine the natural history of LSIL. If the participant in the LSIL 
study is later diagnosed with HSIL, they could potentially be re-screened for the 
ANCHOR study if screening is still in progress and they did not receive any 
treatment for their HSIL within six months before randomization. 
Visit 1 (baseline visit with randomization): Within 1-12 (targeted for 6) weeks of 
the screening visit (Visit 0) we will see participants who were determined to be 
eligible for the study. A questionnaire detailing medical history and behaviors will 
be administered to all randomized participants to determine potential risk factors 
for progression to cancer among those who were enrolled into the study. 
At this point, the visit procedures will depend on the randomization arm. If the 
participant is randomized to the active monitoring arm, we will perform HRA to 
confirm the lesions being followed, counsel the participant, and discharge from the 
clinic with a follow-up appointment. If randomized to the treatment arm, the 
participant will undergo their first treatment at that visit (delay of up to two weeks 
is permitted), unless the plan is for the participant to have TUA. For participants 
randomized to the treatment arm and for whom TUA is planned, planning for the 
surgery will begin at Visit 1. If participants are randomized to the treatment arm, 
and the clinician opts to treat the lesions with patient-applied modalities, such as 
imiquimod or 5-fluouracil cream, the participant will be counseled regarding their 
use. Treatment will begin at that visit (or in no more than two weeks if a delay is 
required). Clinic staff will show the participant how to use the treatment at 
randomization. The participant will continue treatment on his or her own thereafter 
per protocol. Staff will check in regularly with the participant after initiation of 
treatment to determine if there are any problems. A staff member will contact the 
participant between visits in an effort to identify issues that may pose challenges 
for ongoing study participation. This will allow the study staff to intervene as 
necessary to ensure maximal participant retention. 
Participants who consented to the optional A-HRSI scale responsiveness substudy 
(n=100, completed in February 2020) completed questionnaires at 2 additional 
timepoints, targeted for 2 weeks after completing the initial treatment (time 2 
window: 10-70 days after randomization) and after completing all treatment for the 
first six-month block (time 3 window: 71-116 days after randomization) via 
facilitated interview with MSKCC staff or self-administered ePRO questionnaire 
in AdvantageEDC. 


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Participants who consent to the HRQoL objective (n=500, added with protocol 
version 13.0) will complete questionnaires at randomization (discussed above) and 
7 additional timepoints (see Appendix I, HrQOL Objective, for the windows for 
target completion and accepted forms: 
Table 2-C: A-HRSI completion time points for QoL objective 
Time Point 
Target Date 
T1 
Visit 1, before randomization 
T2 
2-7 days after randomization 
T3 
4 weeks after randomization 
T4 – T8 
12, 24, 36, 48, and 60 months 
 
Participants will be encouraged to complete questionnaires as self-administered via 
ePRO in AdvantageEDC; the option to complete the questionnaires via facilitated 
interview with clinical site staff (during visits or via telephone) will be available. A 
separate form collecting contact information updates and the participant’s 
preferences on the next questionnaire completion will also be administered at each 
time point. 
Treatment cycles consist of 6-month blocks. The schedule of interim visits during 
these blocks varies according to the treatment, as specified in the algorithms of 
Figure 4-A and Figure 4-B.. 
Visit 2 and Subsequent Visits: If randomized to the active monitoring arm, the 
participant will be seen every 6 months. At each of these visits, the clinician will 
collect three anal swabs: the first for local cytology and correlative studies, the 
second for DNA, and a third swab for RNA and protein studies. A DARE will be 
performed. HRA will be performed. At HRA the clinician will be asked to 
determine whether a lesion at a given visit is a recurrence in an area previously 
noted, or an incident lesion in a new area. At each visit the clinician will carefully 
map the location of each lesion and where they did their biopsies. Sites will also be 
requested to photograph each lesion at every visit using Second Opinion or other 
software that allows for easy sharing of images between study sites. The purpose 
of the cytology is to serve as quality control measure for the presence of HSIL. 
HSIL on cytology has high predictive value for detection of a HSIL on biopsy (64) 
(65). If the clinician is performing biopsies and the cytology shows HSIL but the 
biopsies do not, then the clinician will ask the participant to return for a repeat HRA 
at 3 months after the visit (± 4 weeks) to try to locate the HSIL. 
If the participant has been randomized to the treatment arm, he or she will follow 
the protocol specific to the treatment modality selected by the clinician. Once the 
participant has been cleared of HSIL, he or she will then be followed every 6 
months as described for those in the active monitoring arm. Further details of the 
procedures in the treatment and active monitoring arms are provided in Section 8.0. 
Participants will be asked to donate samples of blood and anal swabs for research 


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at every 6-month HRA visit. An additional blood sample (processed for plasma and 
whole blood fraction) will be stored for future studies at Visit 2, and serum will be 
stored from each 6-month visit. Anal biopsy tissue will be collected at each 6-month 
visit for those in the treatment arm who are suspected of having HSIL at HRA and 
potentially more often depending on the study arm and treatment modality. HSIL 
lesions will be biopsied annually for those in the active monitoring arm. The 
cytology swabs and formalin-fixed biopsy tissues will be processed as described in 
Section 9.0. Additionally, participants treated with ablative therapies will have one 
additional biopsy taken from the lesion with the most severe appearance of disease 
prior to treatment, no more than once every six months. This biopsy will be placed 
in RNALater for banking only. If a participant is diagnosed with cancer during 
screening or following randomization, an optional cancer biopsy (placed in 
RNALater) may be collected if the participant is willing to return to the clinic for 
biopsy before referral to treatment and study discontinuation. 
2.5 
Correlative Studies 
The ANCHOR study offers many scientific opportunities. We will be screening 17,385 
HIV-infected men and women, to identify and enroll 5,058 with anal HSIL. Approximately 
12,000 individuals will not be enrolled in the study, many of whom because they had LSIL 
or no disease at screening. Studying these individuals may be of great interest scientifically 
for the natural history of incident HSIL, but this is beyond the focus of the ANCHOR study. 
The correlative science studies described in this section were instead selected to focus on 
the issues that make this trial truly unique, i.e., progression of HSIL to cancer. Using the 
data and samples collected in the ANCHOR study, many questions can be asked about the 
molecular mechanisms of progression of HSIL to cancer. This correlative science section 
therefore focuses on three kinds of studies: 1) studies that will elucidate viral and host 
molecular mechanisms of progression from HSIL to cancer, 2) studies that focus on 
identification of biomarkers that will identify individuals at particularly high risk of 
progression from HSIL to cancer, and 3) studies that identify medical and behavioral risk 
factors for progression of HSIL to cancer. 
Any use of biospecimens collected as part of this trial requires specific review and approval 
by NCI CTEP or other mechanisms as will be set up for the NCI Clinical Trials Network 
(NCTN) or by OHAM, and by the ANCHOR Correlative Science Committee. For a request 
for use of specimens to be considered, a protocol for use of banked specimens (or an 
amendment to the protocol if the trial is still active) must be submitted to NCI CTEP; the 
protocol submitted for review must contain a clear statement of the scientific objectives 
and hypotheses, a statistical section that provides a brief description of the statistical design 
and analysis strategy along with sample size/power justification, a description of the assay 
methodology, and identification of the laboratory/individuals that will perform the assays. 
For non-standard assays, information about the assay’s analytical performance (e.g., 
sensitivity, specificity, bias, linearity, reproducibility, as applicable) also may be requested 
by the reviewers. 
The studies described below are not meant in any way to be a complete list; in each 
category there are several other questions that could be addressed, and we fully expect that 
many new questions will arise in the course of the study for each category. We therefore 
plan to ensure that specimen collection will be adequate to address the questions below, as 


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well as provide the opportunity to address new questions in the future as new data become 
available. These studies will be funded by mechanisms external to the ANCHOR study, 
mostly R21 and R01 grants. As such they will also undergo additional peer review prior to 
implementation, as described above. 
The studies described below that involve analysis of HSIL and cancer biopsies include 
three parallel experiments. Please see Figure 2-D on the next page. It is expected that most 
participants with anal cancer will have overlying HSIL, as well as several, anatomically 
discrete areas of HSIL that did not progress to cancer. Tissues shown to have invasive 
cancer will be microdissected to allow for analysis of the invasive cancer specifically, and 
compared with overlying HSIL. By identifying differences between the cancer and 
overlying HSIL, we will elucidate some of the steps involved in progression from HSIL to 
cancer. 
The biopsy that is microdissected for the invasive cancer part of the tissue will allow us to 
analyze the microdissected HSIL portion of the tissue that had not invaded. We will 
therefore have the opportunity to compare HSIL that progressed to cancer with concurrent 
HSIL that did not progress from the same participant, since most participants will have 
multiple foci of HSIL. We will also have the opportunity to compare the HSIL that 
progressed to cancer at a given visit to the same HSIL at one or more visits prior to the 
cancer progression. 
Comparing cancers to overlying HSIL offers different information from comparing HSIL 
that progressed with HSIL that did not progress. Comparing cancers and overlying HSIL 
may identify some of the last steps in progression to cancer. Similar comparisons between 
HSIL that progressed and HSIL that did not progress will provide insight into some of the 
penultimate steps in cancer progression. Steps that are even earlier in the process may be 
identified by comparing HSIL that progressed to cancer to the same lesion at earlier time 
points prior to progression to cancer. Analysis of host and viral factors in these distinct 
subsets of HSIL and in the cancer portion of the tissue will elucidate steps in the 
progression to cancer and will be useful to develop biomarkers that identify individuals at 
high risk of incipient progression to cancer. 
 


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Figure 2-D Paired Lesion Comparisons 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The left side of the figure shows Comparison A, depicted in the blue arrow, between an 
invasive cancer invading through the basement membrane, and the overlying HSIL. The 
tissue is viewed as it would be cut for routine histopathology assessment. The right side of 
the figure depicts two other sets of comparisons. The view is through an anoscope via high 
resolution anoscopy. The participant is on the examining table in the left lateral decubitus 
position, and left, right, posterior, and anterior positions are indicated. Two HSIL lesions 
are seen. One is at the right posterolateral octant (depicted in blue) at the squamocolumnar 
junction, and the other is at the left anterolateral octant at the squamocolumnar junction 
(depicted in red). If the cancer is detected for the first time in the left anterolateral lesion at 
a given study visit, it will be possible to compare the overlying HSIL from that lesion to 
the right posterolateral lesion, which has not changed and which has not progressed to 
cancer (Comparison B). It will also be possible to compare the overlying HSIL at the time 
the cancer is diagnosed at the left anterolateral lesion with HSIL at the same location at an 
earlier time point (Comparison C). For the purpose of this figure we have illustrated a time 
point 8 months before the cancer was first diagnosed, but any study visit prior to the 
diagnosis of cancer at which the HSIL was visualized at that location could be chosen. 
Anal biopsy samples will be collected as described previously. It is presumed that most if 
not all participants who are diagnosed with anal cancer after enrollment will by definition 
have an early cancer since they did not have cancer at baseline and they will have been 
followed closely every 6 months. Many of these cancers will be diagnosed as “at least 
superficially invasive squamous cell carcinoma (SISCCA), and further surgical excision 


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(TUA) is needed to determine if they meet the full definition of SISCCA. This is clinically 
important since they may be treatable with surgical excision instead of CRT. TUA also 
offers the opportunity to collect additional biopsies for research, including frozen biopsies 
if the size of the lesions permits. 
Individuals whose cancer is shown to be beyond the point of SISCCA at the time of initial 
diagnosis will not undergo TUA because there is no clinical indication to do so; they will 
instead be referred immediately for CRT. In this case, there will not be additional biopsies 
obtained for research but the area of invasive cancer will by definition be larger than what 
is available for research in a SISCCA, and it is likely that these fully invasive tumors will 
contain sufficient material for the proposed analyses. 
The first two sets of correlative science studies described below focus on identifying viral 
and host factors important in the pathogenesis of progression from HSIL to cancer, 
respectively. The focus of the third correlative study is to study viral and host proteins, 
genetic changes, etc., already shown in the literature to be of interest in predicting prevalent 
or incident HSIL (primarily cervical) and determining whether these are of value as 
potential clinical biomarkers to predict progression from HSIL to cancer. 
2.5.1 Overview of sample collection 
1) Participants who sign the informed consent and agree to be screened (N= 
17,385) at Visit 0 will provide a sample of blood and three anal swabs for anal 
cytology, DNA and RNA/protein testing. We will create extra monolayer 
cytology slides from the swab used for anal cytology. These specimens will be 
donated to the ANCHOR Biorepository if the participant does not qualify for 
the study and if the participant consents to future testing of his/her specimens. 
These samples will be used for studies that wish to explore risk factors for 
having prevalent anal LSIL or HSIL and HPV infection, as well as studies that 
recruit from among those who did not qualify for the ANCHOR study but who 
will be recruited for other prospective studies. 
2) We will collect serum (N= 5,058) from each participant every 6 months during 
study participation (Visits 2-11). An additional blood sample (processed for 
plasma and whole blood fraction) will be stored for future studies at Visit 2. At 
each 6-month HRA visit, we will collect an anal swab for cytology, a second 
anal swab for DNA studies, and a third anal swab for RNA and protein studies. 
We will create extra monolayer cytology slides from the swab used for anal 
cytology. A sample of blood from each participant (N= 5,058) will also be 
stored for each participant at Visit 2. Serum, blood, and swab samples will be 
stored centrally at the ANCHOR Biorepository but will belong to the ANCHOR 
study until released to outside investigators after completion of the primary 
ANCHOR analyses as described in this section. 
3) Formalin-fixed, paraffin-embedded anal biopsy specimens will be processed 
for routine H and E histopathologic assessment. The remaining tissue block will 
be stored locally if the local institution requires it, as long as they agree to send 
specific blocks to the central ANCHOR study laboratory upon request. 
Alternatively, institutions may choose to send all of their remaining blocks to 
the central laboratory after the initial diagnostic slides are read. Each participant 


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will have at least one biopsy per year, and these will likely span the range from 
normal to LSIL, HSIL and cancer. Biopsy slides will be sent to the Central 
Pathology Laboratory at UCSF upon request for central review. Additionally, 
participants treated with ablative therapies will have one additional biopsy 
taken from the lesion with the most severe appearance of disease prior to 
treatment, no more than once every six months. This biopsy will be placed in 
RNAlater for banking only. Lastly, participants diagnosed with cancer during 
screening or following randomization, may provide an optional cancer biopsy 
(placed in RNALater) if willing to return to the clinic for biopsy before referral 
to treatment and study discontinuation. 
4) Formalin-fixed, paraffin-embedded biopsies shown to have invasive anal 
cancer will be sectioned for laser capture microdissection (LCM). Areas of the 
tissue containing invasive cancer will be removed and processed for the studies 
described below. The HSIL portion of the tissue containing the cancer will be 
similarly processed, as will biopsies of HSIL taken at the same visit that did not 
progress to cancer. HSIL biopsies collected at visits prior to the visit at which 
that HSIL lesion was shown to have progressed to cancer will also be available. 
We can use these samples to study expression of genes and proteins of interest 
at different time points prior to the development of anal cancer, since we will 
have a set of preceding HSIL biopsies obtained at least annually prior to the 
time of cancer diagnosis. A portion of each tissue will be placed on tissue 
microarrays for immunohistochemical analysis, and the remaining tissue will 
be processed for DNA studies and RNA microarray gene expression analysis. 
As described in Section 4.4.2, if a woman participating in the ANCHOR study 
is diagnosed with cervical cancer during the study, we will ask her for 
permission with a separate informed consent form to retrieve tissue blocks of 
the cancer. These may be of value for correlative science and comparison with 
the anal specimens collected as part of the ANCHOR study. 
5) We will maintain an inventory of all of the tissues collected in the study and 
stored at the ANCHOR Biorepository. For any requests to use banked samples 
for other correlative science studies, a full protocol will be submitted for 
approval in accordance with NCI, CTEP policies. No correlative study using 
the specimens collected in this trial will be conducted unless it is explicitly 
approved as part of the main trial protocol or it is approved through mechanisms 
that will be established for the ANCHOR Study. Top priority will be given to 
using the samples to address the correlative science studies that have been 
separately peer-reviewed and funded. Additional studies consistent with the 
ANCHOR study agenda may be proposed by outside investigators, and 
reviewed by ANCHOR study investigators using a letter of intent mechanism. 
Upon approval of the ANCHOR study committee and CTEP, the tissues will be 
released to the outside investigator. Tissues and samples that remain after all of 
the ANCHOR study scientific questions have been addressed will be released 
to the ANCHOR Biorepository. These are available to investigators around the 
world who submit applications to use these specimens for ACSR scientific 
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2.6 
Ancillary Study: SARS-CoV-2 and Anal HPV Infection 
2.6.1 Summary and aims 
The SARS-CoV-2 pandemic has had a dramatic effect impact on the conduct of the 
study, prompting all ANCHOR study sites around the country to cease screening, 
enrollment and follow-up of randomized participants from March 20, 2020 until 
May 18, 2020, with sites resuming normal activity over time through September 
2020. However, apart from affecting normal study activities, SARS-CoV-2 may 
have additional relevance by affecting the natural history of anal HPV infection, 
development and persistence of anal HSIL, and progression from HSIL to anal 
cancer. This is because of the growing body of evidence that SARS-CoV-2 is shed 
in stool and may infect the gastrointestinal tract. Nothing is known at present as to 
whether SARS-CoV-2 can infect the anal epithelium specifically, whether it affects 
the biology of anal HPV infection, how it affects the local immune response, and 
whether it affects the natural history of anal HSIL. Furthermore, nothing is known 
about anal SARS-CoV-2 infection in the group at highest risk of anal cancer, 
PLWH. The ANCHOR study offers an ideal opportunity to begin to address these 
issues, particularly since a high proportion of participants in the ANCHOR Study 
are from medically underserved minority populations who are also at very high risk 
of SARS-CoV-2 infection. 
The focus of this supplemental study is to describe detection of SARS-CoV-2 in 
anal swab samples from PLWH being screened for the ANCHOR study; examine 
its relationship to prevalent anal HPV infection and HSIL in the screening 
population; and determine its effect on the natural history of anal HPV infection 
and HSIL by examining its relationship to regression of HSIL and clearance of 
HPV infection in the subset of enrolled participants randomized to the active 
monitoring arm. Accordingly, our specific aims are outlined in Section 1.6. 
2.6.2 Background 
In early 2020, the global pandemic of SARS-CoV-2 virus spread to the United 
States and as of late October 2020, has led to over 8.7 million cases and over 
225,000 deaths (132). While the main cause of mortality from SARS-CoV-2 is 
respiratory failure, there is growing evidence that SARS-CoV-2 can infect the 
gastrointestinal tract and be associated with gastrointestinal symptoms such as 
diarrhea (133-136). Several studies have demonstrated that SARS-CoV-2 is shed 
in stool samples (133-142). Multiple studies also show that stool shedding may 
persist after oropharyngeal shedding is no longer detectable (134, 138, 140, 142, 
143). In one study from Hong Kong, SARS-CoV-2 RNA was detected in stool 
samples from 48% of patients after respiratory samples tested negative (139). In 
another study from Wuhan, 18 (64%) patients remained positive for viral RNA in 
feces after pharyngeal swabs turned negative. The duration of viral shedding from 
the feces after negative conversion in pharyngeal swabs was 7 (6-10) days, 
regardless of SARS-CoV-2 severity (136). 
Consistent with stool shedding of SARS-CoV-2, a recent study showed that the 
virus can productively infect intestinal enterocytes (144). Another study using 
electron microscopy showed what appeared to be intact viral particles in stool 


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specimens (145) consistent with the possibility that virus shed from the 
gastrointestinal tract is infectious. Furthermore, there are also data showing that 
SARS-CoV-2 may remain viable in environmental conditions that could facilitate 
fecal-oral transmission (146). Thus, there are multiple implications of detection of 
SARS-CoV-2 in the stool. First, if stool shedding persists after oropharyngeal 
shedding, diagnosis of SARS-CoV-2 infection, contact tracing and pandemic 
control may be facilitated by routine anal swab detection of SARS-CoV-2 in 
addition to oropharyngeal sampling. Second, if SARS-CoV-2 infects the 
gastrointestinal tract and viable virus is shed in stool, there is the possibility of 
sexual transmission, particularly through oro-anal contact. 
This supplement focuses on the third important implication of stool shedding, i.e., 
if SARS-CoV-2 infects the anal epithelium as it does in other parts of the 
gastrointestinal tract, there is the possibility that it may affect the natural history of 
anal HPV infection and HPV-associated pre-cancerous lesions, high-grade 
squamous intraepithelial lesions (HSIL). SARS-CoV-2 is known to lead to a large 
inflammatory response in the lungs, but its effect on the gastrointestinal tract is less 
well understood. 
Infection with SARS-CoV-2 of enterocytes elicited a broad signature of cytokines 
and interferon -stimulated genes (144). These data suggest that SARS-CoV-2 may 
affect local immune response. Based on its effect on pulmonary inflammation, it is 
plausible that SARS-CoV-2 has the potential to lead to a pro-inflammatory anal 
environment with exacerbation of existing infection with HPV, impaired clearance 
of HSIL, and increased likelihood of progression to cancer. 
The gastrointestinal tract is a highly diverse organ with different parts exhibiting 
distinct biology and microenvironments. The anal epithelium is the most distal part 
of the gastrointestinal tract and is probably the most under-studied section. It is 
biologically quite distinct from the rectum, colon and small intestine, being 
comprised of stratified squamous epithelium. It has a distinct vascular supply and 
lymphatic drainage from the colon and rectum. We recently showed that the anal 
microbiome is distinct from that of stool specimens, which reflect the entirety of 
the colon and rectum (C. Brickman and J. Palefsky, submitted for publication). 
HPV infects only the anus, but not the rectum or colon. 
Nothing is known at present as to whether SARS-CoV-2 can infect the anal 
epithelium and/or mediate local inflammatory changes that might affect the natural 
history of HPV-related disease. SARS-CoV-2 uses the receptor ACE-2 for entry 
(147). A recent study has shown that, consistent with their susceptibility to SARS-
CoV-2 infection and replication, high levels of ACE-2 mRNA and protein are found 
in the small intestinal enterocytes but not in the goblet cells or intestinal immune 
cells. High expression of ACE-2 on the surface cells may lead to gastrointestinal 
symptoms, mediating SARS-CoV-2 invasion and amplification of the virus and 
activation of gastrointestinal inflammation (148). 
In addition to binding of SARS-CoV-2 spike proteins to ACE-2, entry of 
coronaviruses depends on S protein priming by host cell proteases. In a recent study 
it was shown that SARS-CoV-2 uses the serine protease TMPRSS2 for S protein 


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priming (147). In another recent study, expression of two mucosa-specific serine 
proteases, TMPRSS2 and TMPRSS4, were shown to facilitate SARS-CoV-2 spike 
fusogenic activity, promoting virus entry into host cells (149). In the Preliminary 
Results section we show that anal tissues express both ACE-2 and TMPRSS2. 
2.6.3 
Preliminary results 
Studying anal SARS-CoV-2 infection in the ANCHOR study: There are many 
unanswered questions about the impact of SARS-CoV-2, infection on the risk of 
developing anal cancer in the group at highest risk, people living with HIV (PLWH) 
and there are several reasons to perform this study as a supplement to the ANCHOR 
Study. First, the ANCHOR Study is continuing to screen participants, with 
enrollment currently at 4033 out of a target of 5058. Second, the protocol includes 
collection of all of the specimens and information that we would want to collect for 
this supplement, with the exception of the oropharyngeal swab for SARS-CoV-2 
detection. This includes anal swabs for HPV testing and SARS-CoV-2 testing, and 
high resolution anoscopy (HRA)-guided biopsies to determine the presence or 
absence of anal HSIL. Third, the demographic groups typically enrolled in 
ANCHOR are also known to be among the groups at highest risk for SARS-CoV-
2 infection, with African American and Hispanic men and women comprising well 
over half of our screened and enrolled population. Fourth, all PLWH enrolled into 
ANCHOR have anal HSIL and are followed every 6 months or more often, giving 
us the opportunity to determine the incidence of SARS-CoV-2 infection in this 
high-risk population. Fifth, half of the enrolled participants will be randomized to 
the active monitoring arm, which will allow us to determine if prevalence or 
incidence of SARS-CoV-2 affects persistence of HPV or anal HSIL. 
Impact: Our results could have important implications for development of anal 
cancer in PLWH. SARS-CoV-2 is likely to be circulating for several years, and 
there is a possibility of latency or reinfection. This is not likely to be an acute 
infection like influenza, and if SARS-CoV-2 can infect the anal epithelium an effect 
on the natural history of anal HPV infection and progression from HSIL to cancer 
is very plausible. We need to understand this relationship. Further studies will be 
needed to explore ACE-2 expression, TMPRSS2 expression and SARS-CoV-2 in 
anal epithelium, how expression varies in lesions of different histologic severity, 
the effect of SARS-CoV-2 on local inflammatory response and interaction between 
HPV and SARS-CoV-2 at the molecular level. If we find anal shedding in the 
absence of oropharyngeal positivity, this also has implications for case finding and 
control. 
Studies of anal epithelium and susceptibility to SARS-CoV-2: Nothing has been 
published about ACE-2 or TMPRSS2 expression in the anal epithelium. We 
previously created RNA microarray libraries from a set of anal biopsies, and 
reviewed those data for expression of ACE-2 or TMPRSS2. Our data showed that 
there were detectable levels of expression of both of these genes in the tissues. 
Nothing is known at this time as to where ACE-2 is found in anal epithelium, nor 
its relationship to HPV-related lesions. Given the wide organ tropism of SARS-
CoV-2, evidence for intestinal ACE-2 expression and intestinal SARS-CoV-2 
infection and replication, and our preliminary evidence of ACE-2 and TMPRSS2 


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expression in anal epithelium, it is plausible that SARS-CoV-2 also infects anal 
epithelium. 
We have now had the opportunity to study the natural history of anal HSIL among 
those randomized to the monitoring arm for up to 3 years. Among 390 participants 
examined at the 3-year timepoint, 39% had no HSIL. At the one-year timepoint 
among 1250 participants, 27% had no HSIL. These data indicate that a substantial 
number of PLWH undergo regression of HSIL after entry into the study. Some of 
this may reflect biopsy-associated physical removal of the lesion among those who 
entered the study with small lesions, and some may have missed disease that will 
be diagnosed again at future visits. However, some of these data almost certainly 
reflect true regression. Those with persistent HSIL are the ones who are probably 
the likeliest to progress to cancer over time. 
SARS-CoV-2 testing in anal specimens: The test that we propose to use for SARS-
CoV-2 testing is the Atila AmpFire system, the same system that we will use for 
HPV testing for this study. This is a system that we have installed in our laboratory 
and which we have used for HPV testing and specific genotyping. We have also 
installed the Atila system in Rwanda, Zimbabwe, Mexico and Puerto Rico. We have 
validated the assay against the WHO panel in our laboratory and the test works very 
well. In a recent study, the AmpFire assay detected HPV in clinical samples with 
positive percent agreements of 100.0% for HPV16, 100.0% for HPV18, and 94.7% 
for non-16/18 HR HPV, and 100% negative percent agreements for HPV16, 
HPV18, and non-16/18 HR HPV (150). This same technology had received FDA 
approval for SARS-CoV-2 testing of oropharyngeal swabs. It has not been tested 
yet in anal swab specimens, but based on our experience with HPV testing, we 
expect that this system will work well for this purpose. 
2.6.4 Research plan 
Study summary: To address Aim 1 we will enroll the first 400 PLWH who are being 
screened for the ANCHOR Study. Participants will be recruited at five ANCHOR 
centers: UCSF, Laser Surgery Care (New York), Anal Dysplasia Clinic MidWest 
(Chicago), University of Miami, and Emory University (Atlanta). The only 
additional procedure that participants will need to undergo to participate is an 
oropharyngeal swab for SARS-CoV-2 testing. Each participant in the ANCHOR 
Study has 3 anal swabs collected. The first and second swabs are placed in 
Thinprep, vigorously swished to remove the cellular material, and the swab is 
discarded. The third swab will be stored in RNALater. The anal specimens that will 
be tested this supplement will be retrieved from the ANCHOR Biorepository. An 
aliquot of Anal Swab 2 will be used for HPV testing and SARS-CoV-2 testing. 
To address Aim 2, we will quantify the HPV data in Swab 2 based on the Ct value, 
and categorize the quantitative results into tertiles. The presence or absence of 
HPV, quantity of HPV among those who are HPV-positive, and presence or 
absence of anal HSIL will be correlated with the presence of SARS-CoV-2 in the 
anal swab. 
To address Aim 3, we will repeat these procedures at 6 months among those who 
were enrolled into the active monitoring arm of the study. As at baseline, the only 


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additional procedure that participants would need to undergo to participate is an 
oropharyngeal swab for SARS-CoV-2 testing. Participants will undergo standard 
study procedures including collection of the anal swabs and HRA, and the presence 
of absence of visible HSIL will be noted at that visit. If we screen 400 PLWH we 
expect approximately 160 to be enrolled into either the treatment arm or the active 
monitoring arm and will be re-sampled at 6 months. Anal swab 1 will be studied 
for SARS-CoV-2 and for HPV DNA. 
To address Aim 4, we will focus the analysis on the half of the 160 enrolled 
participants who were randomized to the monitoring arm (N=80). These individuals 
do not undergo treatment of their lesion. The results of the SARS-CoV-2 testing in 
the anal swab will be correlated with the HPV results and presence or absence of 
HSIL. Detection of HPV and SARS-CoV-2 in anal and oropharyngeal specimens. 
To detect HPV, we will use the Atila AmpFire system. This is a real-time reverse 
transcription fluorescent isothermal amplification test, based on isothermal 
amplification technology termed OMEGA amplification (20). The test individually 
detects the 15 most common high-risk HPV genotypes with the quantity 
measurable by determining the threshold cycle (Ct) at which the DNA is detected.  
Like the HPV detection test that we will be using for this study, the iAMP COVID-
19 Detection Kit is a real-time reverse transcription fluorescent isothermal 
amplification test. OMEGA primer sets are designed to specifically detect RNA 
and later cDNA from the N and ORF-1ab genes of the SARS CoV-2 virus in nasal, 
oropharyngeal and/or oropharyngeal swabs from patients with signs and symptoms 
of infection who are suspected of SARS-CoV-2. The test detects 2000 copies of 
viral RNA per swab. 
One of the advantages of the iAMP SARS-CoV-2 assay is that it can detect SARS-
CoV-2 RNA directly from samples without a prior RNA extraction process. To 
detect SARS-CoV-2 from Thinprep solution, we will transfer 1ml Thinprep sample 
from the first swab to a 2 ml Eppendorf tube. We will then centrifuge and pellet the 
cells, discard the supernatant, then lyse the cells to detect SARS-CoV-2 RNA. 
Results are available in approximately one hour. 
We will first determine the analytic sensitivity of the SARS-CoV-2 test in Thinprep 
by spiking known quantities of SARS-CoV-2 RNA into Thinprep specimens. We 
will then extract RNA from the specimens using the Qiagen AllPrep PowerViral 
DNA/RNA Kit. This kit is designed for efficient purification of viral and bacterial 
total nucleic acids from samples high in PCR inhibitors, including stool. Viral 
nucleic acids eluted in RNase-free water are ready to use with the iAMP-CoV-2 
assay. 
If we are able to detect 50 viral copies in material eluted from 1 mL of Thinprep 
material with the RNA extraction step, we will begin to analyze real anal swab 
Thinprep specimens from non-ANCHOR participants to determine if this results in 
loss of analytic sensitivity. If our results show acceptable sensitivity and specificity, 
we will proceed to analyze the specimens obtained from ANCHOR participants in 
this study. If we are not satisfied with the results, we also have the opportunity to 
use Anal Swab 3, which was placed in RNALater. We are confident that this swab 


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in RNALater will work well but have chosen to use Swab 2 in Thinprep because it 
does not require a RNA extraction step and because it is a more abundant specimen 
than the RNALater specimen. 
Oropharyngeal swabs will be collected from participants and placed in a dry tube 
per the protocol approved by the FDA, stored at -20C at the ANCHOR 
Biorepository and shipped on dry ice to UCSF. Once ready for testing 1 ml of 
SARS-CoV-2 sample buffer will be added and incubated at room temperature for 
15 minutes. 10 uL of SARS-CoV-2 master mix are added and the SARS-CoV-2 
isothermal reaction is performed. Results are expressed as positive or negative, but 
we will also have the opportunity to examine quantity of viral RNA by determining 
the Ct at which it is detected. 
 
 


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3.0 
PATIENT SELECTION 
All protocol participants must meet all stated eligibility criteria. Before any participant is 
randomized, a clinician who is certified in HRA by the ANCHOR HRA Committee must 
document that the participant satisfies each eligibility requirement. In compliance with 
CTEP policy, no exceptions to eligibility criteria will be granted under any circumstance. 
3.1 
Eligibility Criteria 
3.1.1 HIV positive. Documentation of HIV-1 infection by means of any one of the 
following: 
• Documentation of HIV diagnosis in the medical record by a licensed health care 
provider; 
• Documentation of receipt of ART by a licensed health care provider 
(Documentation may be a record of an ART prescription in the participant’s 
medical record, a written prescription in the name of the participant for ART, 
or pill bottles for ART with a label showing the participant’s name. Receipt of 
at least two agents is required; each component agent of a multi-class 
combination ART regimen will be counted toward the 2-agent requirement, 
excepting receipt of a PrEP regimen alone [e.g., Truvada], which is 
exclusionary); 
• HIV-1 RNA detection by a licensed HIV-1 RNA assay demonstrating >1000 
RNA copies/mL; 
• Any licensed HIV screening antibody and/or HIV antibody/antigen 
combination assay confirmed by a second licensed HIV assay such as a HIV-1 
Western blot confirmation or HIV rapid multispot antibody differentiation 
assay. 
NOTE: A “licensed” assay refers to a U.S. FDA-approved assay, which is required 
for all IND studies. 
3.1.2 Age 35 years or older. This age restriction is intended to enrich the study population 
at risk for cancer since anal cancer occurs only rarely under this age even among 
HIV-infected individuals. Fewer than 1% of anal cancers occur under the age of 35 
years (1). 
3.1.3 Biopsy-proven anal HSIL at baseline (AIN2 with a positive p16 stain, AIN2-3, or 
AIN3). 
3.1.4 At least one focus of HSIL must be identified that is not within a condyloma that 
may be treated after enrollment into the study. This requirement is to ensure that 
there will still be at least one focus of HSIL among participants in the Active 
Monitoring Arm even if they undergo treatment for condyloma. 
3.1.5 For females, documentation that the participant is being followed with cervical 
cytology and/or HPV testing per current “Guidelines for the Prevention and 
Treatment of Opportunistic Infections in HIV-Infected Adults and Adolescents” 
and American Society for Colposcopy and Cervical Pathology (ASCCP) 


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guidelines.1,2 Cervical cytology must be performed prior to enrollment for women 
who are overdue for screening per the guidelines. Women should also have 
confirmation of absence of cancer or suspected cancer upon visual examination of 
the vulva, vagina, and cervix within 12 months prior to enrollment. 
3.1.6 ECOG performance status < 1 (Karnofsky > 70%, see Appendix II) 
3.1.7 Life expectancy of greater than 5 years 
3.1.8 Participants must meet the following parameters within 90 days before enrollment 
• Absolute neutrophil count: >750/mm3 
• Platelets: >75,000/mm3 
• Hemoglobin ≥ 9.0 g/dL 
3.1.9 Women of childbearing potential (FCBP)† must have a negative urine pregnancy 
test within 7 days prior to randomization enrollment. Female participants enrolled 
in the treatment arm are advised to not become pregnant during study participation 
due to the risks of the study treatments. All women of childbearing potential must 
agree to either commit to continued abstinence from heterosexual intercourse or to 
use a reliable birth control method during heterosexual intercourse (oral 
contraceptive pills, intrauterine device, Nexplanon, Depo-Provera, or bilateral tubal 
ligation, etc., or another acceptable method as determined by the investigator) during 
the entire period of the trial (5 years or more), and must not intend to become 
pregnant during study participation and for 3 months after treatment is discontinued 
if the participant is enrolled in the treatment arm. Female participants, if engaging 
in heterosexual intercourse, must be willing to comply with an acceptable birth 
control regimen as determined by the Investigator. 
† A female of childbearing potential is a sexually mature woman who: 1) has not 
undergone a hysterectomy or bilateral oophorectomy; 2) has not been naturally 
postmenopausal for at least 24 consecutive months (i.e., has had menses at any time 
in the preceding 24 consecutive months). 
3.1.10 Men randomized to the treatment arm should not father a baby while receiving 
topical treatment during this study. Men who could father a child must agree to use 
at least one form of birth control during or continued abstinence from heterosexual 
intercourse if receiving topical treatment during the study, and for 2 weeks after 
stopping topical treatment. 
3.1.11 Participant is willing to be randomized and able to comply with the protocol 
 
1 Panel on Opportunistic Infections in HIV-Infected Adults and Adolescents. Guidelines for the prevention and 
treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from the Centers 
for Disease Control and Prevention, the National Institutes of Health, and the HIV Medicine Association of the 
Infectious Diseases Society of America. Available at http://aidsinfo.nih.gov/contentfiles/lvguidelines/adult_oi.pdf. 
Accessed March 2, 2016. 
2 Massad LS, Einstein MH, Huh WK, Katki HA, Kinney WK, Schiffman M, Solomon D, Wentzensen N, Lawson 
HW; 2012 ASCCP Consensus Guidelines Conference. 2012 updated consensus guidelines for the management of 
abnormal cervical cancer screening tests and cancer precursors. Obstet Gynecol. 2013 Apr;121(4):829-46. 


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3.1.12 Clinician is comfortable that cancer has adequately been ruled out and is willing to 
follow the participant for up to 5 years without treatment of the HSIL. 
3.2 
Exclusion Criteria 
Patients who do not fulfill the criteria as listed in Section 3.1 above, are ineligible. 
Additionally, the presence of any of the following conditions will exclude a participant 
from study enrollment: 
3.2.1 Participant is unable to provide informed consent 
3.2.2 Participants who received any other chronic (defined as more than 50% of the time 
in the last 6 months) systemic immunomodulatory agents (replacement doses of 
steroids for adrenal insufficiency are permitted or treatment with prednisone ≤5 
mg/day). Receipt of investigational agents within the 4 weeks before randomization 
enrollment, other than investigational antiretroviral agents for HIV and 
investigational or approved agents for Hepatitis C, are also exclusionary. 
3.2.3 History of anal cancer, penile, vulvar, vaginal, or cervical cancer, or signs of any 
of these malignancies at baseline. Participants with prior carcinoma in situ will not 
be considered to have prior cancer for eligibility purposes. 
3.2.4 Treatment or removal of HSIL less than 6 months prior to randomization. 
3.2.5 Participant has symptoms related to HSIL and would benefit more from immediate 
treatment than from entry into the study and potential for randomization to active 
monitoring arm 
3.2.6 Current systemic chemotherapy or radiation therapy that potentially causes bone 
marrow suppression that would preclude safe treatment of HSIL 
3.2.7 Participants who only have a single HSIL lesion that is likely to be removed entirely 
with the initial screening biopsy 
3.2.8 Warts so extensive that they preclude the clinician from determining the extent and 
location of HSIL 
3.2.9 Participant plans to relocate away from the study site to a location that does not 
have an ANCHOR study site during study participation 
3.3 
Inclusion Criteria for the HRQoL Substudy 
The HRQoL substudy was completed in February 2020. 
3.3.1 Participant has consented for the ANCHOR Study and the A-HRSI scale 
responsiveness substudy 
3.3.2 Fluent in English 
3.3.3 The participant is willing to conduct a phone interview with MSK if questionnaires 
are not completed at the site or as self-administered 
3.3.4 Participant is in screening for ANCHOR and the investigator can report a target 
randomization date for the participant 


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3.4 
Inclusion Criteria for the QoL Objective 
3.4.1 Participant has consented for the ANCHOR Study and A-HRSI questionnaire 
completion 
3.4.2 Fluent in English or Spanish 
3.4.3 The participant is willing to conduct a phone interview with site staff if 
questionnaires are not completed at the site or as self-administered 
3.4.4 Participant is in screening for ANCHOR and the investigator plans to randomize 
the participant based on screening anal biopsy results 
3.5 
Inclusion Criteria for the SARS-CoV-2 Ancillary Study 
3.5.1 Participant has consented for the ANCHOR Study and SARS-CoV-2 ancillary 
study, and is being enrolled at one of the participating centers for this study 
3.6 
Number of Participants to be Enrolled 
3.6.1 Sample size 
This study will enroll 5,058 participants. 
The optional HRQoL substudy (completed February 2020) enrolled 100 eligible 
participants (50 from the active monitoring arm and 50 from the treatment arm) 
who were enrolled for screening and were randomized to ANCHOR. 
For the QoL objective, up to 500 randomized participants will be enrolled for QoL 
questionnaire completion. 
For the SARS-CoV-2 ancillary study, a total of 400 participants will be enrolled. 
3.6.2 Accrual rate 
Approximately 141 participants per month. 
3.7 
Participant Enrollment Procedures 
Sites must have this protocol approved by their Institutional Review Boards (IRB) and be 
registered for study participation with the ANCHOR Data Management Center (DMC) 
before they may enroll participants. 
3.7.1 Registration for screening 
After an informed consent form has been signed by the participant, the participant 
must be registered for screening on the ANCHOR Protocol, Segment A (on-line 
via AdvantageEDC) no more than one day after signing consent. After successful 
registration into screening, the participant will receive an alphanumeric participant 
ID and will then enter the screening process (screening visit). 
Registration for optional A-HRSI substudy or QoL objective 
Participants will also be advised of the optional HRQoL substudy or QoL objective 
(as available for enrollment) during screening for the ANCHOR protocol and will 
be asked to provide informed consent to the substudy if interested. After the 
participant agrees to participate in the optional HRQoL substudy or QoL objective 
and after enrollment into the ANCHOR protocol screening segment in 


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AdvantageEDC has been completed, the participant must also be registered for the 
optional A-HRSI substudy on-line via AdvantageEDC no more than one day after 
the participant is determined to be eligible for the applicable optional study. 
Registration for optional SARS-CoV-2 ancillary study 
Participants will also be advised of the optional ancillary SARS-CoV-2 study (as 
available for enrollment) during screening for the ANCHOR protocol and will be 
asked to provide informed consent to the study if interested. After the participant 
agrees to participate in the study, the participant will be enrolled at the same time 
as completing the ANCHOR protocol screening segment in AdvantageEDC. 
3.7.2 Enrollment 
After the screening evaluations have been obtained and the participant is 
determined to be eligible, the participating site will complete the protocol-specific 
eligibility checklist and enroll the participant into the ANCHOR Protocol, Segment 
B (on-line via AdvantageEDC). Enrollment should occur on the day of 
randomization and before administration of the first dose of the protocol agent(s). 
Once the eligibility checklist is submitted, a system generated confirmation email 
will be sent to the enroller upon successful completion of the participant enrollment. 
If the on-line system is inaccessible, the site should notify the ANCHOR DMC (via 
email at anchordmc@emmes.com or via phone at 301-251-1161) for further 
instructions. 
Participants must be enrolled into ANCHOR Protocol Segment B to be randomized 
to a study arm. This enrollment must occur before receiving the first dose of the 
protocol agent or treatment. 
 


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4.0 
TREATMENT PLAN 
4.1 
Group 1: Active Monitoring Arm 
Participants in the active monitoring arm will be assessed with HRA every 6 months, but 
will have HSIL only biopsied every 12 months unless the clinician chooses to biopsy a 
lesion sooner because of concern for possible progression to cancer. Biopsies at each 
annual visit may be deferred if the participant was biopsied at a preceding interim visit that 
occurred no more than 3 months before the annual visit target date and the lesion 
appearance has not changed since the prior visit as assessed during the annual visit HRA. 
Every 12 months, all visible lesions suspicious for HSIL, including index and 
metachronous HSIL, will be biopsied. At the end of study, all visible lesions and any prior 
areas of HSIL that appear to have regressed will be biopsied. If this totals less than 4 
biopsies, normal-appearing tissue in remaining quadrants will be biopsied so that a 
minimum of 4 areas are sampled at the final study visit. 
Participants will have anal cytology, additional swabs, and blood obtained at each 6-month 
visit for correlative science studies. 
Participants in both arms will also be biopsied any time there is suspicion of progression 
to cancer. A diagnosis of cancer will lead to immediate referral for treatment. A diagnosis 
of “suspicious for invasion” or “cannot rule out invasion” will require repeat biopsy. If the 
diagnosis remains inconclusive after repeat biopsy the case will be submitted to a Central 
Pathology Review Committee for adjudication. Surgeons performing excisional biopsies 
in participants in the active monitoring arm to rule out cancer should be asked to remove 
as much tissue as needed in their clinical judgment to rule out cancer, but not treat or 
remove any more HSIL than clinically required. If cancer is ruled out, the participant will 
remain on the study. If the diagnosis is inconclusive, the participant will undergo surgical 
excision of the lesion to rule out cancer. If cancer is not diagnosed after surgical excision, 
follow-up will continue. If cancer is diagnosed, the participant will be immediately referred 
for further evaluation and the stage will be recorded. 
4.1.1 Definition and management of drop-in participants 
Participants on the active monitoring arm who have anal HSIL treated at any time 
after randomization will be reported as drop-ins to treatment. Any anal HSIL 
treatment given to active monitoring arm participants will occur as medical care 
outside of the study. Any anal HSIL treatment given to active monitoring arm 
participants will be reported in the HSIL therapy form in AdvantageEDC for each 
relevant 6-month study period in which treatment was administered. Drop-in 
participants shall remain on the active monitoring arm and will receive all active 
monitoring arm study procedures as planned for the duration of the study. 
4.2 
Group 2: Treatment Arm 
Participants in the treatment arm will be assessed with HRA every 6 months and biopsied 
if persistent or metachronous HSIL is suspected, or if visible changes can be seen in areas 
of prior treatment. Areas of prior treatment that appear completely normal (e.g., not 
acetowhite epithelium (AWE), no vascular changes, and strongly Lugol's positive) do not 
need to be biopsied to confirm the success of treatment. Biopsies at each 6-month visit may 
be deferred if the participant was biopsied at a preceding interim visit that occurred no 


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more than 3 months before the 6-month visit target date and the lesion appearance has not 
changed since the prior visit as assessed during the 6-month visit HRA. At the last study 
visit, all visible lesions will be biopsied and additional biopsies should be done of normal-
appearing areas that were previously treated. If this totals less than 4 biopsies, normal-
appearing tissue in remaining quadrants will be biopsied so that a minimum of 4 areas are 
sampled at the final study visit. 
Treatment will be provided from among a list of protocol-approved therapeutic modalities 
at the discretion of the clinician with the goal of eradicating HSIL as completely as 
possible. If a participant has persistent HSIL, then a protocol-approved treatment should 
be continued as clinically indicated or a new protocol treatment should be considered. 
Clinicians may choose to use more than one treatment in a given 6-month block, 
concurrently or sequentially. If a participant misses a treatment visit for the prior 6-month 
block and HSIL remains present at the next 6-month visit, the clinician has the option to 
biopsy, then treat the lesion at the same visit. 
If HSIL is not present, the participant will be re-examined every 6 months. Lesions seen 
on HRA will be biopsied to confirm the success of treatment and to determine the need for 
additional therapy going forward. Lesions suspicious for HSIL will be biopsied. 
Participants will have cytology, additional anal swabs, and blood obtained at every 6-
month HRA visit. Clinicians also have the option of seeing participants in between 6-month 
visits if they are concerned for imminent progression to cancer. 
Participants will also be biopsied any time there is suspicion of progression to cancer. A 
diagnosis of cancer will lead to immediate referral for treatment. A diagnosis of “suspicious 
for invasion” or “cannot rule out invasion” will require repeat biopsy. If the diagnosis 
remains inconclusive after repeat biopsy the case will be submitted to a Central Pathology 
Review Committee for adjudication. If cancer is ruled out, the participant will remain on 
the study. If the diagnosis is inconclusive, the participant will undergo surgical excision of 
the lesion to rule out cancer. If cancer is not diagnosed after surgical excision, follow-up 
will continue. If cancer is diagnosed, the participant will be immediately referred for further 
evaluation and the stage will be recorded. 
Participants with persistent HSIL not responsive to multiple treatment courses: Participants 
with persistent HSIL have the option of being monitored without treatment at the discretion 
of the treating provider in discussion with the participant. Generally, this should be 
considered after at least 3 successive 6-month treatment courses and should be for less than 
one year at a time. 
Clinicians may change therapeutic modalities as needed but are asked to make every effort 
to complete the maximally-allowed number of treatments before concluding that the 
treatment modality needs to be changed. If a participant is unable to tolerate a given 
therapy, that will be sufficient reason to change modality regardless of the number of 
treatments. 
Incident lesions will be considered “not previously treated” and may be treated de novo 
using the approaches described below. In general, study clinicians will be encouraged to 
use only one treatment modality at a time when treating recurrent and metachronous 
lesions. It is expected that these lesions will in general be limited in size if the participant 
is being followed carefully every 6 months or more often if currently under active treatment 


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for pre-existing HSIL. 
The following treatment modalities may be used in the treatment arm of the ANCHOR 
study: 
4.2.1 Patient-applied topical treatments 
For imiquimod and 5% 5-fluorouracil (5-FU) cream, we will re-examine the 
participant at 8 weeks and 16 weeks (± 2 weeks; 16 week visit is optional) after 
initiation of therapy. The primary goal of the 8-week visit is to examine and 
interview the participant for clinical toxicity and treatment tolerability. If the lesion 
appears to be resolved at 8 weeks, the clinician may elect to stop the therapy at that 
time. If lesions are still seen, treatment may continue for up to 16 weeks. The 
clinician may perform a treatment follow-up visit at week 16 at his/her discretion. 
Participants will then be biopsied at the next 6-month visit. This eight-week 
window between 16 and the 6-month visit is built in to allow inflammation to 
subside and healing to occur, since this will facilitate assessment of treatment 
efficacy and biopsy. An algorithm showing the use of imiquimod and 5-FU cream 
is shown in Figure 4-A. 
a) Imiquimod. 5% imiquimod cream will be used. Participants will be instructed 
to insert the cream intra-anally, peri-anally or both, depending on the location 
of the lesion. Imiquimod is applied three times per week on alternate days. Each 
of these weekly cycles may be repeated up to 16 weeks. The imiquimod dose 
will be 750 mg of cream per week, administered as one single use sachet (250 
mg) applied 3 times per week. Participants will be instructed to apply half the 
contents of a sachet onto a right hand glove or a finger cot and insert the cream 
approximately one inch into the anus on the right side. They will then place the 
remaining cream on the left finger, and insert the cream one inch into the anus 
on the left side. Similar instructions apply for perianal application, but without 
insertion of the cream into the anal canal. Participants who are being treated for 
both intra-anal and perianal disease will be given twice the number of sachets 
as those being treated for intra-anal or perianal disease only. In these cases, 
double the maximum dose will be administered to treat both areas. 
b) 5% fluorouracil cream. The UCSF protocol will be used, in which the patient is 
instructed on how to apply the cream and where. The cream is applied for 5 
consecutive days twice per day (i.e., in the morning and at bedtime), and then 
there is a break of 9 days. We will allow up to 8 two-week cycles. Participants 
will be instructed to apply 1 ml of topical 5-fluorouracil 5% cream in the anus. 
One gram of topical 5-fluorouracil is the equivalent of 1 ml. The total daily dose 
will be 1 gram of cream, and the weekly dose is 5 grams of cream. 5% 5-
fluororacil contains 5 grams of 5-fluororacil per 100 grams. The daily dose is 
therefore 50 mg of 5-fluororacil delivered in divided doses of 25 mg BID, and 
the total weekly dose is 250 mg delivered in 5 grams of cream. Participants will 
be given a 40 gram tube of 5-FU cream and instructed to squeeze out the cream 
to the marked line on the applicator (0.5 mL of cream), or onto a right hand 
glove or finger cot. Using a glove or a finger cot they will then squeeze one half 
the amount of cream (0.25 mL) from the syringe (if using an applicator) onto 


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their right index finger and insert the cream approximately one inch into the 
anus on the right side. The participant will then place the remaining cream (0.25 
mL) on the left finger, and insert the cream one inch into the anus on the left 
side. Similar instructions apply for perianal application, but without insertion 
of the cream into the anal canal. Participants who will be treated for perianal 
HSIL will be dispensed an additional tube. In these cases, double the maximum 
dose will be administered to treat both areas. 
4.2.2 Ablative treatments 
The ablative treatment may be repeated if persistent or metachronous HSIL is 
documented. An algorithm showing the use of ablative treatments is shown in 
Figure 4-B. 
1) Infrared coagulation, hyfrecation/electrocautery, and surgical lasers can be used 
interchangeably for in-office ablation. These devices will be used for excision 
or ablation of lesions with anesthetic using HRA (e.g., magnification with a 
colposcope or operating microscope, along with 5% acetic acid and Lugol’s 
solutions) at the discretion of the clinician. Staged ablations are permitted for 
extensive lesions. For large lesions investigators may treat the participant with 
ablative therapies in a staged manner within the 6-month treatment block. For 
example, a large lesion may be partially treated at the beginning of the 6-month 
block, followed by a second procedure to treat the remainder of the lesion 
within 8 weeks. The number of such treatments and timing will be at the 
discretion of the investigator. 
2) Treatment under anesthesia (TUA): Procedures may be performed in an 
operating room as a surgical referral or in an office setting on patients with 
conscious sedation. Referral for surgical excision in an operating room will be 
performed only as a last resort, if the clinician believes that none of the 
treatment approaches described above have the potential to resolve the HSIL, 
or if the participant refuses office ablation. The costs of TUA will only be 
covered by the study with the QA committee’s approval. Patients treated in the 
operating room for extensive lesions will be re-evaluated 8 to 12 weeks after 
surgery and then every 6 months if HSIL is not documented. Clinicians may 
initiate a new 6-month block of therapy if HSIL is still documented at the 8-12 
week visit. We expect TUA to be used in only a small percentage of ANCHOR 
participants, and for these few participants, it is expected that there will be only 
one procedure during the course of the study. However, there may be exceptions 
where more than one procedure is required. The procedure will be done either 
by study clinicians who are surgeons, or by surgeons guided by study clinicians 
in the operating room. 
4.2.3 Recommended treatment algorithms 
As indicated previously the ANCHOR study is a “strategy” study, not a study of 
individual treatment modalities. As such we are examining the effect of making a 
best effort to treat HSIL on the incidence of anal cancer. We therefore leave the 
final choice of treatment to the treating clinician, as would be the case in the real 
world. However, we will provide guidance to the clinicians regarding choice of 


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treatment approaches, and will require that the modalities that are chosen are 
administered per protocol using protocol-specified dosing. The choice of therapy 
will vary depending on the size of lesions, number of lesions, location of lesions, 
clinician preference, and patient preference. Within these parameters, guidance 
given to clinicians will be as follows: 
1) Lesions larger than 1 cm will generally be treated with an ablative method, with 
the choice of method depending on the expertise and preference of the clinician 
2) If there is a limited amount of residual disease after completion of the first 
treatment modality, in terms of number or size of lesions these may be treated 
with patient-applied 5-FU or imiquimod creams. 
3) For lesions larger than 1 cm, the clinician may also opt to start with 5-FU or 
imiquimod, and use targeted ablation of residual disease depending on the 
number and volume of residual lesions. 
4) Metachronous lesions may be treated de novo following the guidelines for 
lesions found at baseline. 
5) In the event that the participant refuses the initial treatment selected for a 6-
month block, the clinician may select an alternative treatment as clinically 
indicated. 
6) As described above, TUA will be performed only as a last resort, if the clinician 
believes that none of the treatment approaches described above have the 
potential to resolve the HSIL. We expect TUA to be used in only a small 
percentage of ANCHOR participants. 
 
 
 


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Figure 4-A: Participant initiates imiquimod/5-FU at visit 1 (randomization visit) 
 
Figure 4-B: IRC, hyfrecation, or electrocautery performed at visit 1 (randomization visit) 
 


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4.2.4 Duration of therapy 
In the absence of treatment delays due to adverse event(s), treatment of participants 
in the treatment arm may continue at the discretion of the clinician until one of the 
following criteria applies: 
• HSIL progression to cancer, 
• Intercurrent illness that prevents further administration of treatment, 
• Other unacceptable adverse event(s), 
• Participant decides to withdraw from the study, or 
• General or specific changes in the participant’s condition render the participant 
unsuitable for further treatment in the judgment of the investigator. 
The reason for discontinuing study treatment will be reported in the HSIL Therapy 
form in AdvantageEDC for the 6-month period in which the discontinuation 
occurred. Any adverse events will be reported as required in Section 6.0. 
4.3 
General Concomitant Medication and Supportive Care Guidelines 
4.3.1 Prohibited medications 
Therapeutic HPV vaccinations and any other treatment for anal HSIL outside of 
this protocol are prohibited. Participants are prohibited from participating in other 
clinical trials for the treatment of HPV-related disease. Prophylactic HPV vaccines 
were removed from the exclusion criteria with version 8.0 of this protocol. Receipt 
of other investigational agents during this protocol (except for investigational 
antiretroviral therapy, Hepatitis C treatments, or for SARS-CoV-2 vaccination or 
treatment) is prohibited. 
4.3.2 Concomitant medication documentation 
During screening, study staff must obtain a listing of all current prescription 
medications taken within the last 45 days. Actual or estimated start and stop dates, 
indication, dosage, and schedule will be collected for the following classes of 
agents below to assess eligibility. These agents must be recorded in AdvantageEDC 
once the participant is randomized: 
• Anticoagulants 
• Antiretroviral therapy 
• Immunomodulatory agents (if receiving, evaluate duration of receipt for 6 
months prior to randomization per 3.2.2) 
• Investigational agents 
• HPV vaccines 
After randomization, all prescription medication received during the study must be 
listed in the source only. Changes to or new administration of agents of the 
following classes will be recorded in the source, documenting actual or estimated 
start and stop dates, indication, dosage, and schedule, and reported in the 


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Concomitant 
Medications 
Form 
as 
changes 
occur: 
anticoagulants, 
immunomodulatory agents, HPV vaccines, and investigational agents. Medications 
that the investigator determines to have a causal relationship to serious adverse 
events must be recorded similarly in the source and in the Concomitant Medications 
Form after screening enrollment. HPV vaccinations should be deferred until study 
participation is completed. If administered while on study, then this must be 
recorded in the source and the Concomitant Medications Form. 
The participant’s antiretroviral medications will be managed by the participant’s 
usual healthcare provider; participants should be referred for care for their HIV as 
medically indicated. Clinicians may opt to defer biopsies or treatment if he/she 
believes that biopsy or treatment may put the participant at unacceptable risk of 
complications due to concomitant medications for any reason. The reason for 
deferral must be listed in the source documents. 
4.4 
Concurrent HPV-related Disease Treatment 
4.4.1 Anal warts and vulvar HSIL in study participants 
Anal warts are commonly found in the populations that will be screened for and 
enrolled in the ANCHOR study. Some will have warts found at the time of 
screening, and incident warts are also expected among some of those who will be 
enrolled. Treatment of warts may be important recruitment and retention tools, as 
some participants may not be willing to enter or remain in the study if their warts 
are not treated. Targeted treatment modalities are the only modalities permitted for 
the warts among participants in the active monitoring arm, including IRC, 
electrocautery, laser, and surgical excision. An additional targeted approach, 
cryotherapy, may also be used to treat concurrent warts in study participants on 
either arm at the discretion of the treating physician. Cryotherapy may not be used 
for the treatment of HSIL on this protocol. Imiquimod, 5-fluorouracil cream or 
other topical treatments, which could “inadvertently” treat HSIL that is physically 
separate from the wart(s) being treated are not permitted for those in the active 
monitoring arm, but may be allowable for those in the treatment arm if these are 
chosen by the clinician to treat the HSIL. FDA-approved topical treatments for 
condyloma may be used to treat perianal warts in treatment arm participants, but 
may not be used in the anal canal. Study-sponsored supplies of 5-fluorouracil 5% 
and topical imiquimod 5% may be used for condyloma treatment of the perianus 
for treatment arm participants, and will only be used in the anal canal when 
administered for concurrent HSIL treatment, adhering to the dose administration, 
modification, and accountability instructions in this protocol if used for condyloma 
treatment. It is recognized that some participants in the observation who are having 
warts treated may also be having small foci of HSIL within the wart being treated 
as well and this may reduce the overall rate of progression from HSIL to cancer in 
the active monitoring arm. 
At screening, participants may report that they have a past or current history of 
internal and/or external warts. If they agree to be screened for the study, they will 
be told that if warts are seen by the clinician, they will need to be biopsied before 
treatment. Of note, this practice will be recommended as standard of care for HIV-


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infected individuals in the upcoming 2014 CDC guidelines for treatment of warts 
(Park, I, personal communication). Participants will be informed that if they qualify 
for the study and are randomized to the treatment arm, all areas of HSIL and warts 
will be treated. Participants will also be informed that if they are randomized to the 
active monitoring arm, warts may be treated at the discretion of the participant and 
the clinician. Potential participants with warts so extensive that they preclude the 
clinician from determining the extent and location of HSIL will not be enrolled in 
the study. 
After enrollment, participants in both study arms with incident warts will have their 
warts treated to the greatest extent possible, at the discretion of the participant and 
the clinician. 
Vulvar HSIL may be commonly found in HIV-infected women who have perianal 
HSIL. Women with vulvar HSIL may be enrolled and vulvar HSIL may be treated 
with targeted ablation. If they are randomized to the treatment arm, both the vulvar 
and perianal HSIL may be treated. If they are randomized to the active monitoring 
arm, vulvar HSIL will be treated per standard of care but HSIL within the boundary 
of the perianal region may not be treated. For the purpose of determining how far 
toward the perianus vulvar HSIL can be treated among those in the Active 
Monitoring arm, the perianus will be defined as extending up to 5 cm from the anal 
verge, but in females not farther than the midway point between the fourchette and 
the anal verge (after the buttocks have been retracted gently). 
4.4.2 Cervical HSIL or cervical cancer in study participants 
All women who participate in the ANCHOR study will undergo screening and 
treatment for cervical HSIL per current standard of care guidelines for HIV-infected 
women. This care will be provided outside the context of the ANCHOR study 
although in some cases, the women may be receiving cervical care by the same 
clinician who is seeing them for their anal HSIL. ANCHOR study staff will work 
closely with the participant and her primary HIV care provider to ensure that she is 
being screened and treated as required. 
Should a female participant be found to develop cervical cancer during the course 
of the ANCHOR study, attempts will be made to ascertain a tissue sample to use 
comparatively in future molecular and/or viral correlative analyses planned. A 
separate consent form for these samples will be given to the participant for her 
agreement before obtaining these samples. 
4.5 
Duration of Follow-Up 
All participants will be followed for up to 5 years after the last participant’s date of 
randomization, diagnosis of invasive anal cancer, or until death, whichever occurs first. 
Participants who develop malignancies other than anal cancer, who are on the monitoring 
arm and drop in to treatment, who are on the treatment arm and discontinue treatment, or 
who develop unacceptable adverse events will remain in follow-up for anal cancer 
outcomes. Participants who cannot attend study visits should be followed for anal cancer 
outcomes via telephone contact, as defined in the protocol Manual of Procedures. Medical 
records will be requested from the participant’s provider in the event that cancer is 


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diagnosed. Participants removed from study for unacceptable adverse event(s) will be 
followed until resolution or stabilization of the adverse event. 
4.6 
Criteria for Removal from the Study (Drop-Out) 
Participants will be removed from the study and reported as drop-outs if one of the 
following events occurs: 
• Participant decides to withdraw from the study 
• Death 
Participants who discontinue the study due to an anal cancer diagnosis will be removed 
from the study but will not be considered drop-outs. 
The reason for study removal and the date the participant was removed from the trial must 
be documented in the Off Study Form in AdvantageEDC. 
 


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5.0 
DOSING DELAYS/DOSE MODIFICATIONS 
5.1 
Dose Modifications Guidelines 
Guidelines for ablative therapy are provided in Section 4.2.2. When administering topical 
treatments, clinicians must follow the algorithms for each treatment modality as shown in 
Figure 4-A and Figure 4-B. Participants experiencing grade 3 adverse events that are at 
least possibly related to treatment should hold the product until the AE resolves or reduces 
in severity to grade 1. The study product should be restarted at the discretion of the site 
investigator using a reduced dose (described in the table below). If the AE continues to be 
resolved or is grade 1 at the reduced dose, then the dose can be escalated again at the 
discretion of the site investigator. 
Participants experiencing grade 2 adverse events that are at least possibly related to 
treatment can either continue the product at the standard dose (preferred) or dose-reduce 
one level. If the participant tolerates the reduced dose after 2 weeks, then the dose can be 
escalated again at the discretion of the site investigator. 
Participants experiencing grade 1 adverse events that are at least possibly related to 
treatment can continue the product at the standard dose. A dose reduction should only be 
done if necessary for continued adherence to study follow-up. 
Table 5-A:1. Dose modifications 
 
Topical 5-fluorouracil 
5% 
Topical Imiquimod 5% 
Standard dose 
Twice daily for 5 days 
followed by 9 days off 
1 single use sachet applied 3 times per week prior to 
normal sleeping hours and left on the skin for 6–10 hours. 
Reduced dose 
Twice daily for 3 days 
followed by 11 days off 
1 single use sachet applied twice per week prior to 
normal sleeping hours and left on the skin for 6–10 hours. 
Minimal dose 
Once daily for 3 days 
followed by 11 days off 
1 single use sachet applied once per week prior to normal 
sleeping hours and left on the skin for 6–10 hours. 
 
 


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6.0 
ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial. The 
following list of AEs (Section 6.1) and the characteristics of an observed AE (Section 6.2) 
will determine whether the event requires expedited reporting in addition to routine 
reporting. All adverse event reporting will be conducted via AdvantageEDC for this 
protocol. 
The descriptions and grading scales found in the revised NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting 
beginning April 1, 2018. The CTEP Version 5.0 of the CTCAE is identified and located 
on 
the 
CTEP 
website 
at 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
All 
appropriate treatment areas should have access to a copy of the CTEP Version 5.0 of 
CTCAE. 
6.1 
Potential Risks Lists 
6.1.1 Expected adverse events related to the diagnostic procedures 
Anal cytology collection, high resolution anoscopy (HRA), and anal biopsy. 
Participants will likely experience pressure and urgency to defecate during the 
cytology collection and HRA. Anal bleeding may occur up to one week after the 
biopsy is taken. 
The risk of infection is less than 1%. 
6.1.2 Adverse event list for topical 5-FU 
Most expected adverse events are local effects where the cream is applied. These 
include pain, burning, irritation, pruritus, erythema, edema, ulceration, bleeding, 
flaking and crusting of the skin, and allergic reactions. Scarring of treated areas has 
also been reported. Participants receiving 5-fluorouracil 5% have also experienced 
emotional upset, medicinal taste, thrombocytopenia, leukocytosis, hair loss, rash at 
other sites, pain/erythema/ulceration at sites adjacent to treated sites (e.g. scrotum), 
eye and nasal irritation, and herpes simplex reactivation. Women who received 5-
fluorouracil 5% have reported miscarriages, and one birth defect (ventriculoseptal 
defect) occurred in an infant born to a woman who was exposed to 5-fluorouracil 
5% during pregnancy. 
6.1.3 Adverse event list for imiquimod 
Most expected adverse events are local effects where the cream is applied. These 
include pain, burning, irritation, pruritus, erythema, edema, ulceration, bleeding, 
flaking, and crusting of the skin, and allergic reactions. Participants receiving 
imiquimod have also experienced headache, rash at other sites, back pain, 
pain/erythema/ulceration at adjacent sites (e.g., scrotum). Sinus infection, nausea, 
fever, and flu-like illness have also been reported. 


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6.1.4 Adverse event list for infrared coagulation, hyfrecation/electrocautery, and laser 
ablation 
Most expected adverse events are local effects where the lesions have been ablated. 
Potential adverse events include minor bleeding (common), textural changes at the 
treatment site, and mild to moderate pain for up to two weeks post-procedure and 
occasionally longer, that is usually well-controlled with pain or anti-inflammatory 
medicines (common). These side effects are generally mild and self-resolve in short 
order. Self-limited bleeding not requiring intervention may occur up to 7-10 days 
post-procedure. Reactivation of herpes simplex has been reported following 
treatment, but a causal relationship to treatment has not been established. Rare 
adverse events (occurring <1% of the time) include heavy bleeding 7-10 days post-
procedure requiring transfusion or operative intervention, severe pain interfering 
with self-care activities of daily living (ADL), stenosis, fissure, fistula, and abscess. 
The rare adverse events described above will be considered unexpected due to 
increased severity if occurring at grade 3 or greater severity. 
6.1.5 Adverse events for surgical excision (TUA) 
Most expected adverse events are local effects where the surgical procedure was 
performed to excise the lesions. Adverse events include minor bleeding (common) 
and pain for up to one to two weeks post-procedure (common). Rare adverse events 
include sustained pain with bowel movements, heavy bleeding, stenosis, fissure, 
fistula, and abscess. In addition, there are side effects related to the anesthesia that 
may be administered for the procedure. These vary with the anesthetic agents used 
and are rare but may be severe including stroke, myocardial infection, and death. 
6.2 
Adverse Event Characteristics and Definitions 
6.2.1 Adverse Event: Any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom or disease temporally associated with the use of a 
medical treatment or procedure regardless of whether it is considered related to the 
medical treatment or procedure (attribution of unrelated, unlikely, possible, 
probable, or definite). 
This includes the following: 
• AEs not previously observed in the participant that emerge during the protocol-
specified AE reporting period, including signs or symptoms associated with 
anal HSIL that were not present prior to study entry. 
• Complications that occur as a result of protocol interventions. 
• Preexisting medical conditions (other than the condition being studied) judged 
by the investigator to have worsened in severity or frequency, or changed in 
character during the protocol-specified AE reporting period. 
The investigator is responsible for ensuring that adequate medical care is provided 
to a participant for all adverse events that occur during the trial, including clinically 
significant laboratory values, related to the trial. All AEs reported in the study 
database will be followed for the participant’s medical care until resolved to the 


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baseline condition or protocol completion; for chronic conditions, resolution may 
be documented when the AE is stable with appropriate medical management. Any 
adverse events that do not require reporting in the study database will be managed 
and followed as appropriate for the participant’s medical care. 
6.2.2 Life-threatening Adverse Event: Any AE that places the participant or participant, 
in view of the Investigator, at immediate risk of death from the reaction. 
6.2.3 Serious Adverse Event (SAE): Any AE occurring at any dose that results in any of 
the following outcomes: Death, a life-threatening AE, inpatient hospitalization or 
prolongation of existing hospitalization, a persistent or significant disability/ 
incapacity, or a congenital anomaly/birth defect. Important medical events that may 
not result in death, be life-threatening, or require hospitalization may be considered 
serious when, based upon appropriate medical judgment, they may jeopardize the 
participant and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. 
6.2.4 Hospitalization: hospitalization for expedited AE reporting purposes is defined as 
an inpatient hospital stay equal to or greater than 24 hours. Hospitalization is used 
as an indicator of the seriousness of the AE and should ONLY be used for situations 
where the AE truly fits this definition and NOT for hospitalizations associated with 
less serious events. (e.g., a hospital visit where a patient is admitted for observation 
or minor treatment such as, hydration and released in less than 24 hours). 
Prolongation of hospitalization is defined as an extension of current hospitalization 
equal to or greater than 24 hours. 
Please note for hospitalization – All hospitalizations (or prolongation of existing 
hospitalization) for medical events equivalent to CTCAE Grade 3, 4, 5 must be 
reported regardless of the requirements for Phase of study, expected or unexpected, 
and attribution. For example, do not report an admission for pharmacokinetic 
sampling, but do report an admission for a myocardial infarction. 
6.2.5 Toxicity: Toxicity is a term NOT clearly defined by regulatory organizations. 
Toxicity has been described as an AE that has an attribution of possibly, probably 
or definitely related to investigational treatment. To minimize confusion the NCI 
would recommend that the term toxicity NOT be utilized for AE reporting 
purposes. The CTCAE continues to use the term ‘toxicity’ because of familiarity. 
6.2.6 Unexpected Adverse Event: Any AE that is not listed in available sources including 
the package insert, the Investigator’s Brochure, or the protocol, or is not consistent 
with the severity or specificity of the risk information described in the available 
sources. 
6.2.7 CTEP Adverse Event Reporting System (CTEP-AERS): An electronic system for 
expedited submission of AE reports. A SAE reporting form in AdvantageEDC will 
be used in lieu of CTEP-AERS for this trial. 
6.2.8 Attribution: An assessment of the relationship between the AE and the medical 
intervention. The CTCAE does not define an AE as necessarily “caused by a 
therapeutic intervention.” After naming and grading the event, the clinical 


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investigator must assign an attribution to the AE using the following attribution 
categories: 
Table 6-A: Mapping adverse event attribution assignments to causal relationship 
designations 
 
RELATIONSHIP 
ATTRIBUTION 
DESCRIPTION 
Unrelated to investigational 
agent/intervention 
Unrelated  
The AE is clearly NOT related to the 
intervention  
Unlikely 
The AE is doubtfully related to the 
intervention  
Related to investigational 
agent/intervention 
Possible  
The AE may be related to the intervention  
Probable 
The AE is likely related to the intervention  
Definite 
The AE is clearly related to the intervention  
NOTE: AEs listed as ‘possibly, probably, or definitely’ related to the 
investigational agent/intervention are considered to have a suspected ‘reasonable 
causal relationship’ to the investigational agent/intervention (ICH E2A). For 
routine adverse event reporting purposes on this protocol, “attribution” defines the 
relationship 
between 
the 
adverse 
event 
and 
the 
investigational 
agent(s)/intervention. 
6.2.9 Unanticipated adverse device effect: Any serious adverse effect on health or safety 
or any life-threatening problem or death caused by, or associated with, a device, if 
that effect, problem, or death was not previously identified in nature, severity, or 
degree of incidence in the investigational plan or application (including a 
supplementary plan or application), or any other unanticipated serious problem 
associated with a device that relates to the rights, safety, or welfare of participants. 
6.3 
Expedited Adverse Event Reporting 
6.3.1 Expedited AE reporting for this study must use the SAE form in AdvantageEDC. 
As a commercial agent study, CTEP-AERS will not be used for this protocol. The 
reporting procedures to be followed in AdvantageEDC are provided in this section 
and will align with the principles for SAE reporting in the “CTEP, NCI Guidelines: 
Adverse Event Reporting Requirements,” which can be downloaded from the 
CTEP home page (http://ctep.cancer.gov). These requirements are briefly outlined 
below (Section 6.3.3). 
A 24-hour notification is to be made to the ANCHOR DMC by telephone at 301-
251-1161, only when Internet connectivity is disrupted. Once Internet connectivity 
is restored, a 24-hour notification phoned in must be entered electronically into 
AdvantageEDC by the original submitter at the site. 
6.3.2 AdvantageEDC is programmed for automatic electronic distribution of SAE reports 
to the following individuals: the ANCHOR DMC, the ANCHOR Medical Monitor, 
Protocol Chairs, and the Principal Investigator at the institution. The ANCHOR 


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DMC is responsible for distribution of SAE reports to other parties on behalf of the 
sponsor. 
6.3.3 Expedited reporting guidelines 
Investigators must report ALL SAEs (as defined in Section 6.2.3) to the ANCHOR 
DMC and IRBs as required by the protocol and local IRB policy. The investigator’s 
initial expedited report to the ANCHOR DMC for the SAE will be made using the 
Adverse Event form in AdvantageEDC within 24 hours of awareness of the event 
(routine report), and unless an exception from expedited reporting applies per 
Section 6.3.4, will be followed by a completed SAE form in AdvantageEDC as 
soon as possible, and in no more than 10 days of awareness. If the event is a grade 
4 or 5 SAE, or determined by the investigator to be an unanticipated adverse device 
effect or grade 3 SAE that is at least possibly attributed to protocol treatment, the 
completed SAE form will be submitted within 5 calendar days of awareness. 
Timeframes for reporting SAEs at any time during trial participation are outlined 
in the flowchart provided below, with exceptions from SAE form reporting noted. 
Figure 6-B: Timeframes and Exceptions for Expedited SAE Reporting 
 
6.3.4 Exceptions to expedited adverse event reporting 
Expedited reports (SAE form in AdvantageEDC) are not required for serious 
adverse events that occur in participants in screening and participants on the Active 
Monitoring arm. On the treatment arm, an expedited report is not required if the 
SAE occurred more than 30 days after administration of the last protocol treatment 
(last dose of topical therapy, ablative treatment, or TUA, whichever occurred last) 
and has an attribution of unrelated or unlikely to the protocol agent(s). SAEs that 
occur in these participants will be submitted as routine adverse event reports only 
(AE form in AdvantageEDC). 


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6.3.5 Reporting to regulatory authorities 
As all agents used on this trial are exempt from the requirements for an IND or an 
IDE, respectively, no serious adverse event reporting to FDA is required. In the 
event that any unexpected and serious adverse events reported on this trial are 
determined to have a reasonable possibility of a causal relationship to the study 
drugs or devices, the SAE or UADE will be reported to FDA using its voluntary 
reporting mechanism (MedWatch Online Voluntary Reporting Form). Voluntary 
SAE/UADE reporting will be performed by the ANCHOR DMC following the 
medical monitor’s determination that the AE meets these criteria. 
6.4 
Routine Adverse Event Reporting 
The following AEs must be reported in an Adverse Event form in AdvantageEDC for 
routine study data submissions: 
• All serious adverse events regardless of relationship to study treatment or study 
procedures 
• All grade 3 events that are at least possibly related to study treatment or study 
procedures 
• All grade 1 or 2 adverse events leading to stopping or changing protocol treatment; all 
other adverse events must be recorded in the source documents only 
• Suspected or confirmed COVID-19 diagnoses (any grade), with information on the 
type of testing performed to confirm the test, if done 
6.4.1 Timeline for routine adverse event reporting 
All adverse events meeting the routine reporting criteria that occur from the time 
of enrollment for screening through the final study visit must be reported in 
AdvantageEDC. Adverse events that are ongoing at the time of protocol 
discontinuation and that are attributed to one of the protocol agents or procedures 
must be followed until resolution or until the investigator deems the event to be 
clinically stable. 
6.4.2 Clinical laboratory abnormalities 
Clinical laboratory abnormalities will be considered AEs if determined to be 
clinically significant by the investigator. In assessing laboratory results, an 
abnormal laboratory value will be considered clinically significant if it is 
characterized by one or more of the following criteria: 
• Is judged by the investigator to have a causal relationship to the 
investigational agent 
• Requires clinical intervention or monitoring, such as: close observation, more 
frequent 
follow-up 
assessments, 
further 
diagnostic 
intervention, 
treatment/therapeutic intervention, or protocol therapy dose modification 
• Is associated with clinical signs or symptoms, which may suggest a disease 
and/or organ toxicity, or may represent a new condition or worsening of a 
baseline condition 


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• Is associated with a serious adverse event, or is otherwise judged by the 
Investigator to be of significant clinical impact 
Laboratory results that are proven erroneous by repeat testing will not be considered 
clinically significant. 
In general, a laboratory abnormality that is not clinically significant will be 
consistent with CTCAE grade 1 (mild) or 2 (moderate) severity, as categorized by 
the relevant severity description in the Investigations System Organ Class (SOC) 
or Metabolism and Nutrition Disorders SOC. Investigators may not designate 
laboratory abnormalities that are consistent with grade 3 or greater severity as not 
clinically significant. 
6.5 
Secondary Malignancy 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (e.g., 
treatment with investigational agent/intervention, radiation, or chemotherapy). A 
secondary malignancy is not considered a metastasis of the initial neoplasm. Three options 
are available in the CTCAE to describe secondary malignancies: 
• Leukemia secondary to oncology chemotherapy (e.g., acute myelocytic leukemia 
[AML]) 
• Myelodysplastic syndrome (MDS) 
• Treatment-related secondary malignancy 
As prior anal cancer is exclusionary and no cancer therapies are provided for this protocol, 
any malignancy possibly related to a cancer treatment preceding protocol participation 
(including AML/MDS) will be reported via the routine reporting mechanisms outlined in 
this protocol. 
6.6 
Second Malignancy 
A second malignancy is one unrelated to the treatment of a prior malignancy (and is NOT 
a metastasis from the initial malignancy). Second malignancies require ONLY routine 
adverse event reporting. 
 


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7.0 
PHARMACEUTICAL AND STUDY DEVICE INFORMATION 
A list of the adverse events and potential risks associated with the investigational or 
commercial agents administered in this study can be found in Section 6.1. 
7.1 
5-Fluorouracil 5% Cream 
NOTE: Please refer to the commercial package insert for more information. 
7.1.1 Other names 
Efudex Cream 
7.1.2 Classification 
Topical 5% fluorouracil cream is an antineoplastic antimetabolite. It is a topical 
preparation, available in a 40 gm tube, containing the fluorinated pyrimidine 5-
fluorouracil, an antineoplastic antimetabolite. Five percent (5%) 5-fluororacil 
cream contains 5 g of 5-fluororacil per 100 g of cream. The cream contains 5% 
fluorouracil in a vanishing cream base consisting of white petrolatum, stearyl 
alcohol, propylene glycol, polysorbate 60 and parabens (methyl and propyl). 
Chemically, fluorouracil is 5-fluoro-2,4(1H,3H)-pyrimidinedione. It is a white to 
practically white, crystalline powder that is sparingly soluble in water and slightly 
soluble in alcohol. One gram of fluorouracil is soluble in 100 mL of propylene 
glycol. The molecular weight of fluorouracil is 130.08 and the molecular formula 
is C4H3FN2O2. 
7.1.3 Mode of action 
There is evidence that the metabolism of fluorouracil in the anabolic pathway 
blocks the methylation reaction of deoxyuridylic acid to thymidylic acid. In this 
manner fluorouracil interferes with the synthesis of deoxyribonucleic acid (DNA) 
and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA 
and RNA are essential for cell division and growth, the effect of fluorouracil may 
be to create a thymine deficiency which provokes unbalanced growth and death of 
the cell. The effects of DNA and RNA deprivation are most marked on those cells 
which grow more rapidly and take up fluorouracil at a more rapid rate. The 
catabolic metabolism of fluorouracil results in degradation products (e.g., CO2, 
urea, α-fluoro-β-alanine) which are inactive. 
7.1.4 Storage and stability 
Store at 25°C (77°F); excursions permitted to 15°C-30°C (59°F-86°F). 
7.1.5 Dose specifics and administration 
Participants will be instructed to apply 1 ml of topical 5-fluorouracil 5% cream. 
One gram of topical 5-fluorouracil is the equivalent of 1 ml. The total daily dose 
will be 1 gram of cream, and the weekly dose is 5 grams of cream. 5% 5-fluororacil 
contains 5 grams of 5-fluororacil per 100 grams. The daily dose is therefore 50 mg 
of 5-fluororacil delivered in divided doses of 25 mg BID, and the total weekly dose 
is 250 mg delivered in 5 grams of cream. Participants will be given a 40 gram tube 
of 5-FU cream and instructed to squeeze out the cream to the marked line on the 
applicator (0.5 mL of cream), or a right hand glove or finger cot. Using a glove or 


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a finger cot they will then squeeze one half the amount of cream (0.25 mL) from 
the syringe (if using an applicator) onto their right index finger and insert the cream 
approximately one inch into the anus on the right side. The participant will then 
place the remaining cream (0.25 mL) on the left finger, and insert the cream one 
inch into the anus on the left side. Similar instructions apply for perianal 
application, but without insertion of the cream into the anal canal. Participants who 
will be treated for perianal HSIL will be dispensed an additional tube to treat the 
perianus (dose doubled). 
7.1.6 Preparation 
Efudex Cream (5-FU) is available in 40-gm tubes containing 5% fluorouracil (NDC 
0187-3204-47) in a vanishing cream base consisting of white petrolatum, stearyl 
alcohol, propylene glycol, polysorbate 60 and parabens (methyl and propyl). It is 
distributed by Bausch Health. FDA-approved generic versions of 5% fluorouracil 
are also permitted on this protocol. 
7.1.7 Side effects 
See Section 6.1.2. Refer to the approved package insert for complete prescribing 
and toxicity information. 
7.2 
Topical Imiquimod Cream (5%) 
NOTE: Please refer to the commercial package insert for more information. 
7.2.1 Other names 
Aldara. 
7.2.2 Classification 
Imiquimod belongs to the chemical class of substances known as 
imidazoquinolinamines. Five percent (5%) imiquimod cream contains 50 mg of 
imiquimod per 1 g of cream, supplied in single-use packets that contain 250 mg of 
cream. 
7.2.3 Mode of action 
Imiquimod works through stimulation of local immune responses, in part activation 
of toll-like receptors. 
7.2.4 Storage and stability 
Store at less than 25ºC (77ºF). Avoid freezing. 
7.2.5 Dose specifics and administration 
The imiquimod dose will be 750 mg of cream per week, administered as one single 
use sachet (250 mg) in cartons of 12 or 24 sachets, applied 3 times per week for up 
to 16 weeks (two 8-week courses). Contents of one packet are applied to the 
affected area at bedtime, three times per week. The cream is left on for 6-10 hours 
and washed off in the morning with soap and water. Twenty-four packets will be 
dispensed for each 8-week course of intra-anal treatment; if perianal HSIL is to be 
treated, an additional 24 packets will be dispensed to the participant for each 8-
week course of perianal treatment (dose doubled to treat the perianus). 


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7.2.6 Preparation 
Chemically, imiquimod is 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-
amine. Imiquimod has a molecular formula of C14H16N4 and a molecular weight 
of 240.3. Imiquimod is available as a white to faintly yellow oil-in-water cream 
base consisting of isostearic acid, cetyl alcohol, stearyl alcohol, white petrolatum, 
polysorbate 60, sorbitan monostearate, glycerin, xanthan gum, purified water, 
benzyl alcohol, methylparaben, and propylparaben. It is marketed by Bausch 
Health. FDA-approved generic versions of 5% imiquimod are also permitted on 
this protocol. 
7.2.7 Side effects 
See Section 6.1.3. 
7.3 
Study Devices 
All study devices will be used in a final form as obtained from the manufacturer. 
7.3.1 Infrared Coagulator 
The Infrared Coagulator 1900 and 2100 are supplied by the Redfield Corporation. 
See Section 2.2.2 for details of IRC treatment. 
7.3.2 Hyfrecation 
The Hyfrecator 2000 is a mobile unit manufactured and serviced by Conmed 
Corporation of Utica, New York. See Section 2.2.3 for details of hyfrecation 
treatment. 
7.3.3 Other ablative devices 
The following classes of devices are permitted for in-office removal of anal HSIL. 
All devices in this category must be reported to the protocol chairs before use to 
confirm these criteria are met. 
• Legally marketed electrocautery devices regulated under 21 CFR 878.4400, 
that are indicated for cutting or coagulating tissue, and that are appropriate for 
use in dermatologic, general, and gastrointestinal surgical procedures. 
• Legally marketed carbon-dioxide lasers regulated under 21 CFR 878.4810, that 
are indicated for the excision, coagulation, or ablation of tissue, and that are 
appropriate for use in dermatologic, general, and gastrointestinal surgical 
procedures. 
Cryotherapy will not be permitted for HSIL treatment on this trial. Concurrent wart 
treatment using targeted cryotherapy may occur at the discretion of the treating 
physician for the participant’s medical care. 
7.4 
Drug Supply and Accountability 
Participating institutions may source topical imiquimod cream (5%) and 5-fluorouracil 5% 
for this protocol through commercial pharmacy supply channels for the institution. For all 
agents sourced by the institution, the institution will maintain records of the inventory and 
disposition of all drugs according to local pharmacy standards of procedure. 


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A supply of Efudex (5-fluorouracil 5%) will be available for use on this protocol for no 
charge (while supplies last). Participating institutions are to refer to the study drug request 
form located on the ANCHOR DMC website (www.anchordmc.com) to order study-
supplied 5-fluorouracil 5% cream. 
For all study-supplied topical agents, the Investigator, or a responsible party designated by 
the Investigator, must maintain a careful record of the inventory and disposition of all drugs 
received or reimbursed by the study using the NCI Investigational Agent Accountability 
Record Form (DARF), available on the CTEP home page (http://ctep.cancer.gov) or by 
calling the Pharmaceutical Management Branch at 240-276-6575. The DARF documents 
the drug delivery date to the site, inventory at the site, use by each study participant, and 
disposal of the drug (if applicable). A site-specific accountability record, either manual or 
electronic, may be used if it includes all the information required on the DARF and if the 
paper printout is identical to the DARF. A separate DARF is required for each protocol 
using the same agent. The investigator will ensure that the drugs are used only in 
accordance with this protocol for the treatment of HSIL or concurrent condyloma, as 
outlined in Section 4.2 and 4.4. 
A tube of study-supplied agent may be designated on the DARF for clinic use for 
demonstrating application of the cream at the randomization visit (Section 8.3.9). 
After ordering, study-supplied topical agents may not be transferred to other institutions. 
Expired drug or drug returned by study participants must be destroyed in accordance with 
the institution’s standards of procedure. 
 


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8.0 
CLINICAL AND LABORATORY EVALUATIONS 
Schedules shown in the study calendar below are provided in Appendix I. 
Treatment cycles are generally organized into 6-month blocks. 
8.1 
Special Instructions and Definitions of Evaluations 
All clinical and laboratory information required by this protocol is to be present in the 
source documents. All data requested by the study should be recorded in the source 
documents. A subset of the data points stated here will be entered on the appropriate CRF 
via the AdvantageEDCSM Internet Data Entry System, as specified in the ANCHOR Study 
Forms Instructions. 
8.1.1 Medical history 
• History of receptive anal intercourse ever 
• History of genital or anal condyloma 
• History of prior anal cytologies or anal biopsies 
• History of CDC HIV risk group and AIDS defining illnesses 
• History of systemic cytotoxic chemotherapy and/or radiation 
• History of hemophilia 
• Other aspects of the medical history including allergies to any medications 
(iodine, lidocaine, or contrast agents) should be in the source documents only 
8.1.2 Medication history 
A medication history must be present in source documents per Section 4.3.2, 
including, including actual or estimated start and stop dates, indication, dosage, and 
schedule. All other prescription medications will be listed in the source. 
• HIV treatment history in the 45 days prior to study entry. 
• Any history of HPV vaccination 
• Anticoagulants 
• Systemic immunomodulatory agents. If receiving any immunomodulatory 
agents at screening, evaluate duration of receipt for 6 months prior to 
randomization. 
• Investigational agents 
• Medications that the investigator determines to have a causal relationship to 
serious adverse events that occur after screening enrollment 
8.1.3 Concomitant medications/antiretroviral medication modifications 
Refer to Section 4.3.2. 


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8.1.4 Study treatment record 
All modifications to study treatment initiated by the investigator, including initial 
doses, dose reductions, and permanent discontinuation of treatment. 
8.1.5 Nadir CD4+ cell count 
The participant’s prior nadir CD4+ cell count (absolute value, and if available, date) 
should be documented, with a copy of the nadir CD4+ cell count report when 
possible, and will be entered on the CRF. If this documentation is not available, 
then participant recollection will suffice. For participants who do not know the 
exact nadir value and for whom there is no source documentation, then recall of the 
categorical nadir (e.g., <50, <100, <200 cells/mm3) will suffice. 
8.1.6 Complete physical exam (at screening visit) 
This examination includes at a minimum an examination of the skin, head, mouth, 
and neck; auscultation of the chest; cardiac exam; abdominal exam; examination of 
the lower extremities for edema; and Karnofsky performance test. The complete 
physical exam will also include signs and symptoms, height, weight, diagnoses, and 
vital signs (temperature, pulse, respiration rate, and blood pressure). 
8.1.7 Targeted physical exam (subsequent visits) 
A targeted physical examination is to include pulse, blood pressure, and review of 
any previously identified or new signs or symptoms, including diagnoses that the 
participant has experienced since the last visit. Staff should inquire about symptoms 
and examine the treatment areas for any adverse events. 
8.1.8 Digital anorectal examination and examination of inguinal nodes 
A digital anorectal examination and an examination of inguinal nodes are to be 
performed to note the presence or absence of any abnormalities. 
8.1.9 Adverse event evaluation 
All signs and symptoms that begin during or after the entry visit must be recorded 
in the source regardless of grade. Any signs and symptoms that began prior to the 
screening visit should be recorded, and will only be considered an adverse event if 
there is a significant worsening of the sign or symptom during the study. All signs 
and symptoms that begin during or after the last dose of protocol treatment must be 
recorded in the source regardless of grade or attribution to treatment. 
All adverse events that require routine reporting per Section 6.4 must be recorded 
by clinical term and/or CTCAE categorization, severity (grade), seriousness, 
attribution to protocol interventions, and event status. AEs that are not serious (as 
that term is defined in Section 6.2), are of grade 1 or 2 severity, and that do not 
require modification to protocol treatment do not require routine AE reporting, and 
may be recorded in the source only by denoting the clinical term, severity, and 
seriousness. 
If known at the time of reporting, a diagnosis should be reported as a single adverse 
event rather than individual signs and symptoms. However, if a constellation of 
signs and/or symptoms cannot be medically characterized as a single diagnosis or 


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syndrome at the time of reporting, it is acceptable to report the information that is 
currently available. If a diagnosis is subsequently established, it should be reported 
as follow-up information. 
8.1.10 Laboratory evaluations 
• Hematology (CBC with differential) may be performed by the enrolling site or 
the results from prior testing may be obtained, provided that the results were 
issued within 90 days prior to randomization from a CLIA-certified laboratory. 
• CD4 count and HIV viral load, to be performed by the enrolling site at baseline 
unless a copy of the results are available from clinical care within 6 weeks prior 
to randomization. The result from routine clinical care may be collected at all 
subsequent visits. 
• Urine pregnancy test for females of childbearing potential (FCBP). After the 
randomization visit, pregnancy tests will only be required for FCBP in the 
treatment arm before treatment procedures, and will not be required at these 
time points if the participant is receiving standard, long-acting contraception 
(e.g., IUDs). 
• For females, a record (from a licensed healthcare provider) of the cervical 
cytology result and documentation of visual examination of the vulva, vagina, 
and cervix to rule out lesions suspicious for cancer within 12 months prior to 
randomization, per Section 3.1.5. 
• Anal cytology for local cytology result, and residual cytology media for 
correlative studies (See Appendix III, Anal Cytology Sampling Procedures). A 
second anal swab will be collected in Thinprep for DNA studies, and a third 
anal swab will be collected in RNAProtect for RNA/protein studies. All three 
swabs will be collected in this order at each visit where required. If a local 
cytology result exists to satisfy protocol requirements at the time of the visit, 
the first anal swab will be recollected with the second and third swabs, and the 
first swab sent to the ANCHOR Biorepository without any local processing. 
• High resolution anoscopy and biopsies (See Appendix IV). 
8.1.11 Blood samples for correlative studies 
A blood sample (one red top tube) will be drawn for storage at the ANCHOR 
Biorepository for future testing. Whole blood (one lavender top tube) will be 
collected at Visit 2 only. Please refer to the ANCHOR Study Manual of Procedures 
for information regarding serum sample collection, preparation, and shipment. 
8.1.12 Oropharyngeal swab collection for ancillary SARS-CoV-2 study 
The swab for oropharyngeal SARS-CoV-2 testing will be collected on the same 
day as the anal swabs. A dry, scored swab will be inserted into the posterior pharynx 
and tonsillar areas. The swab will be rubbed over both tonsillar pillars and posterior 
oropharynx and the clinician collecting the sample will avoid touching the tongue, 
teeth, and gums. He/she will place the swab immediately into the labeled sample 
collection tube and will break the applicator stick off at the score line to permit 


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tightening of the cap. The tube will be stored at -20C or colder until shipment to 
the ANCHOR Biorepository. See the study manual of procedures for shipping 
instructions. 
8.1.13 Retention follow-up 
Site staff may contact the participant between 6-month visits to identify and attempt 
to assist with issues related to visit attendance and study retention. Instructions are 
identified in the protocol Manual of Procedures. 
8.2 
Screening Evaluations (Visit 0) 
Screening evaluations are to be conducted within 12 weeks before randomization (Visit 1), 
except as noted below. It is recommended that screening occur within 4 weeks after the 
participant attends a study information session. Screening evaluations for Visit 0 may be 
performed at Visit 1 (except informed consent, the anal swab for cytology, the screening 
HRA, and anal biopsies), provided that all Visit 0 evaluations are performed before 
randomization. 
8.2.1 Informed consent and comprehension assessment administration at least one week 
before, and no more than 6 weeks before randomization. Participants may be 
verbally re-consented at Visit 1 before randomization if more than 6 weeks have 
elapsed since signing the informed consent document. 
8.2.2 Documentation of HIV status (in compliance with Section 3.1.1), medical history 
(including nadir CD4 count from participant recall), medication review, and all 
eligibility criteria 
8.2.3 Venipuncture for: 
8.2.3.1 Complete blood count with differential and platelets (may occur up to 90 
days prior to randomization) 
8.2.3.2 Serum (red top tube) for the ANCHOR Biorepository for correlative studies 
(may occur up to 12 weeks prior to randomization) 
8.2.4 Urine pregnancy test for females of childbearing potential (FCBP) 
8.2.5 Complete physical examination, height, and weight 
8.2.6 Anal swab for cytology (may occur up to 12 weeks prior to randomization (the local 
cytology result must be known before randomization enrollment) and additional 
swabs for correlative studies. 
8.2.6.1 Leftover material from anal swab cytology suspension (Appendix III). If the 
anal cytology result is available within 12 weeks prior to randomization but 
the residual is not available to ship to the Biorepository, the first anal swab 
may be recollected and submitted directly to the Biorepository for 
correlative studies. 
8.2.6.2 Anal swab for DNA 
8.2.6.3 Anal swab for RNA/protein 
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8.2.8 High resolution anoscopy with biopsies of visible lesions (may occur 1-12 weeks 
prior to randomization, including before the initial screening visit). Participants 
who have only a single lesion that is likely to be removed entirely with the initial 
screening biopsy should be excluded from the trial because the participant cannot 
be followed for 5 or more years after randomization. 
HSIL lesions that were biopsied for clinical care prior to the visit may be used to 
document eligibility, and will not require repeat HRA and rebiopsy during the 
screening visit only if all three criteria below are satisfied: 
• The biopsies were obtained within 12 weeks before the randomization visit; 
• The HSIL lesion(s) are observed by the study clinician during the HRA at Visit 
1 preceding randomization; and, 
• The pathology report and slides from those prior biopsies are available to 
submit for central pathology review. 
8.2.9 Participants who fail screening due to suspected cancer diagnoses or cancer 
diagnoses will undergo central pathology review to confirm the diagnosis, if 
biopsies are available from screening procedures performed by the site. Participants 
diagnosed with cancer during screening may provide an optional cancer biopsy 
(placed in RNALater) only if both of the following criteria are satisfied: 
• Consent is provided for use of the participant’s specimens following screen 
failure 
• The participant is willing to return to the clinic for biopsy before referral to 
treatment and study discontinuation. 
8.2.10 Substudy completed in February 2020: ANCHOR Health-Related Symptom Index 
(A-HRSI; Appendix X) and patient-reported ECOG questionnaire completed via 
telephone-facilitated interview (by MSKCC staff) or electronic patient-reported 
outcomes (ePRO) platform (independently by the participant) at any time following 
screening enrollment until time of randomization (if the participant consents to 
participate in the optional HRQoL substudy). 
8.2.11 QoL objective: If the participant consented and is eligible to complete 
questionnaires for the QoL objective, A-HRSI completion up to 2 weeks before 
randomization (see Section 8.9). 
8.2.12 SARS-CoV-2 ancillary study: If the participant consented for this study, an 
oropharyngeal swab will be collected at the same screening visit as anal swabs 1-
3, as outlined in Section 8.1.12. 
8.3 
Baseline Visit with Randomization Evaluations (Visit 1) 
Visit 1 is to be conducted 1-12 weeks following informed consent (re-consent is required 
if written informed consent occurred more than 6 weeks before randomization, as outlined 
in Section 8.2.1) and may occur up to 12 weeks after the screening HRA was performed. 
8.3.1 Eligibility review and choice of initial HSIL therapy (required for randomization 
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8.3.2 ANCHOR Visit 1 Questionnaire (Risk Factor Questionnaire, Appendix V). The 
questionnaire will be administered to the participant via AdvantageEDC. The 
questionnaire may be administered up to two weeks before a planned randomization 
visit. Paper versions of the questionnaire completed by the participant will be 
submitted to the ANCHOR DMC for data entry. 
8.3.3 Review changes in medications and any adverse events from screening procedures 
8.3.4 Venipuncture for CD4 level and HIV viral load within 6 weeks before 
randomization (copies of test results from clinical care will be acceptable). 
Venipuncture may be performed on the day of randomization, even after enrollment 
in the system. 
8.3.5 Urine pregnancy test for all FCBP (must be performed within 7 days prior to 
randomization) 
8.3.6 Digital anorectal examination and examination of inguinal nodes 
8.3.7 High resolution anoscopy for all participants to verify the presence of HSIL lesions 
to be treated or monitored. Only new HSIL lesions or lesions that are suspicious for 
cancer should be biopsied at this visit. This HRA at the randomization visit must 
precede randomization enrollment in AdvantageEDC (Segment B). The participant 
will discontinue screening if no biopsy-proven HSIL diagnosed from the screening 
HRA is located during the randomization HRA. 
8.3.8 Randomization enrollment in AdvantageEDC 
8.3.9 For treatment arm participants being treated with IRC/electrocautery/laser, the 
initial study treatment should be performed at the randomization visit (within 2 
weeks of the randomization visit will be permitted). New lesions that are 
empirically identified as HSIL and biopsied at the randomization visit should be 
treated as part of the initial study treatment, or at a subsequent interim visit if staged 
treatments will be performed. 
For those being treated with 5-FU or imiquimod, treatment must be initiated within 
2 weeks of randomization. The clinician will demonstrate proper application of 
treatment to the areas affected by HSIL at the randomization visit. Demonstration 
using the first dose of study agent is strongly encouraged; an inert cream will be 
used if treatment initiation will be delayed. If the participant has a biopsy at the 
randomization visit, the initial treatment will be delayed within this window to 
permit healing. 
For those being treated with TUA, the participant will be referred to surgery and 
should be performed within 2 months. The costs of TUA will only be covered by 
the study with the QA committee’s approval. 
8.3.9.1 If the participant is to be treated with an ablative therapy, one additional 
biopsy will be collected prior to treatment from the lesion with most severe 
appearance of disease. This biopsy will be placed in RNAlater for banking 
only. A maximum of one biopsy for banking may be obtained every 6 
months. 


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8.4 
Evaluations between Six Month Visits (Interim Visits) 
For treatment arm participants, evaluations are to be conducted per the treatment 
algorithms in Figure 4-A and Figure 4-B and in the schedule of evaluations in Appendix I 
for each treatment modality. Monitoring arm participants may be seen between six month 
visits as clinically indicated for monitoring of potential progression for cancer. 
8.4.1 Medication review 
8.4.2 Adverse event evaluation 
8.4.3 Urine pregnancy test for FCBP in the treatment arm only before treatment 
procedures, unless otherwise needed at the clinician’s discretion 
8.4.4 High resolution anoscopy, digital anorectal examination, and examination of 
inguinal nodes, as clinically indicated 
8.4.5 For treatment arm participants, initiation of additional cycles of treatment (as 
determined by the investigator) 
8.4.5.1 If the participant is to be treated with an ablative therapy, one additional 
biopsy will be collected prior to treatment from the lesion with most severe 
appearance of disease. This biopsy will be placed in RNAlater for banking 
only. A maximum of one biopsy for banking will be obtained during each 
6 month treatment block, if treatment is administered. 
8.4.6 Completed in February 2020: If the participant consents to participate in the 
optional HRQoL substudy for A-HRSI scale responsiveness, the A-HRSI, Patient 
ECOG PS, and PGIC measures will be completed via telephone-facilitated 
interview (performed by MSKCC staff) or ePRO platform (independently by the 
participant) at the following time points: 
8.4.6.1 Time 2: At least 14 days (2 weeks) post-randomization, and at most 70 days 
(10 weeks) post-randomization (Time 2). 
8.4.6.2 Time 3: At least 71 days (10 weeks + 1 day) post-ANCHOR randomization, 
and at most 112 days (16 weeks) post-randomization. 
8.4.7 QoL objective: If the participant consents and is eligible to complete questionnaires 
for the QoL objective, A-HRSI completion at 2-7 week (+3 days) and 4 weeks (±1 
week) after randomization (see Section 8.9 and Appendix I). 
8.5 
Evaluations at Visit 2 (6 Months) for All Participants 
Evaluations are to be conducted ± 4 weeks. 
8.5.1 Medication review 
8.5.2 Adverse event evaluation 
8.5.3 Serum (one red top tube), and plasma and whole blood fraction (one lavender top 
tube) for the ANCHOR Biorepository for correlative studies 
8.5.4 Urine pregnancy test for FCBP on the treatment arm only before treatment 
procedures, unless otherwise needed at the clinician’s discretion. 
8.5.5 Targeted physical exam (per Section 8.1.7) 


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8.5.6 Anal swab for local cytology and additional swabs for correlative studies 
8.5.6.1 Leftover material from anal swab cytology suspension (Appendix III) 
8.5.6.2 Anal swab for DNA 
8.5.6.3 Anal swab for RNA/protein 
8.5.7 Digital anorectal examination and inguinal lymph node examination 
8.5.8 High resolution anoscopy 
8.5.9 Anal biopsy depending on the assigned arm (Section 4.1 or Section 4.2) 
8.5.9.1 HSIL in the treatment arm may be empirically treated at 6-month visits if 
the lesion is biopsied before treatment at the same visit and if cancer is not 
suspected on HRA. 
8.5.9.2 If the participant is to be treated with an ablative therapy, one additional 
biopsy will be collected prior to treatment from the lesion with most severe 
appearance of disease. This biopsy will be placed in RNAlater for banking 
only. A maximum of one biopsy for banking will be obtained during each 
6-month treatment block, if treatment is administered. 
8.5.9.3 If all biopsies do not show HSIL or are inconclusive, and the local cytology 
result shows high grade disease (ASC-H or HSIL), the investigator should 
bring the participant back for repeat HRA at 3 months (interim visit; 
window of ±4 weeks). If a biopsy is performed at an even-numbered visit 
for a monitoring arm participant and the biopsy results are discordant, the 
participant will not be asked to return for repeat biopsy until the next 6-
month visit unless there is suspicion for progression to cancer. If at least 
one biopsy shows HSIL, repeat biopsies of lesions with inconclusive 
histology may be deferred to the next 6-month visit so long as there is no 
suspicion of progression to cancer. If the visit window for the next 6-month 
visit is within 8 weeks of the last biopsy, repeat HRA and biopsies will occur 
as part of the next 6-month visit. 
8.5.10 
SARS-CoV-2 ancillary study: If the participant consented for this study and 
completed swab collection before randomization, an oropharyngeal swab will be 
collected, as outlined in Section 8.1.12. 
8.5.11 
COVID-19 telephone follow-up: If the 6-month visit cannot be conducted due to 
COVID-19 -related restrictions, a telephone follow-up visit may be conducted in 
lieu of a 6-month visit to obtain follow up on treatment completion (if relevant), 
safety, medical history, medication history, and study arm compliance. See study 
source documents for instructions. If a telephone follow-up occurs, a makeup-6 
month visit must be conducted as soon as an in-person visit is feasible again. If a 
site performs a telehealth visit because the 6-month visit cannot be 
performed, it will still require reporting as a missed visit and is still 
considered a protocol deviation. 
8.6 
Evaluations at Follow-Up (Visits 3-10+), Every 6 Months until Study Completion 
Evaluations are to be conducted ± 4 weeks of the 6-month follow-up visit. 


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Interim visits may occur as specified in algorithms for each specific treatment, or for 
clinicians wanting to examine participants more frequently in the active monitoring arm. 
Evaluations are to occur in both study arms unless otherwise specified. 
8.6.1 Medication review 
8.6.2 Recording of HIV viral load and CD4 count laboratory results (annually, only if 
available from clinical care) 
8.6.3 Adverse events evaluation 
8.6.4 Serum (red top tube) for the ANCHOR Biorepository for correlative studies 
8.6.5 Urine pregnancy test for FCBP on the treatment arm only before treatment 
procedures, unless otherwise needed at the clinician’s discretion. 
8.6.6 Targeted physical exam (per Section 8.1.7) 
8.6.7 Anal swab for local cytology and additional swabs for correlative studies 
8.6.7.1 Leftover material from anal swab cytology suspension (Appendix III) 
8.6.7.2 Anal swab for DNA 
8.6.7.3 Anal swab for RNA/protein 
8.6.8 Digital anorectal examination and inguinal lymph node examination 
8.6.9 High resolution anoscopy 
8.6.10 Anal biopsy depending on the assigned arm. Participants in the treatment arm will 
be biopsied every six months if persistent or metachronous HSIL is suspected, or if 
visible changes can be seen in areas of prior treatment. Areas of prior treatment that 
appear completely normal (e.g., not acetowhite epithelium (AWE), no vascular 
changes, and strongly Lugol's positive) do not need to be biopsied to confirm the 
success of treatment. Participants in the active monitoring arm will have anal 
biopsies of HSIL only every 12 months, per Section 4.1. 
8.6.10.1 HSIL in the treatment arm may be empirically treated at 6-month visits if 
the lesion is biopsied before treatment at the same visit and if cancer is not 
suspected on HRA. 
8.6.10.2 If all biopsies do not show HSIL or are inconclusive, and the local cytology 
result shows high grade disease (ASC-H or HSIL), the investigator should 
bring the participant back for repeat HRA at 3 months (interim visit, 
window of ±4 weeks). If a biopsy is performed at an even-numbered visit 
for a monitoring arm participant and the biopsy results are discordant, the 
participant will not be asked to return for repeat biopsy until the next 6-
month visit unless there is suspicion for progression to cancer. If at least 
one biopsy shows HSIL, repeat biopsies of lesions with inconclusive 
histology may be deferred to the next 6-month visit so long as there is no 
suspicion of progression to cancer. If the visit window for the next 6-month 
visit is within 8 weeks of the last biopsy, repeat HRA and biopsies will 
occur as part of the next 6-month visit. 


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8.6.11 Initiate new treatment cycle as needed or schedule surgery for those being treated 
with TUA 
8.6.11.1 If the participant is to be treated with an ablative therapy, one additional 
biopsy will be collected prior to treatment from the lesion with most 
severe appearance of disease. This biopsy will be placed in RNAlater for 
banking only. A maximum of one biopsy for banking will be obtained 
during each 6-month treatment block, if treatment is administered. 
8.6.12 QoL objective: If the participant consents and is eligible to complete questionnaires 
for the QoL objective, A-HRSI completion at 12, 24, 36, 48, and 60 months after 
randomization (see Section 8.9 and Appendix I). 
8.6.13 COVID-19 telephone follow-up: If the 6-month visit cannot be conducted due to 
COVID-19, a telephone follow-up visit may be conducted in lieu of a 6-month 
visit to obtain follow up on treatment completion (if relevant), safety, medical 
history, medication history, and study arm compliance. See the study source 
documents for instructions. If a telephone follow-up occurs, a makeup-6 month 
visit must be conducted as soon as an in-person visit is feasible again. If a site 
performs a telehealth visit because the 6-month visit cannot be performed, it 
will still require reporting as a missed visit and is still considered a protocol 
deviation. 
8.7 
Final Evaluations, Off Study 
At the completion of the follow-up period for the study, the Off-Study Summary Form 
should be completed in AdvantageEDC. Evaluations are to occur in both study arms unless 
otherwise specified. 
8.7.1 Medication review 
8.7.2 Recording of HIV viral load and CD4 count laboratory results (only if final visit is 
an annual visit and results are available from clinical care) 
8.7.3 Adverse events evaluation 
8.7.4 Serum (red top tube) for the ANCHOR Biorepository for correlative studies 
8.7.5 Targeted physical exam (per Section 8.1.7) 
8.7.6 Anal swab for local cytology and additional swabs for correlative studies 
8.7.6.1 Leftover material from anal swab cytology suspension (Appendix III) 
8.7.6.2 Anal swab for DNA 
8.7.6.3 Anal swab for RNA/protein 
8.7.7 Digital anorectal exam and inguinal node examination 
8.7.8 High resolution anoscopy 
8.7.9 Anal biopsy. Monitoring arm participants will have biopsies of all visible lesions 
and any prior areas of HSIL that appear to have regressed. Treatment arm 
participants will have biopsies of all visible lesions and of normal-appearing areas 
that were previously treated. For participants in both arms, if this totals less than 4 


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biopsies, normal-appearing tissue in remaining quadrants will be biopsied so that a 
minimum of 4 areas are sampled. 
8.7.10 QoL objective: If the participant consents and is eligible to complete questionnaires 
for the QoL objective, A-HRSI completion at 60 months after randomization (if 
applicable given the participant’s visit schedule at study discontinuation; see 
Section 8.9 and Appendix I). 
8.8 
Early Study Discontinuation Evaluations 
All participants who notify the site of the intent to discontinue the study early will be asked 
to consent to be contacted once per year for the remainder of the study for follow-up for 
anal cancer diagnoses only. Instructions for this contact are outlined in the study MOP. 
Medical records will be requested from the participant’s provider if anal cancer is 
diagnosed. If more than 3 months have elapsed since the prior 6-month visit, the participant 
will be asked to complete a final visit before discontinuing participation, as outlined below. 
8.8.1 Adverse events evaluation 
8.8.2 Recording of HIV viral load and CD4 count laboratory results (only if final visit is 
an annual visit and results are available from clinical care) 
8.8.3 Medication review 
8.8.4 Serum (red top tube) for the ANCHOR Biorepository for correlative studies 
8.8.5 Targeted physical exam (per Section 8.1.7) 
8.8.6 Anal swab for local cytology and additional swabs for correlative studies 
8.8.6.1 Leftover material from anal swab cytology suspension (Appendix III) 
8.8.6.2 Anal swab for DNA 
8.8.6.3 Anal swab for RNA/protein 
8.8.7 Digital anorectal exam and inguinal node examination 
8.8.8 High resolution anoscopy 
8.8.9 Anal biopsy. Monitoring arm participants will have biopsies of all visible lesions 
and any prior areas of HSIL that appear to have regressed. Treatment arm 
participants will have biopsies of all visible lesions and of normal-appearing areas 
that were previously treated. For participants in both arms, if this totals less than 4 
biopsies, normal-appearing tissue in remaining quadrants will be biopsied so that a 
minimum of 4 areas are sampled. 
8.8.9.1 Participants diagnosed with cancer may provide an optional cancer biopsy 
(placed in RNALater) if willing to return to the clinic for biopsy before 
referral to treatment and study discontinuation. 
8.8.10 If the participant is discontinuing treatment due to progression to cancer, stage at 
diagnosis must be recorded. 
8.8.11 QoL objective: If the participant consents and is eligible to complete questionnaires 
for the QoL objective, A-HRSI completion at the applicable time point given the 
participant’s visit schedule at study discontinuation (see Section 8.9 and Appendix 


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I). 
8.9 
QoL Objective 
If the participant consents to complete A-HRSI questionnaires for the QoL 
objective, the A-HRSI will be completed via telephone-facilitated interview 
(performed by clinical site staff) or ePRO platform (independently by the 
participant) at the following time points. See Appendix I for the window for 
accepted forms. 
Time Point 
Target Date 
Target Completion 
Window 
T1 
Visit 1, before randomization 
- 14 to 0 days 
T2 
2-7 days after randomization 
2-10 days 
T3 
4 weeks after randomization 
± 1 week 
T4 
12 months 
± 4 weeks 
T5 
24 months 
± 4 weeks 
T6 
36 months 
± 4 weeks 
T7 
48 months 
± 4 weeks 
T8 
60 months 
± 4 weeks 
 
 


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9.0 
BIOMARKER STUDIES 
Any use of biospecimens collected as part of this trial requires specific review and approval 
by NCI CTEP or other mechanisms as will be set up for the NCI Clinical Trials Network 
(NCTN) or by OHAM, and by the ANCHOR Correlative Science Committee. For a request 
for use of specimens to be considered, a protocol for use of banked specimens (or an 
amendment to the protocol if the trial is still active) must be submitted to NCI CTEP; the 
protocol submitted for review must contain a clear statement of the scientific objectives 
and hypotheses, a statistical section that provides a brief description of the statistical design 
and analysis strategy along with sample size/power justification, a description of the assay 
methodology, and identification of the laboratory/individuals that will perform the assays. 
For non-standard assays, information about the assay’s analytical performance (e.g., 
sensitivity, specificity, bias, linearity, reproducibility, as applicable) also may be requested 
by the reviewers. 
This correlative science section contains specific aims, hypotheses, and experimental 
designs to address correlative science questions that could potentially be addressed using 
specimens collected as part of the ANCHOR study. These include studies of viral factors 
in HSIL progression to cancer, identification of host factors in HSIL progression to cancer, 
and viral and host biomarker predictors of risk of progression from HSIL to cancer. The 
purpose of this section is not to describe the exact experiments that will be done, since the 
aims, methods, and choice of reagents will likely change in whole or in part by the time 
sample sets of sufficient size to perform the experiments have been assembled. The 
information provided in this section is therefore meant only to provide examples of 
experiments that could be performed, and to illustrate the thinking behind the approach to 
specimen collection in the ANCHOR protocol. 
9.1 
Viral Factors in HSIL Progression to Cancer 
9.1.1 Study aims 
1) Determine the HPV type in cancer and compare to that of overlying HSIL and 
HSIL biopsies collected concurrently that did not progress to cancer. 
2) Determine the strain variant of HPV 16 in participants who progressed to anal 
cancer and compare to participants with HSIL biopsies who did not progress to 
cancer. 
3) Determine the HPV integration site in overlying anal cancer to that of HSIL 
overlying the cancer and HSIL biopsies collected concurrently that did not 
progress to cancer. 
For Aims 1 and 2, microdissected portions of the tissue will be placed in Eppendorf 
tubes and will be processed for PCR and methylation studies. HPV typing will be 
performed using small amplicon primer sets known to be optimal for studies of 
DNA from formalin-fixed, paraffin-embedded (FFPE) specimens, including Spf-
10 primers (66). Tissues shown to contain HPV will be analyzed further to 
determine the type. Results from those with HPV16 will be confirmed using E6 
sequencing to identify strain variants. Strain variants will be classified as published 
previously and grouped into non-European and European strain variants (67)(68). 


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For Aim 3, HPV integration will be assessed using the Amplification of 
Papillomavirus Oncogene Transcript (APOT) Assay in which HPV-cellular fusion 
transcripts are sought using a 3`RACE protocol. This allows for PCR amplification 
for subsequent sequence analysis (69).  RNA will be isolated from sections of FFPE 
biopsies and treated with DNase treatment for 15 min. The APOT assay will be 
performed as previously described. Total RNA will be reverse-transcribed using an 
oligo(dT) primer coupled to a linker sequence (59-AAG CAG TGG TAT CAA 
CGC AGA GTA CT(30)VN-3’), referred to as CDS-primer (Clontech, Heidelberg, 
Germany). HPV-derived fusion transcripts will be amplified using the APOT assay 
based on a rapid amplification of cDNA ends (RACE) performed in a nested PCR 
format. HPV E7 primers will be used as forward primers. An adapter primer 
complementary to the linker sequence in the CDS primer will be used as first 
reverse primer and the CDS-primer as second nested primer. Cellular fusion 
transcripts will be excised from the gel and extracted using the Zymoclean Gel 
DNA Recovery Kit (Analy-tikJena, Jena, Germany). The isolated products will be 
sequenced and the integration locus will be determined by database alignments 
using National Centre for Biotechnology Information (NCBI) human megaBlast 
tool and the University of California, Santa Cruz (UCSC) genome browser. 
9.1.2 Specific hypotheses 
HPV 16 will be the predominant HPV type in anal cancer even if the participant 
who progressed to cancer has HSIL with multiple HPV types. Single HPV 16 
infection will be found in most anal cancers. HPV 16 has been found to be the 
predominant HPV type in anal cancers overall (18), but little is known about the 
distribution of HPV in anal cancers among HIV-infected men and women. Recent 
data suggest that HPV 16 may play a less prominent role in cervical cancers of 
HIV-infected women compared with HIV-uninfected women (51)(63). It will be 
important to know whether this is also true of anal cancers, and whether other HPV 
types may be playing an important role. 
1) Non-European variants will be associated with cancers and will be found in 
HSILs that progressed to cancer more often than in HSILs that did not progress 
to cancer after matching for age, race/ethnic background, gender, smoking 
history, and nadir CD4 level. Non-European variants have been associated with 
increased risk of developing HSIL compared with European variants 
(67)(68)(70), but little is known about their role in progression from HSIL to 
cancer. 
2) Different concurrent areas of HSIL represent outgrowth of biologically distinct 
foci of HPV infection and this will be manifest in varying patterns of HPV 
integration. HPV integration sites will differ between HSILs that progressed to 
cancer and concurrent HSILs that did not progress to cancer. In the HSIL that 
progressed to cancer the integration sites will be same between the invasive 
cancer and the overlying HSIL, and reflects a site of integration that is 
pathogenetically important in progression to cancer. Although several studies 
show that HPV integration sites are not consistent in different cancers, recent 
studies show some hot spots, including integration at sites that may be 
pathogenetically important (69). Little is known about HPV integration sites in 


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either anal HSIL or anal cancer, and it will be important to determine if and 
how HPV integration is playing a role in anal cancer pathogenesis. 
9.1.3 Statistical design 
The HPV type(s) in invasive cancer portion of the tissue, overlying HSIL and from 
biopsies of concurrent HSIL that did not progress will be described in a descriptive 
analysis. HPV 16 strain variants will be classified as European and non-European. 
The strain variant of HPV 16 in HSIL from participants who did not progress to 
cancer will be compared with the HPV 16 from those who progressed to cancer. 
The results will be controlled for race and ethnic background since non-European 
strain variants are found more frequently in non-White individuals. 
We expect that non-European variants will be found in 75% of HSIL that 
progressed to anal cancers and 25% of those that did not. If we have 18 cancer 
cases, 36 well-matched controls will be sufficient to detect a difference in the 
proportion of non-European variants of 75% in the anal cancer cases vs. 25% in the 
controls, at the two-sided 0.05 significance level with power of 0.89, assuming a 
correlation of 0.25 between matched cases and controls. Similar samples sizes will 
suffice for comparing sets of HSIL biopsies as described above. 
Descriptive statistics will be used to describe the integration locus of HPV in the 
invasive cancers and whether they differ from those of the overlying HSIL. 
Descriptive statistics will also be used to determine if the loci differ in HSIL that 
progressed and concurrent HSIL biopsies that did not progress. In each case we will 
only analyze tissues that contain HPV 16. 
9.2 
Identification of Host Factors in HSIL Progression to Cancer 
9.2.1 Study aims 
We will have access to tissues that contain early invasive anal cancers, with the 
expectation that most will also contain overlying HSIL. This will allow for 
comparison of molecular characteristics of HSIL with overlying invasive disease. 
It will also allow for comparisons between HSIL that progressed to cancer and 
HSIL that did not progress within the same individual, since many participants will 
have multiple foci of HSIL. In so doing we will elucidate some of the steps involved 
in progression from HSIL to cancer, potentially opening up new avenues for 
treatment of HSIL and cancer, and identifying biomarkers that predict progression 
to cancer. Comparing cancers to overlying HSIL offers different information from 
comparing HSIL that progressed with HSIL that did not progress. Comparing 
cancers and overlying HSIL may identify some of the last steps in progression to 
cancer. Similar comparisons between HSIL that progressed and HSIL that did not 
progress and between HSIL that progressed and the same lesion at earlier time 
points will provide insight into some of the penultimate steps in cancer progression. 
The goal of performing gene expression analysis is identify genes involved in 
progression from HSIL to cancer and whose expression may serve as biomarkers 
of progression to cancer. The goal of performing whole exome sequencing is to 
identify genetic changes involved in progression from HSIL to cancer and whose 
expression may serve as biomarkers of progression to cancer. 


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1) Perform gene expression array analysis comparing expression in anal cancer 
with HSIL overlying the cancer. Perform gene expression array analysis 
comparing expression in HSIL biopsies that progressed to cancer with non-
progressing HSIL biopsies at other locations. Perform similar analyses 
comparing expression in HSIL biopsies that progressed to cancer with the same 
lesion at earlier time points prior to progression. 
2) Characterize genetic changes in anal cancers compared with HSIL overlying 
the cancer. Characterize genetic changes in HSIL biopsies that progressed to 
cancer compared with non-progressing HSIL biopsies at other locations. 
Characterize genetic changes HSIL biopsies that progressed to cancer with the 
same lesion at earlier time points prior to progression. 
For Aim 1, RNA will be extracted from sections of microdissected anal cancer and 
HSIL using the Ambion RecoverAll Kit following the RNA protocol. This kit is 
designed for FFPE tissue and removes the secondary structure caused by the 
formalin. RNA will be amplified, fragmented, and labeled using NuGEN 
Technologies WT- Ovation Kits according to the manufacturer’s instructions 
(NuGEN Technologies, San Carlos, CA). Labeled samples will then hybridized to 
Affymetrix Human Gene 1.0 ST GeneChips (Affymetrix, Santa Clara, CA). The 
GeneChips will be washed and stained on Affymetrix 450 Fluidics Stations and 
scanned on an Affymetrix 3000 Scanner according to standard protocols. 
For Aim 2, the projected number of cases of incident invasive anal cancer (n=18) 
makes most genome-wide association approaches untenable. However, the sample 
size of non-progressors for the ANCHOR study in the active monitoring arm is 
considerable (n= approximately 2000). Consequently, the depth of phenotypic 
characterization and the availability of optimally-matched controls (matched on 
age, race/ethnic background, gender, smoking history, and nadir CD4 level) can 
permit the discovery of rare mutations enriched in incident cancer cases compared 
with matched controls using next-generation sequencing. Accordingly, we will 
perform whole exome sequencing of all incident cancer cases available for analysis. 
We will perform several comparisons: 1) compare the genomes of the cancers to 
the overlying HSIL, 2) the cancers to normal tissue from the same individuals will 
serve as controls to identify sequencing artifacts, 3) HSIL from the time of 
progression to cancer to concurrent HSIL at other locations in the same individual 
that did not progress to cancer, 4) HSIL from the time of progression to the same 
lesion at different time points prior to progression; and 5) compare HSIL DNA from 
participants who progressed to cancer with a HSIL from well-matched participants 
who did not. An excellent review of paired tissue analysis was published by 
Meyerson et al. and we will use the approaches described in that publication (71). 


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9.2.2 Specific hypotheses 
1) Tumor promoter and suppressor genes that play a role in some of the last steps 
in progression from HSIL to cancer will differ between cancer and overlying 
HSIL. 
2) Tumor promoter and suppressor genes that play a role in some of the 
penultimate steps in progression from HSIL to cancer will differ between HSIL 
that progressed and concurrent HSIL that did not progress. 
3) Tumor suppressor genes important in some of the last steps in progression from 
HSIL to cancer will have mutations in invasive cancers compared with 
overlying HSIL, with mutations predicted to cause loss of function. Tumor 
promoter genes may also be mutated. 
4) Tumor suppressor genes important in some of the penultimate steps in 
progression from HSIL to cancer will have mutations in HSIL that progressed, 
compared with HSIL that did not progress. Tumor suppressor genes important 
in some of the penultimate steps in progression from HSIL to cancer will have 
mutations in HSIL that progressed, compared with HSIL at the same location 
at time points prior to progression. Tumor promoter genes may also be mutated 
in these tissues. 
Many studies have characterized chromosomal changes and genes that are over or 
under-expressed in cervical HSIL compared with normal tissues, or between 
cervical cancer and normal tissues or cervical HSIL; a few have looked at anal 
cancer and HSIL (72)(73)(74)(75)(76)(77)(78). These studies have also shown that 
cervical swab material can be used to identify markers of increasing grade of 
abnormality (79). In AMC 046 we performed microarray analysis of perianal HSIL 
tissues compared with adjacent normal perianal tissues (Chein A and Palefsky J, 
manuscript in preparation). We found that several members of the immune response 
gene family and cell cycle genes were up-regulated in the HSIL tissues compared 
with normal. There are few studies of changes in gene expression in anal cancer, 
and few at any other anatomic site at which HPV causes cancer that examine 
changes in gene expression as HSIL progresses to cancer. 
Characterization of the genetic risk factors for progression from HSIL to invasive 
cancer is also of great importance. Tumor samples from individuals with invasive 
anal cancer are not well represented in the NCI Cancer Genome Atlas project. An 
understanding of the genomes of incident cancers may provide additional insight 
into the genomic alterations that provide these invasive tumors a proliferative 
advantage. In the ANCHOR study, it is most likely that any cancers will be 
diagnosed in an office-based biopsy on a FFPE tissue, and we will therefore 
perform genetic studies that can be done on FFPE (80). 
9.2.3 Statistical design 
Microarrays from all available (up to 18 anticipated) pairs of invasive cancer tissues 
and overlying HSIL will be compared and analyzed to compare gene expression in 
cancer to that of overlying HSIL. Likewise array data from pairs of 18 HSIL tissues 
that progressed to cancer and matched HSIL that did not progress from the same 


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participant or which were obtained at time points prior to progression. Microarrays 
from HSIL that progressed will also be compared with those from HSIL from well-
matched control participants (matched on for age, race/ethnic background, gender, 
smoking history, and nadir CD4 level) who did not progress. Microarrays will be 
normalized for array-specific effects using Affymetrix’s “Robust Multi-Array” 
(RMA) normalization. Normalized array values will be reported on a log2 scale. 
Average normalized expression is typically around 7.0. For statistical analyses, all 
array probe sets where no experimental groups have average log2 intensity greater 
than 3.0 will be removed. It is a standard cutoff below which expression is 
indistinguishable from background noise. 
Linear models will be fitted for each gene using the Bioconductor “limma” package 
in R (81)(82). Moderated t-statistics, fold-change and the associated p values will 
be calculated for each gene. To account for the fact that thousands of genes will be 
tested, false discovery rate (FDR)-adjusted values will be calculated using the 
Benjamini-Hochberg method (83). FDR values indicate the expected fraction of 
falsely-declared differentially-expressed (DE) genes among the total set of declared 
DE genes, i.e. FDR=0.15 would indicate that 15% of the declared DE genes were 
expected to be false due to experimental noise instead of actual differential 
expression. The target FDR will be a maximum of 0.05 or less. 
To analyze the results, the GO-Elite software tool designed by the J. David 
Gladstone Institutes will be used (http://genmapp.org/go_elite/). This tool was 
designed to identify a minimal non-redundant set of Gene Ontology (GO) 
biological terms or pathways to describe a particular set of genes 
(https://code.google.com/p/go-elite/wiki/AboutUs#CitingGO-Elite). A z-score and 
permuted p value will be calculated to assess over-representation of GO terms and 
pathways in the gene set. The permuted p value represents the chance of randomly 
selecting the same number of regulated probe sets from all probe sets examined and 
recalculating z-scores for all terms 2000 times. 
We will perform whole exome sequencing of DNA for Aim 2 using DNA from the 
matched tissue sets as described. Whenever possible, we will perform these studies 
with approximately 5 controls per 1 case. A ratio of 5:1 was selected to maximize 
the precision of the estimates of association. 
9.3 
Viral and Host Biomarker Predictors of Risk of Progression from HSIL to Cancer 
9.3.1 Study aims 
1) Study different measures of HPV activity as biomarkers of progression from 
HSIL to invasive cancer (HPV DNA methylation, E6 RNA and E6 protein 
quantity, serum E6 antibodies, circulating HPV DNA). 
2) Study host genetic and epigenetic changes as biomarkers of progression from 
HSIL to invasive cancer. 
3) Study host immune and inflammatory response markers (serum inflammatory 
cytokines and IL-10, CXCL 12, HLA-G and CCR-2 polymorphisms) as 
biomarkers of progression from HSIL to invasive cancer. 


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4) Study cell proliferation proteins as biomarkers of progression from HSIL to 
invasive cancer (phosphatase and tensin (PTEN), survivin, U3 small nucleolar 
ribonucleoprotein protein IMP3, epithelial mesenchymal transition proteins). 
For Aim 1, HPV DNA methylation studies will be performed on anal swabs as 
described previously for cervical swabs. Briefly, the extent of CpG methylation will 
be determined by pyrosequencing (Qiagen, Valencia, CA) according to the 
manufacturers’ recommendations. DNA documented to contain HPV 16 DNA will 
be treated with bisulfite using the EZ DNA methylation kit (Zymo Research, 
Orange, CA) according to the suggested protocol. All segments will be amplified 
by Platinum Taq high fidelity DNA polymerase (Invitrogen, Carlsbad, CA) with a 
primer containing biotin, captured with streptavidin-coated beads, and annealed to 
a sequencing primer (available from RDB). Sequence extension will be performed 
with PyroMark Gold Q96 Reagents (Qiagen, Valencia, CA) that uses DNA 
polymerase with sequential addition of each dNTP; the released pyrophosphate will 
be catalyzed by ATP sulfurylase, luciferase, and apyrase sequentially to generate 
light signals, which are detected by a charge-coupled device (CCD) camera on a 
PSQ96 system (Qiagen, Valencia, CA). Because the bioluminometric response is 
linear for the sequential addition of dNTPs, the signal intensity reflects the original 
methylation ratio producing continuous values between 0 and 1. We will examine 
methylation in HPV DNA from anal swabs in individuals who progressed to cancer 
in comparison with swab DNA from matched controls (matched on for age, 
race/ethnic background, gender, smoking history and nadir CD4 level) who did not 
progress. Methylation will also be compared in swabs in a given individual as they 
approach the diagnosis of cancer. 
E6 RNA levels will be measured using the commercially available Aptima test. 
Although this test is currently configured for clinical diagnostic purposes to be 
reported as “positive or negative,” the quantity of E6 RNA can also be measured in 
relative light units (RLU). We have already shown that the test can successfully be 
performed on anal swab material (J. Palefsky, personal communication). 
Participants in the active monitoring arm who develop anal cancer will be compared 
with matched participants who did not develop cancer. Anal swab specimens at 
baseline and at least annually up to the time of development of cancer will be 
analyzed, classified as positive or negative for HPV 16, 18 or 45, and the RLU as 
measure of RNA quantity will also be analyzed. The quantity of E6 RNA from anal 
swabs in individuals who progressed to cancer will be compared with matched 
controls who did not progress. E6 RNA levels will also be compared in swabs in a 
given individual as they approach the diagnosis of cancer. 
Similar analyses will be done for HPV 16, 18, and 45 E6 protein analysis, using the 
Arbor-Vita E6 protein detection assay. We have already shown that the test can 
successfully be performed on anal swab material in pilot studies (J. Palefsky, 
personal communication). Samples are described as positive or negative for E6, and 
quantitation is performed using a densitometer. 
Measurement of serum E6 antibodies will be performed as described previously. 
Results will be classified as positive or negative and titers will be measured and 
analyzed. Participants in the active monitoring arm who develop anal cancer will 


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be compared with matched participants who did not develop cancer. Antibody 
positivity and titer levels will be compared between the groups as will changes over 
time in a given individual as they approach the diagnosis of cancer. 
Measurement of circulating (serum) HPV 16 DNA will be performed as described 
previously. Results will be described as positive or negative and changes over time 
in a given individual will be described as they approach the diagnosis of cancer. 
Levels will be compared among those who progressed to those who did not. 
For Aim 2, approaches similar to those described for analysis of HPV DNA 
methylation in anal swab specimens will be used to study select cellular genes for 
methylation. We will study methylation of nine hypermethylated differentially 
methylated regions (DMRs) shown to be important in cervical HSIL, and zinc 
finger protein 582. Frequency and level of methylation of these genes in anal swab 
specimens from individuals who progressed to cancer will be compared with those 
who did not progress to cancer. Among those who developed cancer we will also 
study changes in anal swab specimens over time leading up to the diagnosis of anal 
cancer. 
For Aim 3, standard ELISAs will be performed for host inflammatory response 
proteins (TNF-alpha, interferon), IL-10, and others. Results will be classified as 
positive or negative and titers will be measured and analyzed. Participants in the 
active monitoring arm who develop anal cancer will be compared with matched 
participants who did not develop cancer. Antibody positivity and titer levels will be 
compared between the groups as will changes over time in a given individual as 
they approach the diagnosis of cancer. 
HLA-G2 and CCR-2 polymorphisms will similarly be studied in progressors and a 
set of matched non-progressors. DNA from normal tissue as well as lesional tissues 
will be available for analysis. 
For Aim 4, standard immunohistochemical protocols will be used to examine HSIL 
tissues that progressed to cancer for proteins involved in stimulation or repression 
of cell proliferation, including PTEN, survivin and U3 small nucleolar 
ribonucleoprotein protein IMP3. To perform immunohistochemistry, antibodies to 
each of the proteins are subjected to a standardized test procedure to develop an 
individual and optimized immunostaining protocol for each antibody (124). 
Initially one dilution, based on antibody stock concentration of the primary 
antibody, is tested. The outcome of this test staining is used to guide further 
dilutions and optimization of the protocol. 
TMA slides are deparaffinized and hydrated in xylene and graded ethanol to 
distilled water prior to immunostaining. A blocking step to quench endogenous 
peroxidase is performed in 0.3 % H2O2 in 95% ethanol for 5 minutes. Heat-induced 
epitope retrieval (HIER) is performed in a retrieval buffer pH 6, using a pressure 
boiler as a heat source for 4 minutes at 125°C. After boiling is completed, slides 
remain in the pressure boiler and are allowed to cool to 90°C. The total processing 
time for HIER is approximately 45 minutes. If no conclusive result is obtained in 
the initial test, further tests are done based on the observed staining pattern. 
Immunohistochemistry (IHC) conditions may be modified, including changing 


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antibody incubation times, antibody dilutions and diaminobenzidine (DAB) 
incubation times. 
For staining all incubations are done at room temperature. Slides are rinsed in wash 
buffer, incubated with Ultra V block for 5 minutes, rinsed again in wash buffer, and 
incubated with primary antibody for 30 minutes. After rinsing twice in wash buffer 
the slides are incubated with labeled horseradish peroxidase-polymer for 30 
minutes. After rinsing twice in wash buffer the slides are developed in DAB 
solution for 10 minutes, rinsed and counterstained in Mayers hematoxylin for 5 
minutes. The slides are rinsed in lithium carbonate water, diluted 1:5 from saturated 
solution for 1 minute, rinsed in tap water for 5 minutes, dehydrated in graded 
ethanol, and cover-slipped. 
Results will be scored as positive or negative, and graded according to intensity 
(weak, moderate, strong) and location of staining. Tissue microarrays will be used 
for this purpose since this will allow for analysis with a small amount of the biopsy 
specimen. Signal in invasive cancers will be compared with overlying HSIL. 
Signals in HSIL that progressed will be compared to HSIL that did not progress, as 
well as arrays of tissues from earlier time points prior to progression. Tissues will 
also be stained for proteins associated with epithelial-mesenchymal transition 
(EMT), including e-cadherin, ZO-1, cyclin-D1 and beta-catenin. 
9.3.2 Specific hypotheses 
Biomarkers of prevalent or incident cervical HSIL will have value as biomarkers 
of risk of progression from HSIL to cancer: 
1) Higher levels of HPV DNA methylation, HPV E6 RNA and HPV E6 protein 
levels will be associated with progression from HSIL to cancer. 
2) Hypermethylation of cellular genes shown to be associated with risk of cervical 
HSIL (nine selected markers) will be associated with progression from HSIL to 
cancer. 
3) Higher levels of inflammatory response proteins or polymorphisms associated 
with higher levels of these proteins, and polymorphisms that lead to reduced 
immune response to HPV will be associated with progression from HSIL to 
cancer. Detection of any level of HPV DNA in serum will be associated with 
progression to invasive cancer. 
4) Higher levels of proteins associated with increased cellular proliferation and 
vice versa, will be associated with progression from HSIL to cancer, as will 
expression of proteins associated with EMT. 
Progression from HPV infection to HSIL and then to cancer is a multistep process 
involving many different pathways. Viral gene expression plays a key role, as do 
the host immune response, cell cycle pathways, and many others. Given the high 
proportion of individuals who become infected with HPV but the relatively small 
proportion who develop cancer, host genetic susceptibility and epigenetic 
regulation of host gene expression play an important role. Many studies of 
biomarkers for cervical cancer risk have been performed examining each of these 
areas (84). Relatively few if any have examined biomarkers for anal HSIL, and 


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more importantly, few have examined biomarkers of progression from HSIL at any 
mucosal to site to invasive cancer. In this correlative study, we will examine a group 
of biomarkers shown to be of interest to predict incident cervical HSIL, and 
determine whether, using similar or different cutoff levels, they may also be useful 
to predict progression from anal HSIL to cancer. To do we will use different kinds 
of samples that will be available through the ANCHOR study. Anal swab material 
and serum are particularly suitable for screening in the community should these be 
shown to be of value in the proposed studies. In general markers that involve 
immunohistochemical analysis of tissue are not as suitable for screening, but 
proteins shown to be interest can then be tested in swab material in future studies. 
Biomarkers determined to be of interest in the ANCHOR study can also then be 
validated for use in the cervix and oropharynx. 
We have framed this study according to the overall nature of the pathways involved. 
Clearly there is some degree of overlap. For example, epigenetic modifications of 
cellular may alter the level of expression of one or more of the genes being studied 
in the other specific aims. Likewise, polymorphisms in host genes may alter the 
function of the proteins or their level of expression. 
We are specifically proposing studies of HPV DNA methylation since it has been 
shown to be important in epigenetic regulation of HPV gene expression, and is a 
marker of progression to cervical HSIL (85)(86)(87)(88). Serum E6 antibodies 
have been correlated with HPV-associated oropharyngeal cancer, but their role as 
markers for prevalent or incident anal cancer is not known (89)(90)(91). HPV only 
infects epithelial cells, and detection of HPV DNA in serum may reflect circulating, 
potentially metastasizing HPV-infected tumor cells. Circulating HPV DNA has 
been detected in patients with oral cancers and some cervical cancers, but not 
patients without cervical cancer (92)(93). Detection of serum HPV DNA may also 
reflect tumor burden, and it is therefore possible that serum HPV DNA tests in 
patients with very early anal cancer will be negative. Nonetheless it is an issue 
worth exploring since serum testing could be a convenient test to predict prevalent 
anal cancer, particularly in patients with very widespread anal HSIL in whom it 
might be difficult to exclude a cancer. E6 RNA has been shown to be nearly as 
sensitive as HPV DNA tests with increased specificity for cervical HSIL (94)(95). 
Similar results have been found in ASIL (96). E6 protein-based tests offer 
additional potential predictive value and are in early stages of development (97). 
We have pilot tested E6 protein testing in anal samples in our clinic and have shown 
that the test can work in anal samples (J. Palefsky, personal communication). Like 
E6 RNA, E6 protein testing data can be quantified as we will explore the value of 
doing so. 
Methylation of cellular genes may be important biomarkers of prevalent cancers or 
potentially, progression from HSIL to cancer (98). Although there are limited data 
on host gene methylation in ASIL (99) its role as a diagnostic test for anal disease 
and its predictive value for progression from HSIL to cancer are not yet known. We 
have identified several genes of interest in cervical disease that will be of value to 
study in the setting of anal disease. These include but are not limited to methylation 
of genes regulating miRNA expression including hsa-miR-203 and -375 (100), 


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differentially methylated regions in cervical cancer (101), zinc finger protein 582 
(102), genes involved in one-carbon metabolism (103) and CADM1 (104). 
The host immune and inflammatory responses likely play a critical role in 
pathogenesis of HPV-related cancers. We will explore host inflammatory markers 
and relevant gene polymorphisms that have been shown to be important in cervical 
HSIL or cancer. These include, but are not limited to serum IL-10 levels, and serum 
IL-6, tumor necrosis factor-alpha, macrophage inflammatory protein-1 alpha, 
granulocyte macrophage colony-stimulating factor, IL-1 beta, and IL-1 alpha (105). 
Immunohistochemical staining for CD32B+ lymphocytes, GATA3+ and T-bet+ 
lymphoid cells (106), CXCL-12 (107), FoxP3+ lymphocytes (106)(108)(109) have 
all been shown to correlate with cervical HSIL or cancer as have HLA-G (108) and 
CCR-2 polymorphisms (110). 
Finally, markers of cellular proliferation may represent important biomarkers of 
progression to cancer. As with the other areas discussed above, little is known about 
their expression in anal HSIL or progression from anal HSIL to cancer. Here again 
we looked to the cervical literature to identify markers shown to be associated with 
cervical HSIL or cancer that may be of value for anal disease. These include 
phosphatase and tensin (PTEN) (111), survivin (111), U3 small nucleolar 
ribonucleoprotein protein Imp3 (112), p16Ink4a (108)(113)(114), MCM 3 and 5, 
CDC6, Geminin, cyclins A-D, TOPO2A, CDCA1, and BIRC5 (116), and protein 
markers of epithelial mesenchymal transition proteins ZO-1, e-cadherin and beta-
catenin (114). 
9.3.3 Statistical design 
DNA methylation will be studied in anal swab specimens at three CpG sites in each 
of the L1, L2, and E2/E4 genomic regions that were previously shown to be 
associated with an increased risk of CIN3. Percent methylation will be recorded 
and the distribution of percent methylation will be compared between those who 
develop cancer and matched controls who do not. Similarly, the frequency and level 
of methylation of 9 selected DMR markers in anal swab specimens from individuals 
who progressed to cancer will be compared with those who did not progress to 
cancer. 
Patients who progress to cancer and matched participants who do not develop 
cancer will be compared with respect to anal swab E6 RNA and E6 protein, and 
serum E6 antibodies. Each will be classified as positive or negative, and quantitated 
with the levels used for data analysis. A sample of all available incident anal cancer 
case patients will be obtained (sample of 18 estimated). For each case patient, a 
matching sample of 2 control patient(s) will also be obtained. If a minimum sample 
of 54 patients is obtained, this will achieve 89% power to detect an odds ratio of 
9.00 versus the alternative of equal odds using a Chi-Square test with a 0.05 
significance level, assuming a correlation of 0.25 between matched cases and 
controls. 
Changes in the levels of methylation, E6 RNA, protein and antibodies will be 
compared between the groups as will changes over time in a given individual as 
they approach the diagnosis of cancer using general estimating equations. The 


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relationship between E6 RNA, protein, and antibody levels will also be explored in 
this way. Serum HPV DNA detection results will be classified as positive or 
negative and their association with the development of cancer will be assessed 
using a proportional hazards model. 
Levels of serum inflammatory response proteins will be measured and analyzed 
using the study design described above. Polymorphisms in HLA-G 2 and CCR-2 
will be described as present or absent. 
Immunohistochemical studies will compare the detection of protein expression 
(positive or negative) between available anal cancer tissues (up to 18 tissues 
estimated) with invasive anal cancer and overlying HSIL. We will also compare 
HSILs that progressed to cancer with HSILs that did not and with tissues from pre-
progression time points. Similar comparisons will include location of staining 
(superficial, basal, diffuse) and weak, moderate or strong) among these tissues. 
9.4 
Medical History and Behavioral Risk Factors for Progression from HSIL to Cancer 
among Those Who Are Enrolled 
9.4.1 Study aim 
1) Identify medical history and behavioral risk factors for HSIL progression to 
cancer 
A brief questionnaire containing questions on medical history and behavioral 
factors that may be risk factors for progression from HSIL to cancer will be 
administered to all participants who are being enrolled. The questions were 
selected from among those used in prior studies of anal LSIL and HSIL and 
shown to be of interest in univariate or multivariate analysis of prevalent or 
incident disease. The questionnaire is shown in Appendix V. Data from this 
questionnaire will be used in a case-control analysis of potential risk factors for 
progression to cancer among those who were enrolled into the study. 
9.4.2 Specific hypotheses 
Smoking and chronic irritation induced by sexual activity and chronic hemorrhoids 
will be risk factors along with anal HPV infection for progression of anal HSIL to 
anal cancer. 
9.4.3 Statistical design 
For each risk factor of interest, Fisher’s exact test or Pearson’s chi-square test will 
be used to determine if there is an association between the risk factor and 
development of invasive anal cancer. Factors associated with invasive anal cancer 
at the 0.10 significance level will be incorporated into a logistic regression model 
to determine if they are independently associated with invasive anal cancer. Cox 
regression analyses will also be used to evaluate the association between risk 
factors and time to diagnosis of invasive anal cancer. 
To further elucidate the risk factors associated with invasive anal cancer, a matched 
control analysis will be done. For each invasive anal cancer case, multiple controls 
(e.g., 2 or 3) matched by age, gender, and race/ethnicity will be randomly selected. 
Using a stratified logistic regression model where each stratum is comprised of a 


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case and its matching controls, risk factors will be assessed to determine whether 
they are associated with the diagnosis of invasive anal cancer. 
 
 


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10.0 
STATISTICAL CONSIDERATIONS 
10.1 
Study Design/Endpoints 
10.1.1 Primary objective 
To determine whether treating anal high-grade intraepithelial neoplasia (HSIL) is 
effective in reducing the incidence of anal cancer in HIV-infected men and women. 
10.1.2 Secondary objectives 
• To determine the safety of infrared coagulation, electrocautery, imiquimod, 
laser, and 5- fluorouracil treatments for anal HSIL. 
• To assess the responsiveness (sensitivity to change) and clinical significance of 
the A-HRSI subscales by comparing change scores within groups of 
participants as defined by participant responses to the PGIC item. 
10.1.3 Exploratory objectives 
Collect clinical specimens and data to create a bank of well-annotated specimens 
that will enable correlative science: a) Identification of viral factors in HSIL 
progression to cancer; b) identification of host factors in HSIL progression to 
cancer; c) identification of biomarkers of HSIL progression to cancer and d) 
identify medical history and behavioral risk factors for HSIL progression to cancer. 
Ancillary study aims are outlined in Section 2.6. 
10.1.4 Quality of Life Objectives 
• Primary QOL Objective: To compare arms in terms of changes in physical 
symptoms and impacts from T2 to T3, adjusting for T1.   
• Secondary QOL Objective: To compare arms in terms of changes in 
psychological symptoms from T3 to T4, adjusting for T1.  
• Exploratory QOL Objective: To assess of long-term HR-QoL changes in 
physical symptoms/impacts and psychological symptoms from T4 through the 
subsequent T5-T8 follow-ups overall and by arm. 
10.2 
Sample Size/Accrual Rate 
For this study we are assuming an incidence rate of anal cancer of 100/100,000 among all 
HIV-infected men and women, which by definition includes those with and without 
prevalent anal SIL. We are assuming that the obligate anal cancer precursor is HSIL, and 
that all cases of cancer develop from HSIL. If half of the population develops HSIL then 
the incidence of cancer among those with HSIL would be expected to be 200/100,000. Of 
note, in their meta-analysis, Machalek et al estimated that 1/377 HIV-infected MSM 
progress from anal HSIL to anal cancer each year (16). This is equivalent to 265/100,000 
per year. All participants must have HSIL to be enrolled in the ANCHOR study and thus 
we estimate that an incidence of 200/100,000 among study participants in the active 
monitoring arm is conservative to perform an intent-to-treat (ITT) analysis of our primary 
objective. The ITT analysis will include all randomized study participants, including those 
who fail treatment of anal HSIL in the treatment arm, and those who develop new, 
metachronous lesions, which may or may not have been fully treated at the end of study 


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follow-up. 
Sample size estimates were based on using a log-rank test to compare the treatment and 
active monitoring arms under the following assumptions: three-year accrual period, five 
years of follow-up, 5% annual drop-out rate for both arms, and 7% annual drop-in rate for 
the active monitoring arm (116)(117). The 7% annual drop-in rate is the rate at which 
participants in the active monitoring arm opt to have treatment for HSIL. The 5% annual 
dropout rate includes individuals who are lost to follow-up or who die over the course of 
the study. For both drop-ins and drop-outs, observation time will be censored at the time 
of drop-in or drop-out. The annual incidence rates of anal cancer were assumed to be 
constant over time. To detect a difference between an annual incidence of anal cancer of 
0.2% (200/100,000) in the active monitoring arm and 0.05% in the treatment arm at the 
two-sided 0.05 significance level with power of 0.90 will require 2529 study participants 
per arm for a total of 5058 study participants. Under these assumptions, the expected 
number of events is 7.0 and 23.7 in the treatment and active monitoring arms, respectively. 
The power that we would attain under a range of assumptions is shown in the table that 
follows. Assuming that we enroll 2529 in each arm (5058 total), power was estimated for 
three levels of incidence rates for the active monitoring arm, and 3 levels of drop-in rate 
from the control to the treatment arm under the following assumptions: 
• Constant hazard rates (incidence rates) over time for both groups 
• Two-sided 0.05 significance level 
• Sample size of 5058 evenly divided between treatment and active monitoring arms 
• 3-year accrual period and minimum follow-up period of 5 years 
• Annual drop-out rate of 5% for both observation and treatment arms 
• No drop-ins from treatment to active monitoring arm. 
• Drop-in rate of 7% from observation to treatment 
• Alternative hypothesis is a 75% reduction in the incidence of anal cancer from the 
active monitoring arm 
Table 10-A: Power associated with varying drop-out rates and drop-in rates from 
the active monitoring arm with fixed sample size (N=5058), two-sided significance 
level of 0.05, incidence rate for the active monitoring arm (200/100,000) and the 
treatment arm (50/100,000) 
Drop-out 
rate 
Drop-in rate from the active monitoring arm to the treatment arm 
7% 
10% 
15% 
5% 
90.0 
84.9 
74.6 
10% 
85.0 
79.4 
69.2 
15% 
79.2 
73.5 
63.7 
20% 
72.8 
67.2 
58.3 
 
Please note that the exact incidence of progression to cancer among HIV-infected 
individuals is not known. We have estimated 200/100,000 as described above (data shown 
in bold in the table), but the estimate of Machalek et al yields an estimate of 265/100,000. 


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We believe that the recruitment goal is a bit larger than it needs to be but should serve to 
ensure that we will have sufficient power for the primary study objective. 
We will monitor the incidence of anal cancer in both arms on a regular basis, and can either 
discontinue the trial early if the numbers warrant it as determined by the DSMB, or extend 
follow-up beyond 5 years if cancer is developing at a rate lower than projected. 
Over the course of the recruitment period, we estimate that we will need to screen 11065 
men to diagnose 4426 (40%) with anal HSIL. We estimate that we will need to screen 6320 
women to find 632 (10%) with anal HSIL. The proportion of HIV-infected men and women 
expected to have HSIL was based on multiple studies of HSIL prevalence demonstrated on 
biopsy, as determined using HRA. An assumption of 40% prevalence of HSIL among HIV-
infected men is conservative based on the literature (118)(119)(120)(121) as is the estimate 
of 10% prevalence among HIV-infected women (122). Although the study of HSIL in 
women showed 9% with anal HSIL, that estimate only included women who were referred 
for HRA because of abnormal cytology. Women in that study with normal cytology were 
not referred for HRA. Since it is well known that anal cytology underestimates the true 
grade of disease, and some high-risk individuals with normal anal cytology may have anal 
HSIL (121)(123), the 9% figure represents a low-end estimate of the prevalence of HSIL 
among HIV-infected women. 
Table 10-B: Sample size = 5058, two-sided significance level = 0.05, 3 year accrual, 5 
year follow-up 
Power 
(%) 
Events 
Active 
Monitoring 
Arm 
Events 
Treatment 
Arm 
Anal cancer incidence per 
100,000 
Dropout 
rate 
(%) 
Drop-in rate from 
the active 
monitoring arm to 
the treatment arm 
Active 
monitoring 
Arm 
Treatment 
Arm 
78.8 
17.8 
5.2 
150 
37.5 
5 
7 
72.1 
15.3 
4.4 
150 
37.5 
10 
7 
72.1 
16.7 
5.2 
150 
37.5 
5 
10 
65.6 
14.4 
4.4 
150 
37.5 
10 
10 
 
 
 
 
 
 
 
90.0 
23.7 
7.0 
200 
50 
5 
7 
85.0 
20.4 
5.9 
200 
50 
10 
7 
84.9 
22.2 
7.0 
200 
50 
5 
10 
79.4 
19.2 
5.9 
200 
50 
10 
10 
 
 
 
 
 
 
 
95.6 
29.6 
8.7 
250 
62.5 
5 
7 
92.4 
25.4 
7.4 
250 
62.5 
10 
7 
92.3 
27.8 
8.7 
250 
62.5 
5 
10 
88.3 
23.9 
7.4 
250 
62.5 
10 
10 
 
The sample size of 5058 evenly divided between treatment and active monitoring arms was 
based on detecting a difference in the incidence of anal cancer of 200/100,000 in the active 
monitoring arm and 50/100,000 in the treatment arm at the two-sided 0.05 significance 
level assuming a 5% annual dropout rate across both arms, and a drop-in rate from the 
active monitoring arm to the treatment arm of 7%. The following table shows the power 
estimates for changing levels of dropout rate (5%, 10%, 15% and 20%) and changing levels 
of drop-in rate from the active monitoring to the treatment arm (7%, 10%, 15%). Study 
participants randomized to the active monitoring arm who drop-in to the treatment will be 


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followed, if feasible, to determine if they develop anal cancer. 
Table 10-C: Power associated with varying drop-out rates and drop-in rates from 
the active monitoring arm with fixed sample size (N=5058), two-sided significance 
level of 0.05, incidence rate for the active monitoring arm (200/100,000) and the 
treatment arm (50/100,000) 
Drop-out rate 
Drop-in rate from the active monitoring arm to the treatment arm 
7% 
10% 
15% 
5% 
90.0 
84.9 
74.6 
10% 
85.0 
79.4 
69.2 
15% 
79.2 
73.5 
63.7 
20% 
72.8 
67.2 
58.3 
10.3 
Stratification Factors 
Randomization will be stratified by study site, nadir CD4 count (less than or equal to 200, 
greater than 200), and lesion size at baseline (greater than 50%, less than or equal to 50% 
of anal canal/perianal region). Nadir CD4 count will be self-reported; if the participant’s 
lowest CD4 count is unknown (specifically or categorically), the site may rely on a positive 
history of opportunistic infections as a surrogate to categorize the participant as having a 
CD4 count less than or equal to 200. In order to document lesion size at baseline, the site 
must report this stratification factor based on the clinician’s evaluation of these results as 
documented in the source. Three zones are to be evaluated for extent: 1) from the 
squamocolumnar junction (SCJ)/transformation zone (TZ) to the dentate line; 2) dentate 
line to the verge; and 3) the verge outward to the perianus. If any one of these zones have 
HSIL involvement of more than 50% , or if two of these zones have more than 25% HSIL 
involvement, the participant is considered to have more than 50% extent of HSIL. Lesions 
do not have to be contiguous to qualify for this definition. The site must report this 
stratification factor based on the clinician’s evaluation of these results as documented in 
the source. 
10.4 
Statistical Analysis 
10.4.1 Primary analysis 
The primary analysis population for this study will be the ITT population which 
will include all randomized study participants. For each study participant, time to 
anal cancer will be defined as the time from randomization to diagnosis of anal 
cancer, and censored at the date of last follow-up. The log-rank test will be used to 
compare the treatment and control arms with respect to time to detection of anal 
cancer. For each arm, the hazard rate and its 95% confidence interval will be 
estimated. The proportional hazards model will be used to assess the association of 
study site, lesion size, and nadir CD4 level with time to detection of anal cancer. 
10.4.2 Analysis of safety of anal HSIL treatments 
Adverse events will be summarized at the event level and at the participant level by 
type of adverse event and severity grade for each of the treatments (infrared 
coagulation, laser, electrocautery, imiquimod, and 5- fluorouracil treatments), for 


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the treatment arm as a whole and for the active monitoring arm over the course of 
study participation. For adverse events that occur in more than 5% of any of the 
treatments, the Poisson rates will be used to estimate the number of adverse events 
per unit time and the binomial proportion and its 95% confidence interval will be 
used to estimate the proportion of participants who reported the event. 
10.5 
Quality of Life Analyses 
10.5.1 A-HRSI scale responsiveness substudy 
This substudy will use the PGIC item to document change between the second and 
third follow-up time points (T2 and T3): “Since the last time you completed a 
questionnaire, how would you rate your OVERALL QUALITY OF LIFE?”, 
marked on a 7-point scale from “Very much worse” to “Very much better” with a 
mid-point of “No Change”. Each patient will be categorized into one of 3 groups: 
change for the worse (a response of “Very much worse”, “Moderately worse”, or 
“A little worse”), no change (“About the Same”), and change for the better (“A 
little better”, “Moderately better”, and “Very much better”). Changes in the A-
HRSI scores will also be calculated (T2 scores minus baseline scores). 
Responsiveness is supported if changes in the A-HRSI scores correspond reliably 
with the anticipated changes between the three groups. This will be analyzed by a 
one-way ANOVA on the changes in the A-HRSI scores across the 3 groups 
(“worse”, “no change”, and “better”). 
The PGIC item has been used with recall periods up to 6 weeks. It may or may not 
work for recall periods exceeding 6 weeks. We will collect the dates of assessments 
and thus can assess for trends in PGIC item. 
A sample size of 90 will support 80% power if the three groups differ by a 
standardized change of 0.46, in a one-way ANOVA. We estimated the statistical 
power by running a simulation where the “worsened” group had a mean change in 
A-HRSI score of -0.46 (simulated from a standard normal of mean=-0.46 and 
standard deviation=1.0), the “no change” group had a mean change of 0.0, and the 
“improved” group had a mean change of +0.46. Additional statistical assumptions 
included a two-sided type-I error rate of 5%, a correlation of 0.35 between the 
assessment scores, and n=30 in each of the 3 groups. We understand that this 
change of ±0.46 may be optimistic. If the changes are only ±0.30, then it will 
support 80% at a type-I error rate of 0.31 (i.e., considerably reduced confidence in 
rejecting the null). To allow for 10% dropout, 100 participants (50 in each study 
arm) will be enrolled in the substudy. 
Additionally, the standardized response mean (SRM) will be computed as the mean 
change in subscale scores divided by the standard deviation of change scores within 
each change category. The purpose of this statistic is to examine the extent to which 
patients’ quality of life change over time. Values greater than 0.8 will be considered 
large and values between 0.5 and 0.8 will be considered moderate. 
The amount of time spent on each ePRO item screen will be descriptively 
summarized in order to evaluate participant behavior in completing the 
questionnaire via the ePRO tool, as well as to verify that the ePRO application is 


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functioning properly. 
10.5.2 QoL objective 
In the final implementation of quality of life assessment as measured by the A-
HRSI will occur at 8 time points: Pre-randomization (T1), within 2-7 days (T2) and 
at 4 weeks of treatment/randomization (T3), and then yearly out to 5 years (T4-T8). 
The primary endpoint for the QoL objective is changes in physical symptoms and 
impacts from T2 to T3, adjusting for T1. It is anticipated that participants assigned 
to the treatment arm will experience significant reduction in physical symptoms 
and impacts from T2 to T3 versus those in the active monitoring arm.  
The secondary endpoint for the QoL objective is changes in psychological 
symptoms from T3 to T4, adjusting for T1. Due to potential uncertainty related to 
lack of treatment for those in the active monitoring arm, it is anticipated that 
participants assigned to the active monitoring arm will experience significant 
increases in psychological symptoms from T3 to T4 versus those in the treatment 
arm. 
The exploratory endpoint is the assessment of long-term HR-QoL changes in 
physical symptoms/impacts and psychological symptoms from T4 through the 
subsequent T5-T8 follow-ups. 
In the table below, we show the minimum difference that can be detected with 150, 
200, and 250 participants per arm in mean A-HRSI physical symptoms and physical 
impacts subscale change scores (T3 minus T2) using an analysis of covariance 
adjusting for the covariate baseline subscale to test for differences between arms at 
a one-sided 0.025 significance level with approximately 90% power. A one-sided 
alpha of 0.025 was selected for each comparison to maintain an overall one-sided 
alpha level of 0.05 after a Bonferroni adjustment for two comparisons. The details 
of the A-HRSI and subscales may be found in Section 2.3.11. 
Table 10-D: Smallest differences that can be detected for comparing change for specified 
time points according to treatment group for various scenarios based on pilot data with 
90% power using a one-sided alpha of 0.025 
 
Endpoint 
Alpha 
(1-sided) 
SD for 
change 
score 
R2 with 
baseline 
covariate 
Minimum difference that can be 
detected with given sample size  
(per arm): 
n=150 
n=200 
n=250 
Primary (T3 vs. T2): 
 
 
 
 
 
 
Physical Symptoms 
0.025 
0.78 
0.29 
0.29 
0.25 
0.23 
Physical Impacts 
0.025 
1.09 
0.22 
0.43 
0.37 
0.33 
Secondary (T4 vs. T3): 
 
 
 
 
 
 
Psychological Impacts 
0.05 
1.34 
0.17 
0.50 
0.43 
0.39 
SD=standard deviation 
Note: Estimates of SD and R2 were from responsiveness substudy. 


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Our plan is to target 250 participants per arm to allow for screen failures in 
consented participants and nonresponse. In the A-HRSI scale responsiveness study 
16% of consented participants were screen failures, and 12% of eligible participants 
did not return at either follow-up. 
The above table shows that with 150-200 participants per arm we would be able to 
detect differences of roughly 0.3 to 0.4 unit change on the two primary endpoints 
of change in physical symptoms and physical impacts. Although the timing of T2 
and T3 are somewhat different compared to the responsiveness substudy, the 
minimum differences that can be detected are smaller than the changes observed in 
the responsiveness substudy for those with worse ECOG PS scores compared to 
those with no change (mean group differences of 0.43 and 0.53 for the two 
subscales, section 2.3.11). 
Data for the QOL primary endpoints (physical symptoms and physical impacts) 
will each be analyzed using an analysis of covariance (ANCOVA) with the 
dependent variable being change in subscale (T3 score minus T2 score) and 
independent variables for arm and baseline subscale score. A similar approach will 
be used for analyzing change (T4 score minus T3 scores) in psychological impacts. 
The exploratory endpoint will be analyzed in a mixed model repeated measures 
ANCOVA with change from baseline calculated at each follow-up visit (T4 through 
T8) for each subscale and baseline subscale included as a covariate. Arms will be 
compared across time points by testing the main effect for arm. Additionally, the 
interaction between time and arm will be examined and trajectories over time, 
according to arm and specific treatments, will be explored using graphs and post-
hoc tests at specific follow-up times using model-based contrasts. 
Procedures to address missing data. As stated above, we are targeting 250 
participants per arm to allow for screen failures and varying survey response rates 
over time with the goal of obtaining evaluable data from n=150-200 per arm, which 
still allows for meaningful effects to be detected with 90% power (Table 10-D). We 
would not anticipate missing questionnaire to be differential with respect to known 
and unknown factors between arms due to randomization. However, as a sensitivity 
analysis, we plan to use regression-based, multiple random imputations for the 
primary and secondary endpoints assuming questionnaire are missing not a random 
(MNAR). We will use participant characteristics and baseline A-HRSI 
measurements in this predictive model. The analysis will then be carried out in 
multiple data sets, and the results combined using standard methods to produce arm 
effect estimates with standard errors that incorporate the imputation error. We will 
also compare participant characteristics for evaluable and unevaluable participants 
for each primary and secondary endpoint. 
10.6 
SARS-CoV-2 Ancillary Study Analyses 
Aim 1: Determine the prevalence of SARS-CoV-2 detection in anal and oropharyngeal 
swabs among people living with HIV (PLWH) being screened for the ANCHOR study. 
The prevalence of SARS-CoV-2 in anal swabs and in oropharyngeal swabs will be 
estimated using the binomial proportion and its 95% confidence interval. With a sample 


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size of 400 for each swab type and a hypothesized prevalence rate of 10%, the 95% 
confidence interval for the SARS-CoV-2 prevalence will be no greater than ±3.0% 
Aim 2: Determine the relationship in the ANCHOR screening population between 
prevalent anal SARS-CoV-2 positivity, anal HPV infection, and anal high-grade squamous 
intraepithelial lesions (HSIL) Fisher’s exact tests will be used to compare those screened 
participants with and without SARS-CoV-2 detected on the anal swab with respect to the 
prevalence of anal HPV infection and anal HSIL. 
Aim 3: Determine the 6-month incidence of SARS-CoV-2 detection in anal and 
oropharyngeal swabs among participants with anal HSIL newly enrolled into the 
ANCHOR study. For each swab type (anal and oropharyngeal), participants will be divided 
into two groups: those who tested positive for SARS-CoV-2 at baseline and those who did 
not. For those who tested positive for SARS-CoV-2 on anal (oropharyngeal) swabs, 
participants will be categorized as persistent if they test positive on the 6-month anal 
(oropharyngeal) swab. For each swab type, persistence will be estimated as the binomial 
proportion of those who tested positive at 6-months divided by those who were positive for 
SARS-CoV-2 at baseline. The point estimate and 95% confidence interval will be 
estimated for the binomial proportion. For those who tested negative for SARS-CoV-2 on 
anal (oropharyngeal) swabs, participants will be categorized as incidence cases if they test 
positive on the 6-month anal (oropharyngeal) swab. For each swab type, the incidence rate 
will be estimated as the binomial proportion of those who tested positive at 6-months 
divided by those who were negative for SARS-CoV-2 at baseline. The point estimate and 
95% confidence interval will be estimated for the binomial proportion. 
Aim 4. Determine the relationship between prevalent or incident SARS-CoV-2 detection 
and regression of anal HPV infection or HSIL among participants newly enrolled into the 
ANCHOR study and who are randomized to the monitoring arm. 
For monitoring arm participants, the incidence of regression of anal HPV infection will be 
estimated for prevalent and incident SARS-CoV-2 cases using the binomial proportion and 
its 95% confidence interval. Similarly, the incidence of regression of HSIL will be 
estimated for prevalent and incidence SARS-CoV-2 cases using the binomial proportion 
and its 95% confidence interval. Study time frame: We will enroll 400 PLWH who are 
being screened for ANCHOR over a 5-month period from the start of the study. This should 
be straightforward since the study typically screens over 200 PLHW/month. To reduce 
costs will perform the study only at the top 5-6 screening/enrolling sites in ANCHOR, and 
collectively these sites screen more than 1100 individuals/year. By completing enrollment 
within 5 months, we will also complete the 6-month follow-up analysis for Aims 3 and 4 
within a year of starting the study. We will analyze the data in the last month of the study. 
10.7 
Data and Safety Monitoring 
10.7.1 Participant safety will be monitored in accordance with the AMC Data and Safety 
Monitoring Plan (Appendix VI). The study will be monitored by a Data and Safety 
Monitoring Board. The DSMB will meet at least annually after study initiation to 
assess enrollment, retention (drop-out and drop-in rates), and safety data, and may 
meet more frequently if needed. The DSMB will also review the outcome-based 
interim analyses described in Section 10.6.2. 


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10.7.2 Interim analysis 
Futility and Efficacy Halting Rules 
Three interim analyses of the primary efficacy outcome are planned to assess the 
futility of achieving a significant result if the study continues and to potentially 
demonstrate efficacy before all participants are enrolled. The Lan and DeMets 
spending function was used to specify the O’Brien-Fleming boundaries based on a 
one-sided log-rank test 0.025. At the final test, an overall two-sided alpha level of 
0.05 (which corresponds to a one-sided 0.025 alpha level) and 90% power will be 
maintained. The boundaries and operating characteristics for the planned analyses 
are found in the table below. The DSMB will be informed of the results of the 
interim analysis. Consideration will be given to halting the study if the futility or 
efficacy boundary is crossed during the interim analysis. 
Table 10-E: Statistical test boundaries and associated operating characteristics. 
Analysis 
Information 
fraction 
Reject H0 
(Efficacy) Bound 
Z  
Reject H1 
(Futility) Bound 
Z  
Overall 
α spent 
Overall β (1-
power) spent 
1 (Interim) 
0.50 
Z > 2.963 
Z < 0.332 
0.002 
0.020 
2 (Interim) 
0.75 
Z > 2.359 
Z < 1.292 
0.010 
0.058 
3 (Final) 
1.00 
Z > 2.014 
Z < 2.014 
0.025 
0.100 
 
At the time of the interim analyses, the ratio of the hazard rate for the treatment arm 
to the active monitoring arm will be estimated using its point estimate and 95% 
confidence interval. The target hazard ratio is 0.25. If the efficacy stopping 
boundary is crossed, then stopping the study for efficacy would be considered if the 
upper bound of the hazard ratio is less than or equal to 0.50 suggesting that HSIL 
treatment reduces the anal cancer incidence rate by at least half. 
10.7.3 Drop-out rate and drop-in rate 
The sample size estimate for this study was based on a drop-out rate of 5% annually 
for both arms, and a drop-in rate of 7% to treatment for participants in the active 
monitoring arm. On a semi-annual basis, the drop-out and drop-in rates will be 
calculated for each year of follow-up. The drop-out rate will be defined as the 
number of participants who drop out in a follow-up year divided by the number of 
person years observed during that follow-up year. The drop-in rate will be defined 
as the number of participants in the active monitoring arm who are treated for HSIL 
(for any reason) in a follow-up year divided by the number of person years observed 
during that follow-up year in the active monitoring arm. 
If the drop-out rate exceeds 5 per 100 person-years for two consecutive 6-month 
periods, the protocol team will be required to implement a corrective action plan to 
decrease the drop-out rate. If the drop-out rate remains above 5 per 100 person-
years for two additional 6-month periods, and would require the accrual duration to 
be extended more than one year, then the trial would be stopped. 
If the drop-in rate exceeds 7 per 100 person-years for two consecutive 6-month 


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periods, the protocol team will be required to implement a corrective action plan to 
decrease the drop-in rate. If the drop-in rate remains above 7 per 100 person-years 
for two additional 6-month periods, and would require the trial duration to be 
extended for more than one year, then the trial would be stopped. 
10.7.4 Stopping rules for accrual 
The planned duration of accrual is 36 months. With the sample size of 5058, accrual 
would be completed in 36 months if the accrual rate is 140.5 enrollees/month. On 
a semi-annual basis, the cumulative accrual will be compared with the projected 
accrual for that timepoint to determine the proportion of the projected accrual that 
has been achieved. After 18 months, the proportion of the projected accrual will be 
calculated as the number of study participants enrolled divided by the cumulative 
projected number of participants for that time period. If it is less than or equal to 
0.20, then the DSMB will be asked to review accrual and to determine with the 
study team if measures can be taken to greatly improve the accrual rate. If the 
proportion of projected accrual that has been achieved is greater than 0.20 and less 
than 0.50, then enrollment will continue for an additional 6 months. At 30 months, 
if the proportion of the projected accrual that has been achieved remains below 
0.50, then the DSMB will consider whether to stop the study due to accrual. 
10.7.5 DSMB safety review rule 
The DSMB will conduct a formal review with recommendations when the rate of 
grade 3 or higher adverse events that are at least possibly related to a study 
treatment or procedure exceeds 5%, by arm; accounting for follow-up this would 
correspond to a rate greater than 5 events per 100 person years. If a safety issue is 
identified, potential actions the DSMB may consider recommending to address a 
safety risk may include recommendations to remove any procedures posing excess 
safety risks from the study, discussing added clinical measures with the protocol 
team to prevent or address the specific risk if feasible, and/or modification of the 
expected severe adverse event frequency in the protocol and consent form. 
 
 


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11.0 
ROLE OF DATA MANAGEMENT 
11.1 
CRF Instructions 
Access to the internet data entry system for this study, AdvantageEDCSM, and instructions 
for recording of study data on CRFs will be provided by the ANCHOR DMC at 
www.anchordmc.com. Participating institutions are responsible for submitting data and/or 
data forms via AdvantageEDC in accordance with the ANCHOR Data Entry Guide and 
specific form instructions, within the timelines specified by the AMC’s Standards of 
Procedure for Site Performance Measures. 
11.2 
Data Quality 
It is the responsibility of the ANCHOR DMC to assure the quality of data for the study. 
See the AMC Data and Safety Monitoring Plan (Appendix VI) for the study plans for 
adverse event reporting and data review. This role extends from protocol development to 
generation of the final study database. 
11.3 
Data Monitoring 
This study will be monitored in compliance with AMC and ANCHOR Study policies and 
by the Clinical Data Update System (CDUS) Version 3.0. Cumulative protocol- and 
patient-specific CDUS data will be submitted electronically to CTEP on a quarterly basis. 
Reports are due January 31, April 30, July 31, and October 31. Instructions for submitting 
data 
using 
the 
CDUS 
can 
be 
found 
on 
the 
CTEP 
Web 
site 
(http://ctep.cancer.gov/reporting/cdus.html). 
The ANCHOR DMC is responsible for compiling and submitting CDUS data to CTEP for 
all participants and for providing the data to the Principal Investigator for review. 
 
 


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12.0 
ETHICAL AND REGULATORY CONSIDERATIONS 
12.1 
IRB Approval and Informed Consent 
The principles of Institutional Review Board (IRB) approval and informed consent 
described in the Food and Drug Administration (FDA) regulations (21 CFR Part 50 and 
56) and Department of Health and Human Services (DHHS) regulations for the Protection 
of Human Participants regulations (45 CFR Part 46) must be followed. IRB approval of 
the protocol and the informed consent form must be given in writing. 
The sponsor’s designee (ANCHOR DMC) must receive a copy of the letter of approval 
from the IRB, which specifically approves the protocol and informed consent, before 
participant enrollment. The IRB must also approve any significant changes to the protocol 
and documentation of this approval must be sent to the ANCHOR DMC. The IRB must 
review the research project at least once every 365 days during the duration of the project. 
Continuing approval of the project must also be given in writing and provided to the 
ANCHOR DMC. 
Records of all study review and approval documents must be kept on file by the 
Investigator and are participant to inspection during or after completion of the study. AEs 
must be reported to the IRB according to local procedures. The IRB should receive 
notification of completion of the study and final report within 3 months of study completion 
and termination. The Investigator will maintain an accurate and complete record of all 
submissions made to the IRB, including a list of all reports and documents submitted. 
Written informed consent will be obtained from the participant. The nature, significance, 
and risks associated with the study must be explained to the participant. The informed 
consent will describe the purpose of the study, the procedures to be followed, the risks and 
benefits of participation, all risks of the investigational agent(s) and/or study participation 
as listed in the model informed consent form, and all other elements of informed consent 
as required by regulation. A copy of the consent form will be given to the participant to 
keep. 
In addition, any institution(s) conducting research according to the guidelines of this 
protocol is required to adhere to local and national laws and regulations governing the 
confidentiality and disclosure of health information. 
12.2 
Changes to the Protocol 
Any change or addition to this protocol requires a written protocol amendment that must 
be approved by CTEP and the Investigator before implementation. All amendments require 
approval by the IRB of the treating institution. A copy of the written approval of the IRB 
must be sent to the DMC. 
12.3 
Women and Minorities 
This study is being conducted by the NCI-sponsored AIDS Malignancy Consortium 
(AMC). As part of their contractual obligations, each participating site within the AMC 
and the AMC as a whole is required to assure that the participation of women and minority 
participants reflects the percentage representation of these populations in their geographic 
region and, for the AMC, the United States as a whole. As such, it is expected that the 


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representation of participants on this trial will reflect the constitution of the respective 
populations. 
Table 12-A: Accrual targets 
Ethnic Category 
Sex/Gender 
Females  
Males 
 
Total 
Hispanic or Latino 
191 
+ 1,074 = 1,265 
Not Hispanic or Latino 
567 
+ 3,226 = 3,793 
Ethnic Category: Total of all participants 
758 
+ 4,300 = 5,058 
Racial Category 
 
American Indian or Alaskan Native 
38 
+ 
215 
= 
253 
Asian 
38 
+ 
215 
= 
253 
Black or African American 
303 
+ 1,720 = 2,023 
Native Hawaiian or other Pacific Islander 
38 
+ 
215 
= 
253 
White 
341 
+ 1,935 = 2,276 
Racial Category: Total of all participants 
758 
+ 4,300 = 5,058 
 
(A1 = A2) (B1 = B2) (C1 = C2) 
Enter actual estimates, whole numbers only (percentages, fractions, or decimals are not acceptable). 
The totals provided for each Ethnic/gender or Ethnic/total combination must match those given for each 
Race/gender or Race/total combination (i.e., A1 must match A2, B1 must match B2, and C1 must match C2). 
 
 


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101. Lendvai Á, Johannes F, Grimm C, Eijsink JJH, Wardenaar R, Volders HH, et al. Genome-
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102. Huang R-L, Chang C-C, Su P-H, Chen Y-C, Liao Y-P, Wang H-C, et al. Methylomic analysis 
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103. Pathak S, Bhatla N, Singh N. Cervical cancer pathogenesis is associated with one-carbon 
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104. Wilting SM, Steenbergen RDM, Tijssen M, van Wieringen WN, Helmerhorst TJM, van 
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105. Kemp TJ, Hildesheim A, García-Piñeres A, Williams MC, Shearer GM, Rodriguez AC, et 
al. Elevated systemic levels of inflammatory cytokines in older women with persistent 
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106. Feng Q, Wei H, Morihara J, Stern J, Yu M, Kiviat N, et al. Th2 type inflammation promotes 
the gradual progression of HPV-infected cervical cells to cervical carcinoma. Gynecol. 
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107. Jaafar F, Righi E, Lindstrom V, Linton C, Nohadani M, Van Noorden S, et al. Correlation of 
CXCL12 expression and FoxP3+ cell infiltration with human papillomavirus infection 
and clinicopathological progression of cervical cancer. Am. J. Pathol. 2009;175:1525–
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108. Zeng C, Yao Y, Jie W, Zhang M, Hu X, Zhao Y, et al. Up-regulation of Foxp3 participates 
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109. Ferguson R, Ramanakumar AV, Koushik A, Coutlée F, Franco E, Roger M, et al. Human 
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110. Chatterjee K, Dandara C, Hoffman M, Williamson A-L. CCR2-V64I polymorphism is 
associated with increased risk of cervical cancer but not with HPV infection or pre-
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111. Lu D, Qian J, Yin X, Xiao Q, Wang C, Zeng Y. Expression of PTEN and survivin in cervical 
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112. Lu D, Yang X, Jiang NY, Woda BA, Liu Q, Dresser K, et al. IMP3, a new biomarker to 
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113. Martin CM, Astbury K, McEvoy L, O’Toole S, Sheils O, O’Leary JJ. Gene expression 
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114. Koay MHE, Crook M, Stewart CJR. Cyclin D1, E-cadherin and beta-catenin expression in 
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116. Lakatos E. Sample sizes based on the log-rank statistic in complex clinical trials. Biometrics. 
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118. Chin-Hong PV, Berry JM, Cheng SC, Catania JA, Da Costa M, Darragh TM, et al. 
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119. Palefsky JM, Holly EA, Efirdc JT, Da Costa M, Jay N, Berry JM, et al. Anal intraepithelial 
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120. De Pokomandy A, Rouleau D, Ghattas G, Trottier H, Vezina S, Cote P, et al. HAART and 
progression to high-grade anal intraepithelial neoplasia in men who have sex with men 
and are infected with HIV. Clin Infect Dis. 2011;52:1174–81. PMID: 21364075 
121. Botes LP, Pett S, Carr A, Marriott D, Cooper DA, Matthews G, et al. Anal cytological 
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122. Hessol NA, Holly EA, Efird JT, Minkoff H, Schowalter K, Darragh TM, et al. Anal 
intraepithelial neoplasia in a multisite study of HIV-infected and high-risk HIV-
uninfected women. AIDS. 2009;23:59–70. PMID: 19050387 
123. Liu H, Wu T. Sample size calculation and power analysis for time-averaged difference. 
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126. Chang, G.J., et al., Surgical treatment of high-grade anal squamous intraepithelial lesions: 
a prospective study. Dis Colon Rectum, 2002. 45(4): p. 453-8. 
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129. Burkhalter, J. E, Atkinson, T. M., Berry-Lawhorn, J. M., Goldstone, S., Einstein, M. H., 
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Feces After Conversion of Pharyngeal Samples in Patients With COVID-19. Ann 
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141. Wu Y, Guo C, Tang L, Hong Z, Zhou J, Dong X, et al. Prolonged presence of SARS-CoV-
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145. Hindson J. COVID-19: faecal-oral transmission? Nat Rev Gastroenterol Hepatol 2020 
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149. Zang R, Gomez Castro MF, McCune BT, Zeng Q, Rothlauf PW, Sonnek NM, et al. 
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Compr. Canc. Netw 2018; 16: 852-71 
 


 
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APPENDIX I: SCHEDULE OF EVALUATIONS 
Participants Randomized to Active Monitoring Arm 
 
Visit 0 
Screening 
 
Within 4 
weeks of 
Information 
Visit 
 
Visit 1* 
Random-
ization 
 
Within 1-
12 weeks 
of 
Screening 
 
Visit 2 
6 
months
1 
 
±4 
weeks 
Visit 3 
12 
months 
 
±4 
weeks 
Visits 4-
10** 
 
Every 6 
months 
 
±4 
weeks 
Final 
study 
visit 
(60 
months 
or later) 
±4 weeks 
Early 
Study 
Discont-
inuation 
Informed consent 
X2 
Demographics 
X 
Medical history 
X 
Concurrent meds 
X 
X 
X 
X 
X 
X 
X 
Physical exam3 
X 
 
X 
X 
X 
X 
X 
Inguinal node exam 
X 
X 
X 
X 
X 
X 
X 
Risk factor questionnaire 
 
X 
Quality of Life 
questionnaires4 
T1 
T2 
T3 
 
Performance status 
X 
CBC w/diff, platelets5 
X 
HIV documentation 
X 
HIV viral load, CD4 level 
X6 
X6 
X6 
X6 
X6 
Anal cytology7 
X 
X 
X 
X 
X 
X 
Digital anorectal exam 
X 
X 
X 
X 
X 
X 
X 
High resolution anoscopy 
X 
X 
X 
X 
X 
X 
X 
Anal swabs8 
X 
X 
X 
X 
X 
X 
Anal biopsy9 
X 
X 
X 
X 
X 
X 
Adverse event evaluation 
X 
X 
X 
X 
X 
X 
Urine HCG10 
X 
Blood for biorepository11 
X 
 
X 
X 
X 
X 
X 
Oropharyngeal swab12 
X 
 
X 
 
 
 
 
* 
Screening evaluations for Visit 0 may be performed at Visit 1 (except informed consent, the anal swab for cytology, the 
screening HRA, and anal biopsies), provided that all Visit 0 evaluations are performed before randomization. 
** Participants who are followed longer than 60 months will follow the same procedures for visits every six months. 
 
1 
Clinicians may elect to perform HRA and biopsy lesions between any of the 6-month visits if cancer is suspected. Visits that 
occur between regularly scheduled 6-month visits will be designated with the appropriate visit number and a, b, c, etc. 
2 
Informed consent will be obtained 1-6 weeks before randomization, and may be given during a preceding information visit, 
if done. 
3 
Complete physical exam will be performed at baseline. Targeted physical exam will be performed at all subsequent 6-month 
visits (see Section 8.1.7). Height and weight will be collected at the screening visit only. 
4 
For participants who consent to the optional HRQoL substudy (completed in February 2020), the A-HRSI and Patient 
ECOG PS will be administered via telephone facilitated interview or ePRO platform between time of enrollment and time of 
randomization (T1). A-HRSI, Patient ECOG PS and PGIC will be administered at least 14 days (2-weeks) post-
randomization, and at most 70-days post-randomization (T2). The third HRQoL follow-up will be scheduled to take place at 
least 71 days (10 weeks + 1 day) post-ANCHOR randomization, and at most 112 days (16 weeks) post-randomization (T3). 
5 
CBC with differential and platelets may occur up to 90 days prior to randomization. 
6 
HIV viral load and CD4 count results are required at Visit 1 (results obtained from routine medical care within six weeks prior 
to randomization will be acceptable). Results will be collected from routine medical care at annual visits through study 
discontinuation, if available. Venipuncture may be performed on the day of randomization, even after randomization. 
7 
Collect per Section 8.1.10. Leftover material is to be shipped to the ANCHOR Biorepository. 
8 
One swab stored for DNA. One swab stored for RNA for correlative science studies. These swabs may be collected at the 
randomization visit if not collected at the screening visit. All swabs will be collected every 6 months on the study. 


 
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9 
Screening biopsy must be performed 1-12 weeks prior to Visit 1. Biopsy of HSIL lesions performed at every other 6-month 
visit (annually). At the final visit, all visible lesions and any prior areas of HSIL that appear to have regressed will be biopsied. 
If this totals less than 4 biopsies, normal-appearing tissue in remaining quadrants will be biopsied so that a minimum of 4 
areas are sampled per Section 4.1. Biopsies may be done at any time if cancer is suspected. 
10 HCG for females of childbearing age potential. 
11 Blood for correlative science studies (serum [red top tube] at all indicated visits, plus plasma and whole blood fraction 
[lavender top tube] at Visit 2 only). 
12 Collect only if consented for participation in the SARS-CoV-2 ancillary study at the 5 participating centers. Swabs must be 
collected at the same visit as anal swab collection. 
 
 


 
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Participants Randomized to Treatment Arm, Treatment with Imiquimod Or 5-
Fluorouracil Cream 
Visit 0 
Screening 
 
Within 4 
weeks of 
Informa-
tion visit 
Visit 1* 
Random-
ization 
Within 1-
12 weeks 
of 
Screening 
Visit 
1A, 1B 
8, 16 
weeks, 
±2 
weeks1 
Visit 2 
6 
months2 
±4 weeks 
Visit 3 
12 
months 
±4 
weeks 
Visits 
4-10** 
Every 6 
months 
±4 
weeks 
Final 
study 
visit 
(60 
months 
or later) 
±4 weeks 
Early 
Study 
Discont-
inuation 
Informed consent 
X3 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
Concurrent meds 
X 
X 
X 
X 
X 
X 
X 
 
Physical exam4 
X 
 
 
X 
X 
X 
X 
X 
Inguinal node exam 
X 
X 
X 
X 
X 
X 
X 
X 
Risk factor questionnaire 
 
X 
 
 
 
 
 
 
Quality of Life 
Questionnaires5 
T1 
T2 
T3 
 
 
 
 
 
 
Performance status 
X 
 
 
 
 
 
 
 
CBC w/diff, platelets6 
X 
 
 
 
 
 
 
 
HIV documentation 
X 
 
 
 
 
 
 
 
HIV viral load, CD4 level 
 
X7 
 
 
X7 
X7 
X7 
X7 
Anal cytology8 
X 
 
 
X 
X 
X 
X 
X 
Digital anorectal exam 
X 
X 
X 
X 
X 
X 
X 
X 
High resolution anoscopy 
X 
X 
X 
X 
X 
X 
X 
X 
Anal swabs9 
X 
 
 
X 
X 
X 
X 
X 
Anal biopsy10 
X 
 
 
X 
X 
X 
X 
X 
Adverse event evaluation 
 
X 
X 
X 
X 
X 
X 
X 
Urine HCG11 
X 
X 
X 
X 
X 
X 
 
 
Blood for biorepository12 
X 
 
 
X 
X 
X 
X 
X 
Oropharyngeal swab13 
X 
 
 
X 
 
 
 
 
* 
Screening evaluations for Visit 0 may be performed at Visit 1 (except informed consent, the anal swab for cytology, the 
screening HRA, and anal biopsies), provided that all Visit 0 evaluations are performed before randomization. 
** Participants who are followed longer than 60 months will follow the same procedures for visits every six months, including 
interim visits for treatment as needed. 
 
1 
If treatment is tolerated at Visit 1A, clinician will continue treatment for an additional 8 weeks or stop if no HSIL is seen. 
Participant will return for an optional visit at 16 weeks for Visit 1B if treatment is continued. 
2 
If biopsies show HSIL clinician may opt to initiate another treatment modality. The schedule for that 6-month block of 
treatment will vary depending on the modality that will be used. Visits that occur between regularly scheduled 6-month visits 
will be designated with the appropriate visit number and an interim visit sequence letter a, b, c, etc. 
3 
Informed consent will be obtained 1-6 weeks before randomization, and may be given during the preceding information visit, 
if done. 
4 
Complete physical exam will be performed at baseline. Targeted physical exam will be performed at all subsequent 6-month 
visits, (see Section 8.1.7). Height and weight will be collected at the screening visit only. 
5 
For participants who consent to the optional HRQoL substudy (completed in February 2020), the A-HRSI and Patient 
ECOG PS will be administered via telephone facilitated interview or ePRO platform between time of enrollment and time of 
randomization (T1). A-HRSI, Patient ECOG PS and PGIC will be administered at least 14 days (2-weeks) post-
randomization, and at most 70-days post-randomization (T2). The third HRQoL follow-up will be scheduled to take place at 
least 71 days (10 weeks + 1 day) post-ANCHOR randomization, and at most 112 days (16 weeks) post-randomization (T3). 
6 
CBC with differential and platelets may occur up to 90 days prior to randomization. 
7 
HIV viral load and CD4 count results are required at Visit 1 (results obtained from routine medical care within six weeks prior 
to randomization will be acceptable). Results will be collected from routine medical care at annual visits through study 
discontinuation, if available. Venipuncture may be performed on the day of randomization, even after enrollment into the 
system. 


 
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8 
Collect per Section 8.1.10. Leftover material is to be shipped to the ANCHOR Biorepository. 
9 
One swab stored for DNA. One swab stored for RNA for correlative science studies. These swabs may be collected at the 
randomization visit if not collected at the screening visit. All swabs will be collected every 6 months on the study. 
10 Screening biopsy must be performed between 1-12 weeks prior to Visit 1. Biopsies of HSIL lesions performed at each 6-
month visit. Areas that appear normal following treatment do not require rebiopsy. At the final visit, HSIL lesions and 
previously treated areas that appear normal will be biopsied (minimum of 4 biopsies per Section 4.2). Biopsies may be done 
at any time if cancer is suspected. 
11 HCG for females of childbearing potential, as needed prior to treatment. 
12 Blood for correlative science studies (serum [red top tube] at all indicated visits, plus plasma and whole blood fraction 
[lavender top tube] at Visit 2 only). 
13 Collect only if consented for participation in the SARS-CoV-2 ancillary study at the 5 participating centers. Swabs must be 
collected at the same visit as anal swab collection. 
 
 
 


 
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Participants Randomized to Treatment Arm, Treatment with Infrared Coagulation, 
Hyfrecation/Electrocautery, or Laser 
Visit 0 
Screening  
 
Within 4 
weeks of 
Informa-
tion Visit 
Visit 1* 
Random-
ization 
Within 1-
12 weeks of 
Screening 
Visit 
1A 
6 
weeks, 
±2 
weeks1 
Visit 2 
6 
months2 
±4 
weeks 
Visit 3 
12 
months 
±4 
weeks 
Visits 
4-10** 
Every 6 
months 
±4 
weeks 
Final 
study 
visit 
(60 
months 
or later) 
±4 weeks 
Early 
Study 
Discont-
inuation 
Informed consent 
X3 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
Concurrent meds 
X 
X 
X 
X 
X 
X 
X 
X 
Physical exam4 
X 
 
 
X 
X 
X 
X 
X 
Inguinal node exam 
X 
X 
X 
X 
X 
X 
X 
X 
Risk factor questionnaire 
 
X 
 
 
 
 
 
 
Quality of life 
questionnaires5 
T1 
T2 
T3 
 
 
 
 
 
 
Performance status 
X 
 
 
 
 
 
 
 
CBC w/diff, platelets6 
X 
 
 
 
 
 
 
 
HIV documentation 
X 
 
 
 
 
 
 
 
HIV viral load, CD4 level 
 
X7 
 
 
X7 
X7 
X7 
X7 
Anal cytology8 
X 
 
 
X 
X 
X 
X 
X 
Digital anorectal exam 
X 
X 
X 
X 
X 
X 
X 
X 
High resolution anoscopy 
X 
X 
X 
X 
X 
X 
X 
X 
Anal swabs9 
X 
 
 
X 
X 
X 
X 
X 
Anal biopsy10 
X 
 
 
X 
X 
X 
X 
X 
Biopsy in RNALater11 
 
X 
Not more than once each 6-month block during study 
Adverse event evaluation 
 
X 
X 
X 
X 
X 
X 
X 
Urine HCG12 
X 
X 
X 
X 
X 
X 
 
 
Blood for biorepository13 
X 
 
 
X 
X 
X 
X 
X 
Oropharyngeal swab14 
X 
 
 
X 
 
 
 
 
* 
Screening evaluations for Visit 0 may be performed at Visit 1 (except informed consent, the anal swab for cytology, the 
screening HRA, and anal biopsies), provided that all Visit 0 evaluations are performed before randomization. 
** Participants who are followed longer than 60 months will follow the same procedures for visits every six months, including 
interim visits for treatment as needed. 
 
1 
As needed, if the participant is having staged procedures or requires an additional treatment of the lesion being ablated in the 
judgment of the clinician. 
2 
If biopsies show HSIL clinician may opt to initiate another treatment modality. The schedule for that 6-month block of 
treatment will vary depending on the modality that will be used. Visits that occur between regularly scheduled 6-month visits 
will be designated with the appropriate visit number and an interim visit sequence letter a, b, c, etc. 
3 
Informed consent will be obtained 1-6 weeks before randomization, and may be given during the preceding information visit, 
if done. 
4 
Complete physical exam will be performed at baseline. Targeted physical exam will be performed at all subsequent 6-month 
visits (see Section 8.1.7). Height and weight will be collected at the screening visit only. 
5 
For participants who consent to the optional HRQoL substudy (completed in February 2020), the A-HRSI and Patient 
ECOG PS will be administered via telephone facilitated interview or ePRO platform between time of enrollment and time of 
randomization (T1). A-HRSI, Patient ECOG PS and PGIC will be administered at least 14 days (2-weeks) post-
randomization, and at most 70-days post-randomization (T2). The third HRQoL follow-up will be scheduled to take place at 
least 71 days (10 weeks + 1 day) post-ANCHOR randomization, and at most 112 days (16 weeks) post-randomization (T3). 
6 
CBC with differential and platelets may occur up to 90 days prior to randomization. 
7 
HIV viral load and CD4 count results are required at Visit 1 (results obtained from routine medical care within six weeks prior 
to randomization will be acceptable). Results will be collected from routine medical care at annual visits through study 
discontinuation, if available. Venipuncture may be performed on the day of randomization, even after enrollment into the 
system. 


 
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8 
Collect per Section 8.1.10. Leftover material is to be shipped to the ANCHOR Biorepository. 
9 
One swab stored for DNA. One swab stored for RNA for correlative science studies. These swabs may be collected at the 
randomization visit if not collected at the screening visit. All swabs will be collected every 6 months on the study. 
10 Screening biopsy must be performed between 1-12 weeks prior to Visit 1. Biopsies of visible lesions performed at each 6-
month visit. Areas that appear normal following treatment do not require rebiopsy. At the final visit, HSIL lesions and 
previously treated areas that appear normal will be biopsied (minimum of 4 biopsies per Section 4.2). Biopsies may be done 
at any time if cancer is suspected. 
11 One additional biopsy of the lesion with the most severe appearance will be collected prior to ablative treatment and stored in 
RNALater for banking. This biopsy may be collected beginning at the randomization and not more than once every six months 
during the study. 
12 HCG for females of childbearing potential, as needed prior to treatment. 
13 Blood for correlative science studies (serum [red top tube] at all indicated visits, plus plasma and whole blood fraction 
[lavender top tube] at Visit 2 only). 
14 Collect only if consented for participation in the SARS-CoV-2 ancillary study at the 5 participating centers. Swabs must be 
collected at the same visit as anal swab collection. 
 
 
 


 
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Participants Randomized to Treatment Arm, Surgery 
Visit 0 
Screening  
 
Within 4 
weeks of 
Informa-
tion Visit 
Visit 1* 
Random-
ization1 
Within 1-12 
weeks of 
Screening 
Visit 
1A, 1B2 
 
Visit 2 
6 months3 
±4 weeks 
Visit 3 
12 
months 
±4 weeks 
Visits 
4-10** 
Every 6 
months 
±4 
weeks 
Final 
study 
visit 
(60 
months 
or later) 
±4 
weeks 
Early 
Study 
Discont-
inuation 
Informed consent 
X4 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
Concurrent meds 
X 
X 
X 
X 
X 
X 
X 
X 
Physical exam5 
X 
 
 
X 
X 
X 
X 
X 
Inguinal node exam 
X 
X 
X 
X 
X 
X 
X 
X 
Risk factor questionnaire 
 
X 
 
 
 
 
 
 
Quality of life 
questionnaires6 
T1 
T2 
T3 
 
 
 
 
 
 
Performance status 
X 
 
 
 
 
 
 
 
CBC w/diff, platelets7 
X 
 
 
 
 
 
 
 
HIV documentation 
X 
 
 
 
 
 
 
 
HIV viral load, CD4 level 
 
X8 
 
 
X8 
X8 
X8 
X8 
Anal cytology9 
X 
 
 
X 
X 
X 
X 
X 
Digital anorectal exam 
X 
X 
X 
X 
X 
X 
X 
X 
High resolution anoscopy 
X 
 
X 
X 
X 
X 
X 
X 
Anal swabs10 
X 
 
 
X 
X 
X 
X 
X 
Anal biopsy11 
X 
 
 
X 
X 
X 
X 
X 
Adverse event evaluation 
 
X 
X 
X 
X 
X 
X 
X 
Urine HCG12 
X 
X 
X 
X 
X 
X 
 
 
Blood for biorepository13 
X 
 
 
X 
X 
X 
X 
X 
Oropharyngeal swab14 
X 
 
 
X 
 
 
 
 
* 
Screening evaluations for Visit 0 may be performed at Visit 1 (except informed consent, the anal swab for cytology, the 
screening HRA, and anal biopsies), provided that all Visit 0 evaluations are performed before randomization. 
** Participants who are followed longer than 60 months will follow the same procedures for visits every six months, including 
interim visits for treatment as needed. 
 
1 
Surgery will be scheduled and should be performed within 2 months. 
2 
Additional visits may occur between 6-month visits if TUA is being performed in stages. 
3 
If biopsies show HSIL clinician may opt to initiate another treatment modality. The schedule for that 6-month block of 
treatment will vary depending on the modality that will be used. Visits that occur between regularly scheduled 6-month visits 
will be designated with the appropriate visit number and an interim visit sequence letter a, b, c, etc. 
4 
Informed consent will be obtained 1-6 weeks before randomization, and may be given during the preceding information visit, 
if done. 
5 
Complete physical exam will be performed at baseline. Targeted physical exam will be performed at all subsequent 6-month 
visits (see Section 8.1.7). Height and weight will be collected at the screening visit only. 
6 
For participants who consent to the optional HRQoL substudy (completed in February 2020), the A-HRSI and Patient 
ECOG PS will be administered via telephone facilitated interview or ePRO platform between time of enrollment and time of 
randomization (T1). A-HRSI, Patient ECOG PS and PGIC will be administered at least 14 days (2-weeks) post-
randomization, and at most 70-days post-randomization (T2). The third HRQoL follow-up will be scheduled to take place at 
least 71 days (10 weeks + 1 day) post-ANCHOR randomization, and at most 112 days (16 weeks) post-randomization (T3). 
7 
CBC with differential and platelets may occur up to 90 days prior to randomization. 
8 
HIV viral load and CD4 count results are required at Visit 1 (results obtained from routine medical care within six weeks prior 
to randomization will be acceptable). Results will be collected from routine medical care at annual visits through study 
discontinuation, if available. Venipuncture may be performed on the day of randomization, even after enrollment into the 
system. 
9 
Collect per Section 8.1.10. Leftover material is to be shipped to the ANCHOR Biorepository. 
10 One swab stored for DNA. One swab stored for RNA for correlative science studies. These swabs may be collected at the 
randomization visit if not collected at the screening visit. All swabs will be collected every 6 months on the study. 
11 Screening biopsy must be performed between 1-12 weeks prior to Visit 1. Biopsies of visible lesions performed at each 6-


 
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month visit. Areas that appear normal following treatment do not require rebiopsy. At the final visit, HSIL lesions and 
previously treated areas that appear normal will be biopsied (minimum of 4 biopsies per Section 4.2). Biopsies may be done 
at any time if cancer is suspected. 
12 HCG for females of childbearing potential, as needed prior to treatment. 
13 Blood for correlative science studies (serum [red top tube] at all indicated visits, plus plasma and whole blood fraction 
[lavender top tube at Visit 2 only). 
14 Collect only if consented for participation in the SARS-CoV-2 ancillary study at the 5 participating centers. Swabs must be 
collected at the same visit as anal swab collection. 
 
 
 


 
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Health-Related Quality of Life (QoL) Objective Schedule of Evaluations 
Sites are to instruct the participant to complete the questionnaire within the target completion 
window. Missed time points will not require submission of a protocol deviation form. 
Time Point and 
Target 
Completion 
T1 
Baseline  
 
Visit 1 
Randomization 
 
T2 
2-7 days post-
randomization 
T3 
4 weeks post-
randomization 
 
T4-T8 
 
12, 24, 36, 48, 
and 60 months 
post 
randomization 
 
Target A-HRSI 
completion 
window 
14 to 0 days 
before 
randomization 
2 to 7 days after 
randomization 
± 1 weeks 
± 4 weeks 
Window for 
accepted forms 
14 to 0 days 
before 
randomization 
2 to 10 days 
after 
randomization 
21 to 60 days 
after 
randomization 
-28 / +56 days of 
target annual 
visit date 
 


 
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APPENDIX II: PERFORMANCE STATUS SCALES 
 
 
Karnofsky Performance Scale 
 
 
ECOG Performance Status Scale 
Percent 
Description 
Grade 
Description 
100 
Normal, no complaints, no 
evidence of disease. 
0 
Normal activity. Fully active, able to 
carry on all pre-disease performance 
without restriction. 
90 
Able to carry on normal activity; 
minor signs or symptoms of 
disease. 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
1 
Symptoms, but ambulatory. 
Restricted in physically strenuous 
activity, but ambulatory and able to 
carry out work of a light or 
sedentary nature (e.g., light 
housework, office work). 
70 
Cares for self, unable to carry on 
normal activity or to do active 
work. 
60 
Requires occasional assistance, but 
is able to care for most of his/her 
needs. 
2 
In bed <50% of the time. 
Ambulatory and capable of all self-
care, but unable to carry out any 
work activities. Up and about more 
than 50% of waking hours. 
50 
Requires considerable assistance 
and frequent medical care. 
40 
Disabled, requires special care and 
assistance. 
3 
In bed >50% of the time. Capable of 
only limited self-care, confined to 
bed or chair more than 50% of 
waking hours. 
30 
Severely disabled, hospitalization 
indicated. Death not imminent. 
20 
Very sick, hospitalization 
indicated. Death not imminent. 
4 
100% bedridden. Completely 
disabled. Cannot carry on any self-
care. Totally confined to bed or 
chair. 
10 
Moribund, fatal processes 
progressing rapidly. 
0 
Dead. 
5 
Dead. 
 
 


 
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APPENDIX III: ANAL CYTOLOGY AND HPV SAMPLING 
 
All anal cytology specimens will be examined at the local institution. 
The participant should undress so that buttocks are exposed, and either bend over the exam table 
or lay on their side in left lateral decubitus. The examiner should use one hand to spread the 
buttocks and expose the anal verge. 
Procedure for obtaining anal swab specimens: 
For anal cytology and correlative science studies: A non-scored polyester swab moistened in tap 
water will then be inserted as far as is comfortable into the anus, a minimum of 1-2 inches. If there 
is difficulty inserting the swab, the participant should also retract their buttocks and the swab 
reoriented in the canal. With pressure on the swab rotate it firmly in a circular fashion for 
approximately 20 seconds and slowly remove from the canal. Do not retract the buttocks when the 
swab is close to the verge to ensure that it is sampled as well. Immediately immerse the swab in a 
liquid-cytology vial agitating vigorously over 20 to 45 seconds to disperse the cells. The liquid 
cytology vial will be sent the local cytopathology lab for processing. The first slide will be used 
for the study. The leftover material from anal swab cytology suspension will be shipped to the 
ANCHOR Biorepository, processed according to the ANCHOR Biorepository’s procedures, and 
stored for future correlative studies. 
For additional correlative science studies: Two additional moistened polyester swabs should be 
obtained. The second swab will be used for DNA studies, and the third for RNA/protein studies. 
These two swabs will be processed for storage in the ANCHOR Biorepository according to the 
instructions in the ANCHOR Study Manual of Procedures (MOP). 
All three swabs will be collected in this order at each visit where required. If a local cytology result 
exists to satisfy protocol requirements at the time of the visit, the first anal swab will be recollected 
with the second and third swabs, and the first swab sent to the ANCHOR Biorepository without 
local processing for the cytology result. 
Procedures and schedules for shipping monolayer slides, Thinprep solution, and swabs to the 
ANCHOR Biorepository are outlined in the ANCHOR Study MOP. 
Guidance regarding inconclusive cytology results: 
Any insufficient or inconclusive cytology at screening must be repeated. Valid results must be 
available within 12 weeks before randomization. 
If a cytology specimen collected after randomization is interpreted as insufficient or inconclusive 
for any 6-month visit, repeat cytology is required. If an interim visit is planned, the repeat specimen 
can be collected at that visit, otherwise the participant should be asked to return specifically for 
sample collection within 3 months. The two additional swabs for correlative science studies will 
be recollected if the local cytology specimen requires recollection. The residual media from both 
cytology specimens should be submitted to the ANCHOR Biorepository for correlative studies, 
denoting which specimen was found to be insufficient or inconclusive. 
Guidance regarding discrepant cytology and histology results: 
During screening, if the local cytology result is ASC-H or HSIL and no lesions were observed or 
no biopsy result showed HSIL, the participant will return for a repeat HRA within 3 months. 


 
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APPENDIX IV: HIGH RESOLUTION ANOSCOPY (HRA) AND ANAL BIOPSIES 
 
Procedure for performing HRA: 
High resolution anoscopy should only be done after the specimens for anal cytology and HPV 
testing are collected. The patient will already be positioned for anal evaluation. A mixture of an 
anesthetic cream (e.g., 4% lidocaine cream) and water-soluble lubricating jelly should be used as 
a lubricant. A digital anal rectal exam should then be performed palpating the entire anal canal, 
distal colon and perianus, noting any masses or areas of induration. The procedure for HRA is as 
follows: 
1. Insert the anoscope, remove obturator, and place a cotton swab wrapped in gauze soaked in 
5% acetic acid into anus. 
2. Remove the anoscope over the swab and leave swab in place for 1 to 2 minutes. 
3. Remove the swab and re-insert the anoscope. Carefully examine the anal canal with a 
colposcope. 
4. Re-apply acetic acid frequently to ensure adequate detection of lesions and verify that all 
aspects of the Anal Transformation Zone (AnTZ) have been visualized. 
5. If acetowhitening is noted, note vascular characteristics, if present. 
6. Lugol’s solution (iodine) may be used as desired to aid in identifying areas of possible LSIL 
and HSIL near the squamocolumnar junction. 
7. At each visit the clinician will carefully map the location of each lesion and where they did 
their biopsies, if any. Sites are requested to photograph each lesion at every visit using Second 
Opinion or other software that allows for easy sharing of images between study sites, for the 
purposes of training and quality control. 
8. Biopsy abnormal appearing areas according to the protocol requirements for the assigned study 
arm. Local anesthetic (e.g., 1% lidocaine with or without epinephrine or .5% bupivacaine) may 
be used at the provider’s discretion prior to biopsy. Biopsies from different quadrants must go 
in separate formalin containers and get separate pathologic interpretations. 
9. Attain hemostasis with pressure prior to removal of the anoscope or by removing the anoscope. 
Monsel’s solution or silver nitrate should be used judiciously and only after all biopsies have 
been obtained because it can interfere with histologic interpretation. Electrocautery or infrared 
coagulation should be used judiciously to stop significant bleeding that does not respond to the 
above measures. It must be documented if used for hemostasis. 
10. Apply acetic acid for one minute to perianal area and examine carefully with colposcope. 
11. Biopsy any external (perianal) areas per the assigned study arm using a local anesthetic (e.g., 
1% lidocaine with or without epinephrine or 0.5% bupivacaine) prior to biopsy. 
12. Participants with signs or symptoms consistent with proctitis or sexually transmitted infections 
other than HPV should be referred for appropriate diagnosis and treatment. 


 
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Procedures for Biopsy Review: 
All pathology specimens will be reviewed by the designated pathologist on site. 
p16 IHC staining is not required on all biopsies diagnosed as HSIL by the local laboratories for 
the ANCHOR Study. If performed, the immunostained slides are also requested to be sent for 
Central Pathology review. p16 IHC is useful to adjudicate differential diagnoses on H&E 
particularly for the following indications: 
• To differentiate between the H &E morphologic diagnosis of HSIL (–IN 2 or –IN 3) and a 
mimic of precancer (e.g., processes known to be unrelated to neoplastic risk such as 
immature squamous metaplasia, reparative epithelial changes, tangential cutting). Strong 
and diffuse block-positive p16 results support a categorization of HSIL. Negative or non–
block-positive staining strongly supports a non–HPV-associated pathology. 
• To clarify a diagnosis of –IN2 when the lesion is clearly HPV-associated, but on H&E 
morphologic interpretation the diagnosis of –IN 2 (under the old terminology) is 
considered. –IN2 is a biologically equivocal lesion falling between the morphologic 
changes of a productive HPV infection (LSIL) and precancer (HSIL). Strong and diffuse 
block-positive p16 results support a categorization of HSIL. Negative or non–block-
positive staining strongly favors an interpretation of LSIL. 
A p16 stain must be obtained if the only area of HSIL among all biopsies taken is AIN 2. At 
any visit, if other lesions show AIN2-3 or more severe histology, p16 staining is highly 
recommended for AIN2 biopsies to adjudicate the diagnosis, but is not mandatory. If, after 
evaluation of screening biopsies, the only remaining focus of HSIL at randomization is AIN2, p16 
staining is required for that lesion. 
The local pathology lab relied upon for this trial must be willing to send slides and tissue blocks 
as requested by this study. Stained H&E slides from all biopsies with a result of AIN2 or greater 
will be sent from participants who are biopsied as part of the screening process and randomized at 
the baseline visit. If p16 IHC staining is performed, the immunostained slides are also requested 
to be sent for Central Pathology review. Participants who fail screening due to suspected cancer 
diagnoses or cancer diagnoses will undergo central pathology review to confirm the diagnosis, if 
biopsies are available from screening procedures performed by the site. The protocol team may 
request additional slides undergo central pathology review. Slides will be submitted in real time 
for any cancer diagnoses, as well as all biopsies collected prior to progression to cancer, and any 
cases that are suspicious for progression to cancer during local pathology review. Tissue blocks 
may be requested for correlative studies, for participants who develop cancer, and for matched 
controls. Blocks will not be exhausted and will be returned to the local pathology lab. 
The baseline pathology review will serve to identify the discrepancy in HSIL vs. no HSIL readings, 
and HSIL vs. LSIL readings between the local pathologist’s finding and the central pathology 
result. If a significant discrepancy is identified at a study site, 100% of the slides will be reviewed 
from the site from all subsequent biopsies, and additional slides will be required from all other 
participating sites. Slides will be batched for shipment for central pathology review at least every 
six months according to the instructions in the ANCHOR Study Manual of Procedures. The 
pathology slides will be reviewed later at UCSF, and results of the central pathology review will 
not be available in real time. 


 
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Guidance regarding repeat biopsy and inconclusive biopsy results/discordant cytology results: 
• After the screening visit, if all biopsies do not show HSIL and the local cytology result shows 
high grade disease (ASC-H or HSIL), the investigator should bring the participant back for 
repeat HRA. If the biopsies at screening do not show HSIL and the local cytology result is 
inconclusive, repeat HRA with biopsy should only be performed if the investigator’s clinical 
impression of the participant’s disease is strongly suggestive of HSIL. If the repeat biopsies do 
not show HSIL, the participant should discontinue screening. 
• After randomization, if all biopsies at a 6-month visit do not show HSIL or are inconclusive, 
and the local cytology result shows high grade disease (ASC-H or HSIL), the investigator 
should bring the participant back for repeat HRA at 3 months (interim visit, window of ± 4 
weeks). If a biopsy is performed at an even-numbered visit for a monitoring arm participant 
and the biopsy results are discordant, the participant will not be asked to return for repeat 
biopsy until the next 6-month visit unless there is suspicion for progression to cancer. If at least 
one biopsy shows HSIL, repeat biopsies of lesions with inconclusive histology may be deferred 
to the next 6-month visit so long as there is no suspicion of progression to cancer. If the visit 
window for the next 6-month visit is within 8 weeks of the last biopsy, repeat HRA and biopsies 
will occur as part of the next 6-month visit. 
• The investigator may request that a participant return for repeat biopsy at any time there is 
suspicion for cancer. 
 


 
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APPENDIX V: RISK FACTOR QUESTIONNAIRE 
 
ANCHOR Visit 1 Questionnaire 
 
Participant ID#: 
__________ - ________ - ___________ 
Visit#: _____________ 
 
Date completed: ______/______/____________ 
 
Interviewer’s name (if administered by study staff): ________________________________ 
 
Background: 
Thank you for agreeing to be part of the ANCHOR Study. Before starting the study, we are giving 
all volunteers this ANCHOR Visit 1 Questionnaire. This questionnaire is a survey that asks 
questions about your medical history, including medical conditions related to anal HSIL, your 
sexual history, and whether you smoke or have used drugs. This questionnaire will take about 25 
minutes to complete. Please read the information below before you start the questionnaire. 
• Your completion of this questionnaire is voluntary. All of your responses will be confidential. 
• Some people may find some of the questions embarrassing. It is important that you answer all 
questions honestly. 
• No one will force you to answer any questions if it is too uncomfortable for you. If you do not 
feel comfortable answering a question, you may leave the question blank and move on to the 
next one. 
• If you feel that you do not have privacy to fill out this questionnaire, talk to a study team 
member immediately. 
• When you are finished completing the questionnaire, place the questionnaire in the envelope 
provided by the study staff. After you submit the questionnaire, the study team will not be able 
to see your answers. 
 
1) What was the month and year of your positive HIV test result? (IF MORE THAN 1 POSITIVE 
TEST, RECORD THE EARLIEST TEST DATE. IF YOU RECALL THE YEAR BUT NOT 
THE MONTH, ENTER “06” (JUNE) FOR THE MONTH.) 
 
 
 
 
 
 
 
 
     Month 
 
 
Year 
 
2) Have you ever had anal condyloma or warts, also known as venereal warts? 
Yes (1) 
No (2) 
 


 
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3) Have you ever had anal herpes? 
Yes (1) 
No (2) 
 
4) Have you ever had anal gonorrhea? 
Yes (1) 
No (2) 
 
5) Have you ever had syphilis? 
Yes (1) 
No (2) 
 
6) Have you had chlamydia infection of the anus? Chlamydia infection of the anus is also 
known as chlamydia proctitis. 
Yes (1) 
No (2) 
 
7) Have you ever had any of the following conditions? 
a. Inflamed hemorrhoids or piles? 
Yes (1) 
No (2) 
 
b. Diarrhea, 2 or more loose stools per day for at least 2 weeks? 
Yes (1) 
No (2) 
 
c. A draining or crack-like sore or hole in the anal wall, fissure or fistula? 
Yes (1) 
No (2) 
 
d. An infection or abscess in your anal area? 
Yes (1) 
No (2) 
 
e. Swollen or tender lumps around your crotch area? 
Yes (1) 
No (2) 
 
f. Hardening or narrowing of the anal passage, or stenosis? 
Yes (1)  
No (2) 
 
 


 
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8) At what age was your first sexual experience with another person? 
 
  
Age 
 
9) Have you ever had receptive anal intercourse, that is, anal intercourse in which a man’s penis 
was inserted into your anus? 
Yes (1) 
No (2) If no, skip to question 12. 
 
10) Using the letter codes below, please tell me the letter that best describes the total number of 
sexual partners you have had receptive anal intercourse with over your lifetime. ________ 
 
Letter Codes for Question 10 
1 to 10…………….. B 
11 to 50………….... C 
51 to 100………….. D 
101 to 200………… E 
201 to 500………… F 
501 to 1000……….. G 
over 1000…………. H 
 
11) Using the letter codes below, please tell me the letter that best describes how often you had 
receptive anal intercourse during the past 12 months? ___________ 
Letter Codes for Question 11 
Never……………………………. A 
once/month or less ……………… B 
about once a week ………………. C 
about 3 or 4 times per week …….. D 
about 5 to 10 times per week ……. E 
about 11 to 20 times per week …... F 
more than 20 times per week ……. G 
 
12) During the past 5 years did you have any objects inserted into your anus, other than a penis, 
fingers, or a scope by a medical provider? 
Yes (1) 
No (2) 
 
13) If you replied “yes” to question 12, which letter code below best describes how many times 
an object was inserted into your anus (other than by a medical provider) during the past 5 
years? __________ 
 


 
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Letter Codes for Question 13 
Less than 10 times total……….. A 
10 to 100 times ……………….. B 
101 to 500 times ……………… C 
more than 500 times ………….. D 
 
14) Overall, does your sexual partner wear condoms when he inserts his penis into your 
anus/rectum? 
 
Letter codes for Question 14 
Never………………………………………………………….A 
Some of the time (<50% of the time)…………………………B 
Most of the time ( >50% but less than 100% of the time)…….C 
All of the time…………………………………………………D 
Not applicable…………………………………………………E 
 
15) For women: have you ever had an abnormal cervical Pap smear? 
Yes (1) 
No (2) 
 
What age? 
 
 
Age 
16) For women: have you ever been diagnosed with pre-cancer or high-grade dysplasia of the 
cervix, vulva, or vagina? 
Yes (1) 
No (2) 
 
If yes, what age? 
 
   Age 
17) Have you smoked more than 100 cigarettes in your lifetime? 
Yes (1) 
No (2) If no, skip to question 23 
 
18) At what age did you first start smoking? 
 
19) At that time, how many cigarettes did you smoke per day? 
20) How many years did you smoke this amount of cigarettes? 


 
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21) Do you currently smoke? 
22) Currently, how many cigarettes/day do you smoke? 
23) Have you ever used any of the following substances in the past year? Please indicate how 
many times you have used the substance using the letter codes below. 
 
Letter Codes for Question 23 
Never………………………………..A 
Less than 10 times total……………..B 
10 to 100 times ……………………..C 
101 to 500 times ……………………D 
more than 500 times ………………..E 
 
a. Marijuana or hashish _____ 
b. Cocaine, (blow, snow) or crack cocaine _____ 
c. Amyl or Butyl nitrite or poppers _____ 
d. LSD or Acid _____ 
e. Angel dust, PCP, Ketamine, Special K, or Super K _____ 
f. Speed, crank, crystal, or amphetamines _____ 
g. Ecstasy or MDMA _____ 
h. MDA or Adam _____ 
i. Ethylchloride _____ 
j. Molly_____ 
k. Heroin ______ 
 
 


 
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APPENDIX VI: AMC DATA AND SAFETY MONITORING PLAN 
(Version 9.0  October 6, 2020) 
 
Introduction 
The AIDS Malignancy Consortium (AMC) Data and Safety Monitoring Plan (DSMP) outlines the 
measures employed by the group to monitor the safety of participants and ensure the data validity 
and integrity for all clinical trials it conducts. This includes methods to: 1) monitor the progress of 
trials and the safety of participants; 2) comply with regulatory requirements for adverse event (AE) 
reporting; 3) processes for trial termination or temporary suspension and major modifications; and 
4) plans for ensuring data accuracy and protocol compliance. As the AMC conducts protocols of 
varying research phase, region of conduct (which may include trials conducted in the U.S., 
international sites, or both), IND sponsor (AMC investigator, CTEP, or industry-sponsored) and 
clinical data entry system use, this plan addresses broad processes applying to the range of trial 
designs and requirements. Refer to the individual AMC protocol to identify the applicable study 
characteristics for the relevant requirements described in this plan. 
Monitoring the Progress of Trials and the Safety of Participants 
Routine and expedited AE reporting 
All AMC protocols that collect safety data adhere to the National Cancer Institute (NCI), Cancer 
Therapy Evaluation Program (CTEP) Guidelines: Adverse Event Reporting Requirements 
(https://ctep.cancer.gov/protocolDevelopment/adverse_effects.htm), as applicable to the clinical 
protocol. AEs are to be recorded in the source documents, assessed by a clinical investigator for 
the AE reporting criteria, and promptly reported in the clinical data entry system as required by 
each protocol. For AMC trials conducted under a CTEP IND and AMC trials conducted within the 
U.S., all AEs that meet the NCI’s expedited reporting requirements are reported to the NCI via the 
CTEP Adverse Event Reporting System (CTEP-AERS) web application, either directly or through 
integration with Medidata Rave where this system is employed for AMC protocols. Use of this 
system ensures notification to the protocol chair and Investigational Drug Branch (IDB) at CTEP, 
as required for trials conducted under a CTEP IND, and a uniform expedited reporting and safety 
review process for AMC domestic trials. The system may also be programmed to include sponsor 
notification as required for trials with industry support. Alternate process for expedited AE 
reporting to the AMC protocol chairs and AMC Operations and Data Management Center 
(ODMC) within the clinical data entry system (AdvantageEDC or Advantage eClinical only) may 
be defined in the protocol for select trials (international studies and The ANCHOR Study). 
All serious adverse events (SAEs) received by the AMC ODMC will be reviewed by the AMC 
medical monitor at the AMC ODMC for consideration of individual participant safety, safe trial 
conduct, data reporting quality for AE term selection, and appropriate application of the regulatory 
criteria for seriousness, expectedness, and relatedness to the investigational therapy. If alternate 
procedures are followed for SAE review, the process for adequate medical monitoring will be 
defined in the AMC protocol and the Transfer of Regulatory Obligations (TORO) with the sponsor. 
AMC medical monitor review includes review of the CTEP-AERS report before CTEP submission 
for IDB review (if applicable), or review of the SAE report in the data entry system for trials not 
using CTEP-AERS for expedited reporting. The IND sponsor or its designee will issue the 
determination as to whether the AE requires IND safety reporting to FDA as a serious and 
unexpected suspected adverse drug reaction (SUSAR). For protocols not conducted under an IND, 


 
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in the event of disagreement between the reporting physician and the AMC medical monitor 
regarding the relationship of the AE to the investigational agent(s) (i.e., determination of whether 
the attribution is unrelated or unlikely, or possible, probable, or definite), the AMC medical 
monitor will provide the final determination of the relationship. IND safety reporting to FDA is 
performed by CTEP for trials conducted under a CTEP IND; IND safety reporting is performed 
by the sponsor or sponsor’s designee (AMC ODMC or other party defined in the study agreement 
or TORO) for IND studies sponsored by AMC investigators or industry sponsors. 
Expedited reporting to the Institutional Review Board (IRB) 
The requirements for IRB review will be identified in the protocol section on ethical and regulatory 
obligations. All AMC trials initiated before September 1, 2020 and all international sites for all 
AMC studies are subject to local IRB review; only U.S. sites are subject to the NCI requirement 
to use a single IRB for protocols initiated on or after September 1, 2020. For trials subject to local 
IRB review, the site principal investigator is responsible for ensuring that expedited AE reports 
for its trial participants and any unanticipated problems that affect the local institution only are 
submitted to the local IRB of the reporting institution, per the local IRB’s requirements for such 
reporting. For studies reviewed by the single IRB, the protocol chair will render a determination 
as to whether a SAE or other problem constitutes a trial-wide unanticipated problem that requires 
reporting to that IRB, in accordance with its standards of procedure.  
To comply with investigator notification requirements for IND studies under 21 CFR 312.32 and 
312.55, IND safety reports from all trials the AMC conducts and reports from external sponsors 
investigating the same agents are made available to all investigators upon receipt from the sponsor 
or its designee, either via the password-protected section of the AMC Operations web site (AMC 
trials subject to local IRB review only) or the CTSU website (U.S. trials subject to single IRB 
review/CTEP IND agents). The site clinical investigator responsible for the applicable AMC 
protocol(s) is responsible for reviewing any IND safety reports received and documenting 
submission to the IRB of record (if required by local policy) within the timeline defined by the 
Clinical Trials Monitoring Branch (CTMB) audit guidelines. 
Procedures for monitoring trial progress and pharmacovigilance 
For trials using AdvantageEDC or Advantage eClinical for clinical data entry, the AMC ODMC 
provides on demand tabular listings of all reported AEs and SAEs on a participant level to the 
protocol chair and co-chair(s) for review via the password-protected section of the AMC 
Operations web site, www.AIDScancer.org. For trials using OPEN and Medidata Rave for clinical 
data collection, data listing will be made available using that system. Summary reports of AEs by 
frequency and relationship to the investigational agent(s) are provided to all AMC investigators 
and their staff It is the responsibility of each site to provide trial-specific AE listings to their 
respective IRB, if required by its policies. For blinded studies, the AE and SAE listings are 
reviewed and tabulated without treatment assignment. 
Accrual summaries for each AMC trial are updated nightly on the password-protected section of 
the AMC web site. The progress of each AMC trial is reviewed regularly by the protocol chair and 
also by the appropriate Scientific Working Group (SWG) during scheduled conference calls 
(monthly SWG calls and as required, protocol-specific monitoring conference calls). Summary 
accrual, summary AE, and individual SAE reports are provided to SWG leadership and protocol 
chairs to monitor participant safety during these monthly calls. 


 
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The AMC medical monitor reviews listings of all reported AEs on a quarterly basis for assuring 
compliance with the protocol requirements for AE reporting and the identification of any safety 
concerns (individual AE or increased frequency/severity of expected AEs) for the agents under 
investigation. Findings from these reviews are communicated to the protocol chairs and all AMC 
investigators, and posted to the AMC Operations web site. 
Data and Safety Monitoring Board Review (DSMB) review 
The AMC has formed an independent Data and Safety Monitoring Board (DSMB) for AMC trials 
and for the ANCHOR Study. As required by NCI policy, the AMC requires DSMB review for all 
phase III randomized trials. All other clinical trials that the AMC initiates will be reviewed by the 
AMC ODMC and AMC Statistical Center during protocol development to issue a recommendation 
as to whether the study requires DSMB oversight, which will require the approval of the AMC 
Executive Committee. This determination will be based on the phase of the study, experimental 
design, risk posed by the investigational approach, extent of data available on the safety of an 
investigational agent, risk posed by the natural course of the health condition under research, and 
the categories of vulnerable populations involved. The involvement of a DSMB in reviewing an 
AMC protocol will be identified in each clinical protocol as approved by CTEP and, as applicable, 
required by the IRB of record. 
Regarding the composition of the AMC DSMB, voting members usually include physicians, 
statisticians, an ethicist, and a patient advocate. All voting members have no other affiliation to 
the AMC and are appointed by the AMC Executive Committee with the approval of the OHAM 
Director. Nonvoting members are the AMC group statistician, the protocol statistician, an AMC 
ODMC staff member, two representatives (normally a clinician or statistician) from CTEP, and 
the grant program directors from the NCI Office of HIV and AIDS Malignancy (OHAM). 
The DSMB reviews all applicable AMC studies in accordance with the National Cancer Institute’s 
Policy for Data and Safety Monitoring. Confidential reports of all trials under review are prepared 
by the AMC group statistician with support from the AMC ODMC. A written report containing 
the current status of each trial monitored, and when appropriate, any toxicity and outcome data, 
are sent to DSMB members by the AMC ODMC within the timelines specified by the DSMB 
charter. This report addresses specific toxicity issues and any other concerns about the conduct of 
the trial, as defined by the protocol plan for DSMB review. The report may contain information 
for the DSMB to render determinations for participant safety, early trial termination, results 
reporting, or continuing accrual or follow-up. 
The results of each DSMB meeting are summarized in a formal report sent by the DSMB chair to 
the AMC group chair and AMC ODMC. The DSMB report contains recommendations on whether 
to close each study reviewed, whether to report the results, and whether to continue accrual or 
follow-up. A primary recommendation (e.g., continue with no change; recommended or required 
modification; stop) must be included in the document. The group chair or designee is then 
responsible for notifying the protocol chair and relevant SWG chair before the recommendations 
of the DSMB are carried out. In the unlikely event that the protocol chair does not concur with the 
DSMB, then the OHAM program directors and the NCI division director or designee must be 
informed of the reason for the disagreement. The protocol chair, relevant SWG chair, group chair, 
DSMB chair, and NCI division director or designee will be responsible for reaching a mutually 
acceptable decision about the study. CTEP approval of a protocol amendment will be required 
prior to any implementation of a change to the study. 


 
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Following a DSMB meeting, the DSMB’s recommendations are provided to all AMC investigators 
and staff. It is each site principal investigator’s responsibility for conveying this information to its 
local IRB as relevant for its protocol participation. For trials reviewed by a single IRB, the AMC 
ODMC will support notification to the IRB as required per its procedures. 
Cohort trial reviews not subject to DSMB review 
For phase I dose escalation trials, dose escalation (or dose de-escalation) is based on the rules in 
the protocol and the protocol chair, AMC medical monitor, and protocol statistician determine 
whether these criteria have been met based on a review of all safety data for the protocol-defined 
evaluation period. If applicable for phase II trials, stopping the trial for toxicity or efficacy, or 
suspending enrollment pending observation of responses in a multi-stage phase II trial, is based on 
meeting criteria stated in the protocol, and the protocol chair, AMC medical monitor, and protocol 
statistician determine whether these criteria have been met. 
Plans for Assuring Compliance with Requirements Regarding AE Reporting 
The protocol chair, AMC group chair, and the AMC ODMC share responsibility in assuring that 
participating investigators comply with applicable regulatory and protocol requirements for AE 
reporting. The AMC site principal investigator certifies compliance with NCI and FDA 
requirements for trial conduct by signing the site subaward agreement for the grant and the AMC 
Adherence Statement for site membership; clinical investigators also certify compliance in 
completing the protocol signature page for each protocol active at the site, and Form FDA-1572 
for CTEP investigator registration, and also for AMC IND studies sponsored by AMC 
investigators or industry sponsors. Protocol compliance with AE identification, assessment and 
reporting requirements is assessed by the AMC ODMC using several methods: 1) programmed 
system checks and messages to instruct the site to complete routine and/or expedited reporting 
when certain criteria are reported in the clinical data entry system; 2) programmed data reports 
provided to the protocol chairs that identify reports requiring expedited AE reporting; 3) remote 
review of data entry or data reports to ensure compliance with protocol and NCI AE reporting 
requirements; 4) AMC medical monitor review described in the section above; and, 5) routine site 
audits by reviewing the site’s source documentation. 
The clinical data entry systems used for AMC studies include the Oncology Patient Enrollment 
Network, OPEN for enrollment, and Medidata Rave for clinical data entry for enrolled 
participants; trials activated before September 1, 2020 or that involve only AMC international sites 
may be reported in AdvantageEDC/Advantage eClinical, a web-based data entry and enrollment 
system. These data entry systems are programmed to notify the site investigator, protocol chair, 
AMC medical monitor, and AMC ODMC via email in the event that a site reports an AE that 
meets expedited reporting criteria to NCI and/or FDA. Additional reporting conditions may be 
programmed depending on the sponsor reporting requirements of a given protocol (e.g., adverse 
events of special interest [AESI]). If the site does not follow with an expedited report, the AMC 
ODMC contacts sites to request compliance with reporting requirements. Additionally, the 
protocol chair, AMC ODMC, and the AMC medical monitor review reported AEs on a routine 
basis to identify AEs reported by sites that require expedited reporting. The protocol chair, AMC 
SWG chairs, AMC group chair, and IND sponsors have general oversight for assuring that routine 
and expedited adverse reporting requirements are met by the responsible parties. 
For studies monitored by CTEP using the Data Mapping Utility (DMU), cumulative protocol- and 
patient-specific data will be submitted weekly to CTEP electronically via the DMU. For trials 


 
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monitored by the NCI’s Clinical Data Update System (CDUS), AE information is transmitted 
electronically to NCI on a quarterly basis. For trials monitored by NCI’s Clinical Trials Monitoring 
Service (CTMS), AE information is transmitted electronically to NCI every two weeks. 
Plans for Assuring that any Action Resulting in a Temporary or Permanent Suspension of 
an NCI-Funded Clinical Trial is Reported to the NCI Grant Program Director Responsible 
for the Grant 
In the event that temporary or permanent suspension of a trial, or major modification to the 
protocol is under consideration, the protocol chair will convene the AMC ODMC, AMC Statistical 
Center, and SWG chair by conference call to discuss the options. Suspension actions will also be 
reviewed by the AMC Executive Committee for program oversight and direct communication of 
the action with the OHAM program directors. For phase III trials, closure decisions are typically 
rendered by the AMC DSMB; if the trial in question is under AMC DSMB oversight but rendered 
by the AMC investigators, the AMC DSMB will be notified of the suspension and the reason. For 
phase I and II trials, the protocol chair also has the option of asking the DSMB to review the study. 
The AMC ODMC will inform the CTEP Protocol Information Office (PIO), with copy to OHAM 
Directors, when studies are temporarily or permanently closed. In the event of major trial 
modification, CTEP must approve all protocol amendments prior to distributing to the AMC sites. 
Plans for Assuring Data Accuracy and Protocol Compliance 
All study data for AMC clinical trials are entered directly by AMC clinical site staff into the 
applicable clinical data entry system for the trial. During data entry, the system performs validation 
checks on many fields and performs consistency checks between select fields. Range checks are 
placed on each field to eliminate entry of out-of-range values. Edit check programs are run on the 
database on a set schedule to identify and resolve inconsistencies between forms or data collected 
at different points in time. Submitted data entry forms are reviewed for compliance with the 
protocol and data entry instructions according to the AMC ODMC’s standards for data quality 
processes. AMC ODMC staff routinely interacts with site staff to resolve any data submission 
problems. 
In accordance with NCI guidelines, the AMC ODMC conducts audits at the AMC sites to evaluate 
compliance with regulatory issues, and to review data for specific cases by checking source 
documents. These reports are sent to the site principal investigator and to the NCI. In the event 
that major violations are identified, sites are asked to provide a written corrective and preventative 
action plan to correct deficiencies. If needed, a repeat site audit is conducted. In the event that a 
site does not correct deficiencies in a pre-determined time frame, the AMC Executive Committee 
has the option to implement remedial action(s) for the site. Possible actions include, but are not 
limited to, suspending enrollment of new patients to AMC trials until deficiencies are corrected; 
recommending a decrease in funding to the site; and requiring specific training for site 
investigators or staff members. 
 
 


 
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APPENDIX VII: PARTICIPANT INSTRUCTIONS FOR APPLYING IMIQUIMOD 5% 
CREAM 
You have been prescribed to receive topical imiquimod 5% cream three times a week at night for 
8 weeks. You will receive imiquimod for up to 16 weeks if you are tolerating the treatment. Please 
follow the directions below for applying the cream you have been given. 
The imiquimod dose will be 750 mg of cream per week, administered as one single use sachet (250 
mg) applied 3 times per week. You have been given at least 24 sachets with 250 mg of imiquimod 
for 8 weeks of treatment. If you have perianal HSIL, you will be given extra sachets. 
When applying the cream, a latex glove or other protective covering must be used on your hand. 
Apply the imiquimod cream at night before bed or before your longest period of rest once a day. 
You will place half of the contents of the packet onto your right index finger and insert the cream 
approximately one inch into the anus on the right side, rubbing from side to side as far along the 
right side as your finger can reach. 
Then place the remaining cream on the left index finger, and insert the cream one inch into the 
anus on the left side, rubbing from side to side as far along the left side as your finger can reach. 
You may find it is easier to lie on your side then turn over. Or, you may crouch, or place your right 
and left leg alternately on the toilet or chair. Use whatever position allows you to reach into the 
anus. 
If perianal HSIL is present, the study clinician will tell you to apply one additional packet of cream 
using a gloved finger to the perianus. When applying the cream to the perianus, you will place half 
of the contents of the packet onto your right finger and rub the cream on the right side of your 
perianus. You will place the remaining cream on the left index finger and rub the cream on the left 
side of your perianus. 
Please speak to the study clinician if you have any side effects that are more than mild. The study 
clinician will tell you if you should reduce the dose. 
If you miss a dose please apply the next dose as scheduled. Do not try to make up for it by adding 
the contents of more than one sachet at your next dose. 
Store the packets of imiquimod at room temperature, away from sunlight. 
 
 


 
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APPENDIX VIII: PARTICIPANT INSTRUCTIONS FOR APPLYING EFUDEX (5-
FLUOROURACIL) 5% CREAM 
You have been randomized to receive topical 5-fluorouracil (5-FU) cream for 8 weeks. You will 
receive 5-FU for up to 16 weeks if you are tolerating the treatment. Please follow the directions 
below for applying the cream you have been given. 
You have been given a 40 gram tube of 5-FU cream. If you have also been given a syringe or 
applicator, you will join the syringe tip onto the end of the tube. You will squeeze out the cream 
to the marked line on the applicator (0.5mL of cream). Please continue to do this until you can 
confidently squeeze the correct volume of cream. If you do not have a syringe or applicator, you 
may also measure out one inch of cream to apply. 
Using gloves or a finger cot, squeeze one half the amount of cream (0.25mL) from the syringe (if 
using) onto your right index finger. 
You will then insert the cream approximately one inch into the anus on the right side, rubbing from 
side to side as far along the right side as your finger can reach. 
Then place the remaining cream (0.25mL) on the left finger, and insert the cream one inch into the 
anus on the left side, rubbing from side to side as far along the left side as your finger can reach. 
You may find it is easier to lie on your side then turn over. Or, you may crouch, or place your right 
and left leg alternately on the toilet or a chair. Use whatever position allows you to reach into the 
anus. 
Clean any excess cream from the outside of the anus unless you are treating the outside area. The 
study clinician will let you know if you should treat the outside. 
The 5-FU cream will be inserted twice a day (it does not need to be exactly 12 hours apart) for 5 
days in a row. You will then stop for 9 days. Each cycle of 5-FU consists of 14 days. You will then 
repeat the cycle continually for 16 weeks, or 8 cycles. 
If you miss a dose please apply the next dose as scheduled. Do not try to make up for it by using 
extra 5-FU cream at your next dose. 
Please speak to the study clinician if you have any side effects that are more than mild. The study 
clinician will tell you if you should reduce the dose. 
 
 


 
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APPENDIX IX: REGULATORY STATUS OF INVESTIGATIONAL AGENTS 
This appendix serves to outline the regulatory status of the topical drugs and medical devices that 
will be used in the treatment of anal high grade squamous intraepithelial lesions for the ANCHOR 
Study: Anal Cancer/HSIL Outcomes Research Study. Questions pertaining to this justification for 
the regulatory status of these agents may be directed to the ANCHOR Study Chair, Dr. Joel 
Palefsky, and the ANCHOR Data Management Center via email at anchordmc@emmes.com. 
IND Exempt Topical Treatments 
Topical imiquimod 5% and topical 5-fluorouracil 5% meet all of conditions of exemption from the 
requirement for an Investigational New Drug (IND) application at 21 CFR 312.2(b). This 
investigation will not be used to support a change in the labeling, marketing authorization, 
advertising, or the indications for use for any of the study agents. ANCHOR Study investigators 
will use a supply of these drugs that are sourced as legally marketed in the U.S. Participants will 
not be charged for any of the agents. The protocol will be conducted at participating U.S. AMC 
member sites in compliance with the requirements for informed consent and institutional review 
set forth in 21 CFR 50 and 56, respectively. The investigation will not involve an exception from 
informed consent (21 CFR 50.24). 
By review of an IND application for a related AMC trial of topical imiquimod and 5-fluorouracil 
creams, the Food and Drug Administration (FDA) determined that these agents are exempt from 
the requirement for an IND when used in the treatment of anal HSIL in persons with HIV infection. 
(Protocol AMC-088, PI: Dr. Timothy Wilkin, IND 122,224 for Efudex and Zyclara 2.5%). The 
sponsor-investigator believes that the exemption status of the agents used in protocol AMC-088 
apply to the ANCHOR Study as the agents will be used for the same indication for use, patient 
population, and route. The use of the 5% imiquimod cream in this trial as compared to the 2.5% 
strength imiquimod cream used in AMC-088 is addressed by a less frequent dosing schedule in 
the ANCHOR Study and does not significantly increase the risks or decrease the acceptability of 
the risks associated with the use of imiquimod. The FDA has concurred with this determination of 
IND exemption for these agents in discussions related to this trial. 
IDE Exempt Study Devices 
By review of this investigation, FDA has confirmed that the classes of medical devices used in the 
ANCHOR Study, described in protocol Section 7.3.3, are exempt from the requirement for an 
Investigational Device Exemption (IDE). A copy of FDA’s exemption letter is available upon 
request. 
As a Federally-funded cancer research initiative, the ANCHOR Study protocol chairs and the 
collaborating consortium, the AMC, have no intention to demonstrate safety and effectiveness of 
the study devices for clearance or approval in the prevention of cancer, nor will the results of this 
study be submitted to the FDA to support a change in the labeling or the marketing of the study 
devices. 
Redfield infrared coagulator 
FDA regulates the Redfield Infrared Coagulator as an endoscopic electrosurgical unit (21 CFR 
876.4300) intended for use in electrosurgical procedures. It is a class II medical device 
(intermediate risk) that FDA has cleared for use in the treatment of hemorrhoids, tattoo removal, 
chronic rhinitis, genital condyloma (condyloma acuminata), and general warts. It is 


 
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contraindicated in the use of invasive cancer, vulvar or vaginal intraepithelial neoplasia, and in 
patients with severe photosensitivity reactions. 
Hyfrecator and electrosurgical devices 
Electrosurgical devices are class II medical devices that are regulated as electrosurgical cutting 
and coagulation devices intended to remove tissue and control bleeding by use of high-frequency 
electrical current (21 CFR 878.4400). Specific indications for use vary by device. These devices 
are cleared for use in general and plastic surgery, in applications including dermatology, 
gynecology, and proctology, including the removal of hemorrhoids, condyloma, Bowen’s disease, 
basal cell carcinoma, and squamous cell carcinoma. 
Laser surgical devices 
The generic type of medical devices regulated under 21 CFR 878.4810 includes laser surgical 
instruments for use in general and plastic surgery and in dermatology, which are regulated as class 
II medical devices. This classification includes devices intended to cut, destroy, or remove tissue 
by light energy emitted by carbon dioxide, or argon lasers intended to destroy or coagulate tissue 
in dermatology by light energy emitted by argon. Laser surgical devices in this classification are 
indicated for use to cut, destroy, remove, or coagulate tissue, generally soft tissue, for general 
surgical purposes in medical specialties such as general and plastic surgery, dermatology/aesthetic 
surgery, podiatry, otolaryngology, gynecology, neurosurgery, orthopedics (soft tissue), dental and 
oral surgery, and dentistry. Legally marketed carbon-dioxide lasers that are indicated for use in 
general surgical procedures, including the excision or ablation of tissue, are permitted for use on 
this trial. 


 
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APPENDIX X: ANCHOR HEALTH-RELATED SYMPTOM INDEX 
 
Below is a list of statements that other people diagnosed with anal HSIL (pre-cancer lesion in the anus) and treated or actively 
monitored have said are important. Please check the box to select your answer based on your experiences in the past 7 days. 
 
PHYSICAL SYMPTOMS 
Check if 
Not 
Applicable 
Not at 
all 
0 
A little 
bit 
1 
Somewhat 
2 
Quite a 
bit 
3 
Very 
much 
4 
1. I have anal pain 
2. I have pain other than anal pain 
3. I have pain during bowel movements 
4. I have constipation 
5. I have bleeding from the anus 
6. I have itching in or around the anus 
7. I have discharge (wetness) in my anal area 
8. I have burning sensations in the anal area 
9. I have urgency for bowel movements 
PHYSICAL IMPACTS 
Check if 
Not 
Applicable 
Not at 
all 
0 
A little 
bit 
1 
Somewhat 
2 
Quite a 
bit 
3 
Very 
much 
4 
10. I have problems with my physical ability to move around 
11. I have problems with sitting 
12. I have problems completing daily household chores (e.g., 
cleaning, cooking, laundry, house maintenance) 
13. I have problems taking care of myself (e.g., bathing, 
dressing, shaving) 
14. I have problems participating in leisure activities (e.g., 
watching television, relaxing) 


 
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15. I have problems participating in social activities (e.g., going 
out to eat, visiting friends) 
 
16. I have problems with work productivity 
PSYCHOLOGICAL SYMPTOMS 
Check if 
Not 
Applicable 
Not at 
all 
0 
A little 
bit 
1 
Somewhat 
2 
Quite a 
bit 
3 
Very 
much 
4 
17. I have difficulty concentrating 
 
18. I have a decreased enjoyment of anal sexual activity 
19. I have a decreased enjoyment of forms of sexual activity 
other than anal sexual activity 
20. I have a decreased desire for anal sexual activity 
21. I have a decreased desire for forms of sexual activity other 
than anal sexual activity 
22. I am worried about my condition getting worse 
23. I have anxiety 
24. I have depression 
25. I have problems with my intimate relationships 
 
 
 


 
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APPENDIX XI: ANCHOR HEALTH-RELATED SYMPTOM INDEX – SPANISH LANGUAGE VERSION 
ÍNDICE DE SÍNTOMAS RELACIONADOS CON LA SALUD ANCHOR 
 
A continuación presentamos una lista de afirmaciones que otras personas diagnosticadas con HSIL (lesión precancerosa en el ano) y 
que han sido tratadas o monitoreadas activamente han dicho que son importantes. Por favor seleccione la casilla con su respuesta 
basándose en sus experiencias en los últimos 7 días.  
SÍNTOMAS FÍSICOS 
 
Escoja si 
no aplica 
Nada 
0 
Un poco 
1 
Algo 
2 
Bastante 
3 
Muchísimo 
4 
1. Tengo dolor anal  
 
 
 
 
 
 
2. Tengo dolor pero no en el área anal  
 
 
 
 
 
 
3. Tengo dolor cuando entro al baño a defecar 
 
 
 
 
 
 
4. Tengo estreñimiento  
 
 
 
 
 
 
5. Tengo sangrado del ano  
 
 
 
 
 
 
6. Tengo picazón en el ano o alrededor del ano  
 
 
 
 
 
 
7. Tengo una secreción (mojado) en el área anal  
 
 
 
 
 
 
8. Tengo sensación de ardor en el área anal   
 
 
 
 
 
 
9. Siento urgencia de entrar al baño a defecar 
 
 
 
 
 
 
IMPACTOS FÍSICOS  
 
Escoja si 
no aplica 
Nada 
0 
Un poco 
1 
Algo 
2 
Bastante 
3 
Muchísimo 
4 
10. Tengo problemas con mi capacidad física para 
moverme   
 
 
 
 
 
 
11. Tengo problemas para sentarme 
 
 
 
 
 
 
12. Tengo problemas manteniendo la casa (por ej., 
limpiar, cocinar, preparar el café, administrar la casa) 
 
 
 
 
 
 
13. Problemas con mi arreglo personal (por ej., 
bañándome, vistiéndome, afeitándome) 
 
 
 
 
 
 
14. Tengo problemas participando en actividades de relajo 
(por ej., mirar televisión, relajarme) 
 
 
 
 
 
 


 
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15. Tengo problemas participando en actividades sociales 
(por ej., salir a comer, visitar amigos)   
 
 
 
 
 
 
16. Tengo problemas siendo productivo en el trabajo 
debido a mis síntomas  
 
 
 
 
 
 
SÍNTOMAS PSICOLÓGICOS  
Escoja si 
no aplica 
Nada 
0 
Un poco 
1 
Algo 
2 
Bastante 
3 
Muchísimo 
4 
17. Tengo dificultad concentrándome  
 
 
 
 
 
 
18. Me ha disminuido el disfrute de la actividad sexual 
anal   
 
 
 
 
 
 
19. Me ha disminuido el disfrute de cualquier forma de 
actividad sexual diferente a la actividad sexual anal  
 
 
 
 
 
 
20. Me ha disminuido el deseo por la actividad sexual anal   
 
 
 
 
 
21. Me ha disminuido el deseo por formas de actividad 
sexual diferentes a la actividad sexual anal  
 
 
 
 
 
 
22. Me preocupa que mi enfermedad empeore 
 
 
 
 
 
 
23. Tengo ansiedad  
 
 
 
 
 
 
24. Tengo depresión 
 
 
 
 
 
 
25. Tengo problemas con mis relaciones íntimas 
 
 
 
 
 
 


 
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APPENDIX XII: PATIENT VERSION OF THE EASTERN COOPERATIVE GROUP PERFORMANCE STATUS 
Questionnaire only required for A-HRSI Scale Responsiveness Substudy (completed February 2020) 
 
Toxicity/Grade 
Original Clinician Language 
Patient Language Adaptation 
ECOG Performance Status 
 
 
0 
Fully active, able to carry out all predisease performance 
without restriction 
I am fully active and able to carry out activities the same 
as before my cancer diagnosis, without any 
restrictions 
1 
Restricted in physically strenuous activity but ambulatory 
and able to carry out work of a light or sedentary 
nature, eg, light house work, office work 
I have difficulty with physically strenuous activity but I 
am able to walk and carry out work that is light or 
based in one location; such as light house-work or 
office-work 
2 
Ambulatory and capable of all selfcare but unable to carry 
out any work activities; up and about more than 50% 
of waking hours 
I can walk and take care of myself, but I am not able to 
carry out work activities; I am up and about more 
than half the hours that I am awake 
3 
Capable of only limited selfcare, confined to bed or chair 
more than 50% of waking hours 
I am capable only of limited self-care and spend more 
than half of the hours that I am aware in bed or in a 
chair 
4 
Completely disabled. Cannot carry on any selfcare; totally 
confined to bed or chair 
I am completely disabled, cannot carry on any self-care, 
and am totally confined to a bed or chair 
 


 
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APPENDIX XIII: PARTICIPANT GLOBAL IMPRESSION OF CHANGE (PGIC) 
Questionnaire only required for A-HRSI Substudy (completed February 2020) 
 
 
Very 
much 
worse 
(-3) 
Moderately 
worse 
(-2) 
A little 
worse 
(-1) 
No 
change 
(0) 
A little 
better 
(+1) 
Moderately 
better 
(+2) 
Very 
much 
better 
(+3)  
Since you last 
completed a 
questionnaire, 
how would 
you rate your 
OVERALL 
QUALITY 
OF LIFE? 
 
 
 
 
 
 
 
 
 

ICF_001.pdf: 
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SUMMARY OF CHANGES 
 
ANCHOR Study: Anal Cancer Prevention Study 
Anal Cancer/HSIL Outcomes Research Study 
Version 15.0 
 
NCI Protocol #: AMC-A01 
Local Protocol #: ANCHOR 
 
NCI Version Date: 22JAN2021 
Protocol Date: 22JAN2021 
 
 
I. 
Scientific and Substantive Changes 
# 
Section 
Description of Change 
1.  
Additional 
Studies, Health-
Related Quality 
of Life Study, 
Attachment 1 
The ICF was revised to implement the A-HRSI instrument as a formal 
QoL aim, among participants who opt in to complete this 
questionnaire. The questionnaire will be given to up to 500 
participants in English or Spanish at 8 time points. Participants will 
be paid $25 for each completed time point. 
2.  
Additional 
Studies, Oral 
and Anal 
COVID 
Infection 
The ICF was revised to add an optional study for investigating SARS-
CoV-2 in oropharyngeal and anal swab samples from PLWH being 
screened for the ANCHOR study at 2 time points. Participation is 
limited to 5 centers: Laser Surgery Care, Anal Dysplasia Clinic 
MidWest, University of Miami (including Jackson Memorial 
Hospital), Grady Memorial Hospital, and University of California, 
San Francisco; centers not participating in this substudy must not 
offer this optional study in the local informed consent form. 
Participants will be paid $10 for each completed time point. 
3.  
Are there any 
payments? 
Information shared with ClinCard was updated to remove protected 
health information (SSN) that is not added to the system. 
 
II. 
Administrative and Editorial Changes 
# 
Section 
Description of Change 
4.  
Footer 
The footer was updated to version 15.0, dated 22JAN2021. 
5.  
Who will see 
my medical 
information? 
The manufacturer name for topical medications was updated from 
Valeant Pharmaceuticals to Bausch Health. 


 
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MODEL INFORMED CONSENT FORM 
Study Title for Study Participants: 
ANCHOR: Anal Cancer Prevention Study 
 
Official Study Title for Internet Search on http://www.ClinicalTrials.gov: 
ANCHOR: Anal Cancer/HSIL Outcomes Research Study 
______________________________________________________________________________ 
What is the usual approach to preventing anal cancer? 
You are being asked to take part in this study because you are an adult with HIV infection and you 
may have anal high-grade squamous intraepithelial lesions (HSIL). Anal HSIL is tissue in the anal 
canal that has been damaged by human papillomavirus (HPV) and is at risk for turning into anal 
cancer. 
There is no standard approach for preventing anal cancer. Some doctors try to prevent anal cancer 
by looking for and treating anal HSIL. The treatments may vary. 
What are my other choices if I do not take part in this study? 
If you decide not to take part in this study, you have other choices. For example: 
• You may choose not to be screened or treated for anal HSIL. 
• You may choose to be screened or treated for anal HSIL outside of this study. 
Why is this study being done? 
This goal of this study is to see whether treating anal HSIL will prevent anal cancer compared with 
watching HSIL carefully without treatment (active monitoring). We will compare the number of 
people who develop anal cancer in both groups. We know that treating HSIL in the cervix can 
prevent cervical cancer in women. However, we don’t know if treating anal HSIL can prevent anal 
cancer. For this reason, most doctors in the United States don’t look for and treat anal HSIL. Anal 
cancer is more common in women and men with HIV than in the general population. If caught 
early, anal cancer is often cured, but treatment-related side effects can be severe and some can last 
for several years. Anal HSIL occurs before cancer develops and is quite common in HIV-infected 
people (between 10% and 50% have anal HSIL). Most cases of anal HSIL will never become 
cancer. 
What are the study groups? 
5,085 men and women will take part in this study. To be in the study, you need to be 35 years of 
age or older, have HIV infection, have not been treated for anal HSIL in the last 6 months, and 
have never had cancer of the anus, vulva, vagina, or cervix. If you have anal HSIL and you meet 
the other eligibility criteria, you can join this study. A computer will assign you by chance (like 
tossing a coin) to join one of the two study groups: Active Monitoring (Group 1) or Treatment 
(Group 2). This is called randomization. You have an equal chance (50/50) of being assigned to 
either group. We do not know if one group is better than the other and that is why you are being 
assigned to one of the groups. 
• Group 1 (Active Monitoring) will not have any HSIL treatment 


 
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• Group 2 (Treatment) will have treatment of anal HSIL chosen by you and your study 
provider 
Another way to find out what will happen to you during this study is to read the chart below. Start 
reading at the left side and read across to the right, following the lines and arrows. 
 
 
 
 
 
 
 
 
 
 
 
 
 
What are the advantages of being in Group 1 or Group 2? 
The researchers estimate (an educated guess) that the risk of developing anal cancer if your HSIL 
is not being treated is about 1 in 100 over the course of the study.   
Advantages to Group 1 (Active Monitoring) 
• Treating anal HSIL may not be better than active monitoring to reduce the risk of anal cancer, 
and if so, you would have avoided uncomfortable treatments that provide little benefit to you. 
Advantages to Group 2 (Treatment) 
• If you are in the treatment arm your HSIL may be reduced in size or removed entirely. 
How long will I be in this study? 
The researchers plan to recruit volunteers for the study over a period of three years or more. We 
will follow all participants for up to five years after the last participant joins the study. Participants 
who join the study in the first year may be in the study for up to eight years or more. If anal cancer 
is found at any time during the study, your participation in the study will end, and you will 
be immediately referred for the necessary treatment. If at any time during the study, the results 
show that treatment is helpful in preventing anal cancer, and you still have anal HSIL, you will be 
referred for treatment. 
What extra tests and procedures will I have if I take part in this study? 
• Most of the exams, tests, and procedures you will have are part of the usual approach for anal 
HSIL among those doctors who routinely choose to screen and treat this disease. Samples   
collected only for experimental laboratory research studies are shown in bold. All research 
samples are required for this study. The samples will be tested at the end of the study for 
different factors related to HPV, anal HSIL and anal cancer. The results of the research tests 
will not be given to you or your doctor. 
You agree 
to take 
part in the 
study 
If eligible, you will 
be enrolled in the 
study. 
 
The computer will 
randomly put you 
in a study group. 
Group 1 
Active 
Monitoring 
Group 2 
Treatment 
We perform 
tests to see if 
you are 
eligible for the 
study 
If not eligible, 
you will not be 
enrolled in the 
study. 


 
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Before you begin the study: 
• You will take a brief questionnaire (Attachment 3) to be sure the information in this consent 
form is clear. The questionnaire will take about five minutes to complete. 
• You will have a screening visit. During this visit, we will ask you about your medical history 
and perform tests to confirm that you can be in this study. We will ask you to bring in your 
HIV medication(s). You should avoid receptive anal intercourse, enemas, or inserting anything 
in your anus for 24 hours before all study visits. 
• A physical exam including a genital exam. 
• Three anal swabs. The researcher will put a swab (similar to a Q-tip™) into your anus. The 
end of the swab will be rubbed against the skin inside the anus. This swab is for anal cytology 
(Pap test) to test for abnormal cells in the anus. The leftover cells from the first swab, and 
all of the cells from the second and third swabs are for future research studies. 
• An anal/rectal exam with a finger. 
• An anal exam called high resolution anoscopy (HRA) that uses a special microscope to find 
abnormal areas of the anus. A lubricated plastic anoscope will be inserted into your anus. Then, 
a piece of gauze moistened with acetic acid (i.e., vinegar) is placed in your anus so that 
abnormal areas will show up. The researcher puts the anoscope back into your anus. A 
colposcope (a machine with a magnifying lens) is used to see the skin inside the anus. Iodine 
may be used to help make lesions show up. 
• Anal biopsies: These are pieces of anal skin about the size of a sesame seed that are cut out to 
look for HSIL under a microscope. Your provider may inject a small amount of local anesthesia 
(numbing medicine) before the biopsy. Your biopsy may be re-read by another doctor at 
another institution to check your diagnosis. If the provider finds anal cancer, we will ask you 
to return to the clinic for an extra anal biopsy for future research studies before you stop 
the study. 
• Blood will be drawn (about 4 teaspoons) to check your blood cell counts, to test for HIV unless 
we have a copy of your HIV test results, and for future research studies. 
• A pregnancy test, if you are a woman who could become pregnant. 
If you are not eligible for the study, with your permission, we may use the information and samples 
that we have collected for future research. If you are eligible then you will be randomized at your 
next visit to Group 1 (Active Monitoring) or Group 2 (Treatment). A study calendar that shows 
what will be done during the study for both groups is attached. 
For all participants who enroll in the study: 
• We will ask about your medical history and sexual behaviors at your Visit 1 
• At each 6-month visit you will have three anal swabs for anal cytology and future research 
studies 
• Digital anal/rectal exam, and HRA 
• Depending on which group you are in, you may or may not have one or more anal biopsies at 
any one visit. A part of your biopsies may also be used for future research studies. 


 
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• 1½-3 teaspoons of blood will be collected for future research studies 
• We will call you between visits to ask about any side effects from study procedures, answer 
questions about the study, and what the study team can do to make future study visits easier. 
If the provider finds anal cancer at any visit, we will ask you to return to the clinic for an 
extra anal biopsy for future research studies before you stop the study. 
If you are in Group 1 (Active Monitoring): 
• You will have biopsies of your anal HSIL every year. Your provider may choose to perform 
biopsies every 6 months or see you in between 6-month visits at any time if there is any concern 
that cancer has developed. 
• It is important that you do not receive any other treatment for anal HPV-related disease outside 
of this study. If you have anal warts and want to have them treated, the study doctors will treat 
them and you may continue in the study. 
• If at any time during the study, the results show that treatment is helpful in preventing anal 
cancer, and you still have anal HSIL, you will be referred for treatment. 
If you are in Group 2 (Treatment): 
• The study doctor will talk to you about your treatment options for HSIL. Please see the 
attachment for the description of treatments available in this study and the follow-up required 
for each of these treatments. You will receive treatment as soon as you start the study. 
• You may have additional visits after treatments to watch for any side effects and extra biopsies 
to see how the treatment is working. You will have biopsies at least once per year and probably 
more often. 
• Participants receiving some of the treatments in the clinician’s office may have one additional 
biopsy for future research studies every 6 months, just before getting treatment. 
• During the study, the study doctor may try different treatments if your HSIL does not go away 
or comes back. 
Treatments used in the Treatment Arm include: 
• Imiquimod 5% cream, which is also known as Aldara™. This treatment is an approved 
treatment of genital warts (skin growths caused by HPV). You apply this cream yourself to the 
inside or outside of your anus, depending on where your HSIL is found. 
• Topical 5-fluorouracil (5FU) cream, also known as Efudex™. 5FU cream is approved for 
treating actinic keratosis (precancerous conditions of the skin not related to HPV), and for 
superficial basal cell carcinoma (a skin cancer not related to HPV). You apply this cream 
yourself to the inside or outside of your anus, depending on where your HSIL is found. 
• Imiquimod and 5FU are experimental when used to treat anal HSIL. 
• Infrared coagulation, or IRC, which is a method that uses a device to destroy or remove a 
specific area of skin using a focused beam of light. IRC is approved for treating anal warts and 
removing tattoos. It is experimental for treating anal HSIL. 


 
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• Electrocautery, also known as hyfrecation, involves the use of an electric probe that is used to 
destroy or remove a specific area of skin. A laser may also be used. These are approved by the 
FDA for use in surgical procedures. 
• Surgery in the operating room to cut out the HSIL (this is an uncommon approach (less than 
10% of the time) and is only used if none of the other treatments can be used. 
What possible risks can I expect from taking part in this study? 
If you choose to take part in this study, there is a risk that: 
• You may lose time at work or home and spend more time in the hospital or doctor’s office than 
usual. 
• You may be uncomfortable being asked sensitive or private questions  
• The study treatments for anal HSIL may not be better, and could possibly be worse, than doing 
nothing for your anal HSIL. 
• The researchers estimate (an educated guess) that the risk of developing anal cancer if your 
HSIL is not being treated is about 1 in 100 over the course of the study. We do not know what 
the risk is among those whose anal HSIL is being treated. 
Anal swabs for cytology and research material: Putting a swab into the anus may cause some 
discomfort. Minor bleeding (less than a quarter of a teaspoon) occurs in some people due to the 
insertion of the swabs. The bleeding stops almost right away.  
High resolution anoscopy: Insertion of an anoscope will likely cause some discomfort. You may feel 
pressure and the urge to have a bowel movement. Putting acetic acid (vinegar) in the anal canal may 
cause minor burning and irritation. 
Anal biopsies: You may have pain with the anal biopsies. You may have some bleeding for up to 
a week after biopsies, especially when you have a bowel movement. There is a rare chance of very 
heavy bleeding that may require extra treatment. There is a very slight risk of infection (<1%). 
Contact the study clinic if you have symptoms of heavy bleeding or infection (fever, pain, redness, 
or swelling). 
Local anesthetic (numbing medicine): You may have a pinching or burning feeling from the shot 
of numbing medicine. There is a very slight chance of reaction to the numbing medicine including 
rash, flushing, rapid heartbeat, and dizziness. 
Blood drawing (venipuncture) risks: In many people, obtaining blood from a vein may cause some 
discomfort. This may include infection, bruising, and/or tenderness at the site where the blood is 
taken, and fainting or feeling faint. 
Questionnaire: You will be asked questions about any sex you have had recently. Some people 
may find these questions embarrassing. It is important that you answer these questions honestly. 
However, no one will force you to answer any questions if it is too uncomfortable for you. 
Additional risks for those in Study Group 1 – Active Monitoring 
COMMON 
You may be anxious if you are concerned that your HSIL may progress to cancer and you are not 
undergoing active treatment 


 
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Additional risks for those in Study Group 2- Treatment 
In addition to the side effects outlined above, people in Group 2 may also experience the possible 
side effects of the study treatments received. The tables below show the most common and the 
most serious side effects that researchers know about for each agent. There might be other side 
effects that researchers do not yet know about. If important new side effects are found, the study 
doctor will discuss these with you. 
Risks of topical imiquimod 5% 
COMMON, MILD TO MODERATE 
In 100 people receiving imiquimod, more than 20 and up to 100 may have: 
• Pain, burning, irritation, and itching 
• Redness 
• Swelling 
• Ulcerations (i.e., sores on the skin) and bleeding 
• Flaking and crusting of the skin 
• Allergic reactions near treatment area 
• Pain, redness and sores on skin near the treatment area (for example on the scrotum in men) 
 
OCCASIONAL, MILD TO MODERATE 
Of 100 people receiving imiquimod, from 4 to 20 may have: 
• Headache  
• Rash at other sites  
• Back pain 
• Sinus infection, nausea, fever, and flu-like illness 
 
RARE, AND SERIOUS 
Of 100 people receiving imiquimod, 3 or fewer may have: 
• The common skin reactions described above may be severe enough that your study provider 
may stop the medication for a while or try a lower dose of the medication. You may also need 
other treatments such as pain medications to treat this. 
 
Risks of topical 5-fluorouracil 5% (5-FU) 
COMMON, MILD TO MODERATE 
Of 100 people receiving 5FU, more than 20 and up to 100 may have: 
• Pain, burning, irritation and itching 
• Redness 
• Swelling 
• Ulcerations (i.e., sores on the skin) and bleeding 
• Flaking and crusting of the skin 
• Allergic reactions in the skin 
• Pain, redness and sores on skin near the treatment area (for example on the scrotum in men) 
• Scarring of the skin 
 


 
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OCCASIONAL, MILD TO MODERATE 
Of 100 people receiving 5FU, from 4 to 20 may have: 
• Emotional upset, trouble sleeping, irritability 
• Medicinal taste in your mouth 
• Low platelet counts, high white blood cells counts, and other blood count problems. 
• Loss of hair, and other rashes. 
• Red irritated eyes, and irritated nose 
• If you have had herpes infection in the past, it may cause it to return. 
 
RARE, AND SERIOUS 
Of 100 people receiving 5FU, 3 or fewer may have: 
• Extensive skin reactions. The common skin reactions described above may be severe enough 
that your study provider may stop the medication for a while or try a lower dose of the 
medication. You may also need other treatments such as pain medications to treat this. 
• There have been miscarriages (loss of a pregnancy), and one birth defect (a serious heart 
condition) in women who have used 5FU during pregnancy. 
 
Risks of infrared coagulation (IRC), laser, and electrocautery 
COMMON, MILD TO MODERATE 
Of 100 people receiving IRC/electrocautery/laser, 50 or more may have: 
• Slight pain during the treatment 
• Some spotting or drops of blood on toilet tissue paper for up to 2 weeks after the treatment.  
• Mild to moderate pain that may continue for up to 2 weeks after the treatment, usually well 
controlled with pain or anti-inflammatory medicines. There may be a change in the texture of 
the treated area for 1 to 2 weeks. 
 
RARE, SOME MAY BE SERIOUS 
Of 100 people receiving IRC/electrocautery/laser, 1 or fewer may have: 
• Fistula formation (a tunnel between two skin surfaces) 
• Severe bleeding (typically 7-10 days after the procedure) which may need additional 
treatment 
• Abscess (an infection under the skin resulting from the procedure) 
• Scarring, which could keep stool from passing normally 
• Pain that is severe and lasts longer than 2 weeks. 
Additional risks: If you have had herpes infection in the past, the treatment may cause it to return. 
Risks of surgical excision (cutting out the lesion), or treatment under anesthesia 
COMMON, SOME MAY BE SERIOUS 
Of 100 people receiving treatment under anesthesia, from 50 or more may have: 
• Bleeding after the procedure 
• Pain, sometimes severe for up to 2 weeks after the procedure 
• Difficulty having a bowel movement 
 


 
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OCCASIONAL, SOME MAY BE SERIOUS 
Of 100 people receiving treatment under anesthesia, fewer than 20 may have: 
• Changes in the coloring (more pigment or darkening, or less darkening) of their skin when 
treated for disease on the outside of the anus 
 
RARE, SOME MAY BE SERIOUS 
Of 100 people receiving treatment under anesthesia, 1 or fewer may have: 
• Abscess (an infection under the skin resulting from the procedure) 
• A tear or hole in the anus or rectum that may require surgery 
• A bad reaction to the anesthesia, up to and including death 
• Scarring, which could keep stool from passing normally 
 
If you have a biopsy or are treated with IRC, electrocautery, laser, or surgery, you should not have 
receptive anal sex for up to 2 weeks after treatment. This is to allow you to heal. You may have 
some bleeding with receptive anal sex until healing is complete. 
If you are selected to participate in Group 2 (Treatment arm), you may need more than one kind 
of treatment to treat your HSIL over the course of the study. It is possible that the doctors may not 
be able to remove all of the HSIL. It is possible that anal HSIL treatment may not prevent cancer. 
Reproductive risks: The study drugs used in this study could be very damaging to an unborn baby. 
• Female participants should not get pregnant while in this study. Women who could get 
pregnant should use at least one form of birth control during the study and for 3 months after 
stopping all study treatment. 
• Male participants should not father a baby while receiving topical study drugs (creams). Men 
who could father a child should use at least one form of birth control during treatment with 
topical study drugs and for 2 weeks after stopping all treatment. 
Check with the study doctor about what types of birth control, or pregnancy prevention, to use 
while in this study. Please tell the study doctor immediately if you become pregnant or if your 
partner becomes pregnant during this study. If you get treatment with 5-FU or imiquimod, your 
partner should wear a condom if you have receptive anal sex to avoid exposure to these drugs. 
Here are important points about side effects: 
• The study doctors do not know who will or will not have side effects. 
• Some side effects may go away soon, some may last a long time, or some may never go away. 
Here are important points about how you and the study doctor can make side effects less of a 
problem: 
• Tell the study doctor if you notice or feel anything different so they can see if you are having 
a side effect. 
• The study doctor may be able to treat some side effects. 
• The study doctor may adjust the study drugs to try to reduce side effects. 
Let your study doctor know of any questions you have about possible side effects. You can ask the 
study doctor questions about side effects at any time. 


 
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What possible benefits can I expect from taking part in this study? 
It is not possible to know at this time if treating anal HSIL is better than the usual approach (not 
treating it) in reducing the risk of anal cancer so this study may or may not help you. This study 
will help researchers learn which approaches could help people in the future. 
Can I stop taking part in this study? 
Yes. You can decide to stop at any time. If you decide to stop for any reason, it is important to let 
the study doctor know as soon as possible so you can stop safely. We would like you to continue 
in the study even if you decide to stop HSIL treatments or undergoing study procedures. If you 
agree to stay in the study but cannot come to the clinic, we will contact you once a year until the 
study is over to ask whether you were diagnosed with anal cancer. The study doctor will tell you 
about new information or changes in the study that may affect your health or your willingness to 
continue in the study. 
The study doctor may take you out of the study: 
• If your health changes and taking part in the study is no longer in your best interest. 
• If new information becomes available. 
• If you do not follow the study rules. 
• If the study is stopped by the sponsor, Institutional Review Board (IRB), National Cancer 
Institute (NCI), or the Food and Drug Administration (FDA). 
What are my rights in this study? 
Taking part in this study is your choice. No matter what decision you make, and even if your 
decision changes, there will be no penalty to you. You will not lose medical care or any legal 
rights. 
For questions about your rights while in this study, call the ________________________ (insert 
name of center) Institutional Review Board at __________________ (insert telephone number). 
(Note to Local Investigator: Contact information for patient representatives or other individuals 
at a local institution who are not on the IRB or research team but take calls regarding clinical 
trial questions can also be listed here.) 
What are the costs of taking part in this study? 
Your health plan/insurance company will need to pay for all of the standard costs of treating or 
watching your anal HSIL while in this study. This includes the cost of study tests (high resolution 
anoscopy, anal cytology (Pap smear)), and anal biopsies, the costs of the study treatments, and the 
cost of any medicines needed to manage any side effects. At the screening visit, if your health 
insurance plan will not cover these costs, the study will pay for your HRA, anal biopsies, and anal 
cytology at screening. Neither you nor your insurance company will pay for any of the research 
specimens or tests. Before you decide to be in the study, you should check with your health plan 
or insurance company to find out exactly what they will pay for, and what you may be personally 
responsible for in the form of a co-pay or deductible. The study may be able to reimburse you for 
any out-of-pocket costs that you may have for study visits, including copays and deductibles. You 
should discuss this with the study staff to see if you are eligible for reimbursement for these 
expenses, including what paperwork and documents you will need to provide. If you and/or your 


 
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health insurer are unable to pay for the associated standard of care costs, you can meet with a 
financial counselor to determine if you are eligible for the research to cover these costs. If you do 
not have insurance, then all costs related to study procedures, visits, and laboratory tests will be 
covered by the study. The study cannot cover any costs for medical care outside of study visits. If 
the study ends early, the study will not be able to continue to cover the costs of your care. 
Are there any payments for taking part in this study? 
You will not be paid for taking part in this study. However, the study will reimburse you $100 for 
your time and expenses for every scheduled study visit where HRA is performed. You will be 
reimbursed $25 for study visits where only swabs are collected. Also, you may receive a small 
thank-you gift when you reach a milestone in the study, such as a one-year visit. (Instruction to 
site: modify as required for local limits on participant payments. Add any additional retention 
incentives or gifts based as required by local IRB policy). 
(Omit paragraph if site is not using ClinCard for reimbursement) You will receive reimbursement 
for this study using a “ClinCard.” ClinCard is managed by a company called Greenphire. ClinCard 
works like a prepaid credit card and can be used where MasterCard is accepted. At a minimum, 
your name, initials, and date of birth will be given to Greenphire to load funds on your ClinCard. 
If you agree, you may also provide your email and/or cell phone number to Greenphire to receive 
email or text notifications when funds are loaded to your ClinCard. If you sign up for text 
reminders, you may be charged by your cell phone carrier for the texts, depending on your plan. 
Greenphire will not be given any of your medical information. Let the research staff know if you 
have concerns about using ClinCard. 
The study staff will do everything possible to make your visits as easy as possible and may provide 
additional assistance with transportation and childcare. 
What happens if I am injured or hurt because I took part in this study? 
If you are injured or hurt as a result of taking part in this study and need medical treatment, please 
tell your study doctor. The study sponsors will not offer to pay for medical treatment for injury. 
Your insurance company may not be willing to pay for study-related injury. If you have no 
insurance, you would be responsible for any costs. 
If you feel this injury was a result of medical error, you keep all your legal rights to receive 
payment for this even though you are in a study. 
Who will see my medical information? 
Your privacy is very important to us and the researchers will make every effort to protect it. Your 
information may be given out if required by law. For example, certain states require doctors to 
report to health boards if they find a disease like gonorrhea. However, the researchers will do their 
best to make sure that any information that is released will not identify you. Some of your health 
information, and/or information about your specimen, from this study will be kept in a central 
database for research. Your name or contact information will not be put in the database. 
There are organizations that may inspect your records. These organizations are required to make 
sure your information is kept private, unless required by law to provide information. Some of these 
organizations are: 


 
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• The University of California, San Francisco, and the AIDS Malignancy Consortium (AMC), 
the organizations that are conducting this study 
• The Institutional Review Board, IRB, is a group of people who review the research with the 
goal of protecting the people who take part in the study 
• The Food and Drug Administration and the National Cancer Institute 
• The pharmaceutical collaborator, Bausch Health, and other companies who provide support 
for the study 
• The AIDS and Cancer Specimen Resource at the University of Arizona, the specimen bank for 
the ANCHOR Study 
• If you request to be seen at a new ANCHOR Study site, we will share your study records with 
the new ANCHOR site 
Where can I get more information? 
You may visit the NCI Web site at http://cancer.gov/ for more information about studies or general 
information about cancer. You may also call the NCI Cancer Information Service to get the same 
information at: 1-800-4-CANCER (1-800-422-6237). 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by U.S. Law. This Web site will not include information that can identify you. At most, the Web 
site will include a summary of the results. You can search this Web site at any time. 
Who can answer my questions about this study? 
You can talk to the study doctor about any questions or concerns you have about this study or to 
report side effects or injuries. Contact the study doctor __________________ (insert name of study 
doctor[s]) at __________________ (insert telephone number). 
Additional Studies Section 
This section is about optional studies you can choose to take part in: 
Optional Donation of Leftover Tissue Samples 
After all of the tests for the ANCHOR study are finished, there may be unused blood, anal swab, 
and biopsy tissue left over. We would like your consent to donate your leftover samples for future 
research studies. If you agree, your samples will be stored in the AIDS and Cancer Specimen 
Resource (ACSR). The ACSR is funded by the National Cancer Institute (NCI) to collect, store, 
and distribute samples from research study participants to qualified scientists. This research may 
be about cancers and pre-cancers associated with HIV disease. This future research will be 
approved by the study researchers and the NCI. 
Donating your leftover samples will not require any extra tests or procedures. You are free to 
choose if you want to donate your leftover samples. You do not have to donate your leftover 
samples to take part in this study. If you do not give your consent, we will destroy your leftover 
samples at the end of the study. 
The researchers will send the ACSR some medical information from your study records. This 
information may be useful to researchers who perform studies using your samples. The researchers 
will not give the ACSR your name or any information that could personally identify you. The 


 
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researchers will make every effort to protect your privacy. The ACSR will label your samples with 
codes and store them for future testing. 
You may withdraw your samples from the ACSR at any time. You may contact the study 
coordinator if you would like to withdraw your samples. The coordinator can ask in writing that 
your sample be removed from research use and that any identifiable sample and information still 
in their possession be destroyed. However, if any research has already been done using some of 
your samples, the data will be kept and analyzed as part of those studies. 
You may not have any direct benefit from donating your leftover samples to the ACSR. However, 
research done using your samples may help other people in the future. These studies may help us 
learn more about how to prevent and treat HIV-associated cancers. You will not be paid for 
donating your leftover samples to the ACSR. 
Genetic Testing 
Some of your samples may be used for genetic testing to understand how anal HSIL progresses to 
anal cancer. Someone might be able to trace this information back to you. While the ACSR and 
researchers who study your samples for any genetic testing will have no information that could 
identify you, there is a risk that someone could use information from genetic studies to trace your 
samples back to you. The researchers believe the chance that someone will identify you is very 
small, but the risk may change in the future as people come up with new ways of tracing 
information. In some cases, this information could be used to make it harder for you to get or keep 
a job. There are laws against misuse of genetic information, but they may not give full protection. 
None of the results of any genetic tests done with your samples will be reported back to you or the 
study doctor. 
You may contact the research team if you have any questions about donating your leftover samples. 
Anal Health-Related Symptom Index (A-HRSI) Quality of Life Questionnaire 
You may choose to participate in optional questionnaires to measure how monitoring or treating 
your anal HSIL in this study affects your quality of life. This study is being run with researchers 
at Memorial Sloan Kettering Cancer Center (MSKCC). This study involves taking the A-HRSI 
questionnaire with twenty-five questions at eight different times, asking you to rate symptoms of 
your HSIL. These questions ask about such things as pain, problems moving, and enjoyment of 
sexual activity. The A-HRSI questionnaire may only be taken in English or Spanish. Up to 500 
participants may take part. 
You may take the questionnaire using a computer, tablet, or smartphone without going to the clinic. 
You will be asked to provide your phone number to take part in this study. If you do not have a 
phone but want to take part, you can go to the clinic to take the questionnaire over the phone.  
If you agree to take part and if you are eligible to be in the ANCHOR Study, you will take the A-
HRSI questionnaire once before you are randomized in the main study, and at 7 other times after 
randomization (2-7 days, 4 weeks, and 12, 24, 36, 48, and 60 months after randomization). After 
each questionnaire, we will ask you to confirm your phone number and email for reminders, how 
you want to take the questionnaire next time, and if you want to get text or email reminder 
messages for the next questionnaire. 
If you agree to get reminder messages, we will also collect the name of your cell phone carrier and 
email address to send you text or email reminders to take the questionnaires. This contact 


 
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information will be shared with ANCHOR Study staff at The Emmes Company to send you these 
reminder messages. The study team at the clinic will also use your contact information for reminder 
calls, to help you access the electronic questionnaire, or to ask you to complete it over the phone. 
The research team will not share your contact information with any other parties. 
Answering the questions should take about 6-10 minutes each time. There is a risk you may be 
embarrassed by the questions asked. You will not receive any direct benefits from this study. It 
will help the researchers measure how treating or watching anal HSIL affects patients’ quality of 
life. If you wish to stop participating, you may do so at any time. 
If you do not wish to take the questionnaire on a computer you may complete the questionnaire by 
phone. If you take the questionnaire by phone, you will use some of your data or minutes on your 
phone plan, and may risk running out. If you do not have a phone, or do not wish to use your phone 
for this study, you may complete the questionnaire at the site on a computer or using a site phone. 
If you have a change in phone number or phone carrier, you will need to contact the study team to 
let them know. 
Participants will be paid $25 for each of the eight times that they take the A-HRSI questionnaire 
($200 total). This payment will be made using your ClinCard. [Site to specify method of payment 
if different from ClinCard.] 
You may contact the research team if you have any questions about participating in the quality of 
life questionnaire. 
SARS-CoV-2 (COVID-19) oral and anal infection 
(Note to sites: only the 5 participating ANCHOR sites may include this section in the local 
informed consent form) 
We would like your consent to take part in an optional study to research how often PLWH have 
oral and/or anal infection with SARS-CoV-2, the virus that causes COVID-19. We will also study 
if there is any relationship between SARS-CoV-2 and anal HPV infection. Lastly, we will study if 
SARS-CoV-2 infection has any effect on anal HSIL or anal HPV persisting or going away. Up to 
400 participants will take part in this optional study. 
If you agree, we will collect an oropharyngeal swab (a swab of the back of the throat, like a swab 
to test for strep throat) before you start the study. If you are enrolled into the study, we will collect 
another oral swab at visit 2 (6 months). This must be done at the same visit that anal swab samples 
are collected. We will use a portion of your anal swab samples from the Biorepository from these 
two visits for this study. Everyone who comes to an ANCHOR Study site for a study visit is asked 
about symptoms or possible exposure to COVID-19 and if you are participating in this optional 
COVID-19 study, we may use that information in our study analysis. 
These samples will be sent to UCSF for testing. You will not get the results of your tests since they 
will not be run until the end of this substudy and will not be used for your medical care. If you 
think you have COVID-19 symptoms or have been exposed to someone with COVID-19, please 
talk to a healthcare provider immediately to make sure that you get the necessary medical care. 
You are free to choose if you want to take part in this substudy. If you agree to take part and we 
collect your samples, but your screening evaluations show that you are not eligible for ANCHOR, 
you can still take part in this study. Any samples we have collected will still be used for this study. 


 
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You may withdraw your samples from this substudy at any time. You may contact your study 
coordinator if you would like to withdraw your samples. The coordinator can ask in writing that 
your sample be removed from research use and that any identifiable sample and information be 
destroyed. However, if any research has already been done using some of your samples, the data 
will be kept and analyzed as part of this study. 
There are no costs to take part in this substudy. You will receive $10 via your ClinCard at the visits 
where oral swabs are collected for participating in this optional study. 
You may not have any direct benefit from participating in this optional study. However, research 
done using your samples may help other people with COVID-19 and/or anal HSIL and anal HPV 
in the future. 
Samples for Future Research Studies 
If I am enrolled in the ANCHOR study, my leftover samples and related information may be 
donated to the ACSR for use in future health research. (circle one) 
 
YES 
 
NO 
I agree to have my samples undergo genetic testing to learn about, prevent, diagnose, or treat HIV-
related diseases and cancer. (circle one) 
 
YES 
 
NO 
Research Use of Your Screening Visit Samples 
If I am ineligible for the study, or choose not to participate in the study, the researchers may use 
my study data and samples collected during the screening visit for future research. (circle one) 
 
YES 
 
NO 
Future Studies 
We may want to call you in the future to see if you may be interested in being in other studies.  
If you are called, you can decide if you want to be in any of the other studies and would sign 
another form to be in those studies. You are free to decide whether or not you want us to call you. 
Your decision about being called later for other studies will not change your being in this study. 
The researchers may call me about taking part in future studies. (circle one) 
 
YES 
 
NO 
Anal Health-Related Symptom Index (A-HRSI) Quality of Life Questionnaire 
I agree to participate in the A-HRSI questionnaires. I understand that I need to provide a phone 
number or go to the clinic to participate in this substudy. I acknowledge that clinic staff will contact 
me to complete the questionnaires over the phone if I do not take the questionnaires at the site or 
online (circle one). 
 
YES 
 
NO 
If yes, I prefer to complete questionnaires (circle one) 
 
ONLINE BY MYSELF 
SITE STAFF CALLS ME 
AT THIS CLINIC 
If yes, I prefer to complete questionnaires (circle one) 


 
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IN ENGLISH  
IN SPANISH 
My preferred phone number for clinic staff to contact me for this study is:  
 
__________________________________ 
I agree to receive email reminders for taking questionnaires (circle yes and provide email address 
if you agree to these reminders) 
 
YES TO EMAIL REMINDERS 
 
NO EMAIL REMINDERS 
Email address (for sending computer-generated email reminders):  
 
_________________________________________________________ 
 
I agree to receive text message reminders for taking questionnaires (circle yes and provide cell 
phone information if you agree to these reminders) 
 
YES TO TEXT REMINDERS 
 
NO TEXT REMINDERS 
 
Cell phone number (check here if number is the same as preferred phone number above ______):  
 
___________________________________ 
 
Cell phone carrier name (for sending computer-generated text message reminders): 
 
_________________________________________________________ 
 
CONTACT BETWEEN STUDY VISTS (optional, include only if site has a Mosio business 
agreement) 
 
This ANCHOR study site would like to send you text reminders to your cell phone about upcoming 
ANCHOR appointments and to check in with you between ANCHOR study visits. Receiving these 
texts is optional and you can request that we stop sending you reminders at any time. We have a 
contract with Mosio.com, a secure, HIPAA-compliant texting service to help us send these text 
messages and act on your responses to them. As a reminder, anyone with access to your phone text 
messages will be able to see our message. If you consent to receive these messages, you will be 
asked to provide a cell phone number to receive text messages from the ANCHOR study 
coordinator. If you sign up for text reminders, you may be charged by your cell phone carrier for 
the texts, depending on your plan. 
I agree to be contacted via text message for the ANCHOR study (circle one) 
YES 
 
NO 
My preferred cell phone number for receiving texts from this ANCHOR site is: 
(_______) __________ ________________ 


 
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SARS-CoV-2 Optional Study 
(Note to sites: only the 5 participating ANCHOR sites may include this section in the local 
informed consent form) 
I agree to take part in the optional SARS-CoV-2 study. (circle one) 
 
YES 
 
NO 
This is the end of the section about additional research studies. 
______________________________________________________________________________ 
My Signature Agreeing to Take Part in the Main Study 
I have read this consent form or had it read to me. I have discussed it with the study doctor and my 
questions have been answered. I will be given a signed copy of this form. I agree to take part in 
the main study and any additional studies where I circled ‘yes’.  
 
 
____________________________________ 
___________________________________ 
Participant’s Signature 
 
 
 
Date of Signature 
 
 
____________________________________ 
___________________________________ 
Signature of Person(s) conducting the 
 
Date of Signature 
Informed Consent Discussion 


 
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Attachment 1: ANCHOR: Anal Cancer Prevention Study 
Group 1 Study Calendar for Participants, Active Monitoring 
The list below describes the required study visits and the tests and procedures that will be done for 
each visit on the Active Monitoring Arm (Group 1). The researchers will not perform any study 
procedures unless you give your consent to take part and the tests and procedures from the 
screening visit show that you can take part in the study. 
VISIT 1 (Month 0) – Baseline Visit, Enrollment, Randomization (1-12 weeks after screening) 
• Review of any signs or symptoms or changes in your medications. 
• The anal health-related quality of life questionnaire (A-HRSI), if you agreed to take this 
survey. This questionnaire asks about how your anal HSIL symptoms affect you. It takes less 
than 10 minutes to complete. 
• The ANCHOR Visit 1 Questionnaire, which asks questions about your smoking history, drug 
use, sexual history, and medical conditions and diagnoses related to anal disease. This 
questionnaire will take approximately 25 minutes to complete. 
• Blood sample for HIV viral load and CD4 count. 
• Urine pregnancy test if you are a woman who could become pregnant. 
• An anal exam with a gloved finger and a groin exam for lumps or tenderness. 
• High resolution anoscopy. 
• The study team will tell you your randomization assignment. The procedures below are only 
for participants assigned to Group 1, the Active Monitoring arm. 
• After visit 1, and all other study visits, a member of the study team will call you to remind you 
of your next visit. The study staff will ask if there is anything that the study team can do to 
make it easier for you to attend the study visits. (Note: insert information on local activities 
and support for retention if relevant to the site) 
• If you agreed to take the A-HRSI questionnaire, you will take it home on the internet or by 
phone call at 2-7 days and 4 weeks after Visit 1. See the information sheet on the A-HRSI for 
more instructions. 
VISITS 2, 4, 6, 8, and 10 – Six-Month Visits (Months 6, 18, 30, 42, 54, and later visits after 
Month 60 as required) 
• Review of any signs or symptoms or changes in your medications. 
• Questions about your HIV viral load and CD4 count from routine medical care. 
• Physical exam, including vital signs. 
• Anal swab for anal cytology and research. 
• Two more anal swabs for research. 
• An anal exam with a gloved finger and a groin exam for lumps or tenderness. 


 
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• High resolution anoscopy. The study doctor will only collect anal biopsies at this visit if there 
are any areas that show signs of getting worse or progressing to cancer. A part of your biopsies 
may also be used for research studies. 
• Blood sample for research studies. 
VISITS 3, 5, 7, 9, and 11 – Annual Visits (Months 12, 24, 36, 48, 60, and later visits after 
Month 60 as required) 
• All of the items listed above. In addition you will have biopsies of all anal HSIL lesions. 
• If the provider finds anal cancer at any visit, we will ask you to return to the clinic for an extra 
anal biopsy for future research studies before you stop the study. 
• If you agreed to take the A-HRSI questionnaire, you will take it at month 12, 24, 36, 48, and 
60. 
Final Study Visit (Month 60 or later) 
• You will have all of the evaluations listed above. 
• You will have biopsies of all anal HSIL lesions and areas that used to have HSIL but now 
appear to be normal. If this totals less than 4 biopsies, normal-appearing tissue will be biopsied 
so that at least 4 areas are sampled. 
• If you still have anal HSIL after the final study visit is complete, the researchers will contact 
you. You will be referred for appropriate medical care outside of this study. 


 
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ANCHOR: Anal Cancer Prevention Study 
Group 2 Study Calendar for Participants, Treatment 
The list below describes the required study visits and the tests and procedures that will be done for 
each visit on the Treatment Arm (Group 2). The researchers will not perform any study procedures 
unless you give your consent to take part and the tests and procedures from the screening visit 
show that you can take part in the study. 
VISIT 1 (Month 0) – Baseline Visit, Enrollment, Randomization (1-12 weeks after screening) 
• Review of any signs or symptoms or changes in your medications. 
• The anal health-related quality of life questionnaire (A-HRSI), if you agreed to take this 
survey. This questionnaire asks about how your anal HSIL symptoms affect you. It takes less 
than 10 minutes to complete.  
• The ANCHOR Visit 1 Questionnaire, which asks questions about your smoking history, drug 
use, sexual history, and medical conditions and diagnoses related to anal disease. This 
questionnaire will take approximately 25 minutes to complete. 
• Blood sample for HIV viral load and CD4 count. 
• Urine pregnancy test if you are a woman who could become pregnant. 
• An anal exam with a gloved finger and a groin exam for lumps or tenderness. 
• High resolution anoscopy. 
• The study team will tell you your randomization assignment. The procedures below are only 
for participants assigned to Group 2 (the Treatment arm). 
• The study doctor will show you how to apply any topical treatments, or will perform or 
schedule your first treatment for anal HSIL. 
o Imiquimod 5% cream: you will insert the cream inside or outside your anus, depending on 
where the HSIL was found, with a gloved finger three times per week for up to 16 weeks. 
o 5% FU cream: you will apply the cream inside or outside your anus, depending on where 
the HSIL was found, with a gloved finger twice per day for five days, then take a break 
from treatment for 9 days (14 day cycle). You will have up to 8 of these 14-day treatment 
cycles (up to 16 weeks total). 
o Infrared coagulation (IRC), hyfrecation/electrocautery, or laser ablation: during an HRA 
exam, the study doctor will apply the treatment tip of the medical device to your HSIL 
lesion. Before this treatment, the researcher will inject the lesions with a local anesthetic 
(lidocaine or other anesthetic). You may have one additional biopsy for future research 
studies every 6 months, just before getting treatment. You may have multiple treatments 
on any given lesion that will last for a second or two each. Any dead tissue from the 
treatment will be cut away from the treatment site. The researcher may also treat other HPV 
lesions such as anal warts with the device. You may have repeat treatments within the next 
8 weeks depending on the amount of disease. 


 
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o Surgery to remove HSIL (treatment under anesthesia): If the study doctor believes that 
none of the treatment approaches above will help to get rid of HSIL, you will be referred 
to a surgeon and will have surgery with anesthesia to remove the HSIL. You may have 
repeat procedures after 8 or 12 weeks for follow-up if the HSIL is still present. This 
treatment will only be recommended as a last option. 
• After Visit 1, and all other study visits, a member of the study team will call you to remind you 
of your next visit. The study staff will ask if there is anything that the study team can do to 
make it easier for you to attend the study visits. (Note: insert information on local activities 
and support for retention if relevant to the site.) 
• If you agreed to take the A-HRSI questionnaire, you will take it home on the internet or by 
phone call at 2-7 days and 4 weeks after Visit 1. See the information sheet on the A-HRSI for 
more instructions. 
Visits 1A and IB – Interim Treatment Visits for Imiquimod 5% or 5-FU (8 weeks, and 
possibly 16 weeks after Visit 1) 
• At 8 weeks, you will have Visit 1A 
o Review your signs and symptoms and any changes to your medications. 
o An anal exam with a gloved finger and a groin exam for lumps or tenderness. 
o High resolution anoscopy exam to see how you are responding to treatment. 
o If you are a woman who could become pregnant, you will have a urine pregnancy test. 
• If the study doctor recommends that you continue treatment for 8 more weeks, you may be 
asked to return at 16 weeks (Visit 1B) for another follow-up visit with the same procedures 
listed above. 
• You may have one additional biopsy every 6 months for research purposes. 
Visit 1A – Interim IRC, Electrocautery, or Laser Ablation Treatment Visits (up to 8 
weeks after Visit 1) 
• Within 8 weeks after randomization, you will have Visit 1A 
o Review your signs and symptoms and any changes to your medications. 
o An anal exam with a gloved finger and a groin exam for lumps or tenderness. 
o High resolution anoscopy exam to see how you are responding to treatment. 
o If you are a woman who could become pregnant, you will have a urine pregnancy test. 
o If the study doctor recommends that you continue treatment, or there are more lesions 
to treat, you will have another ablation treatment at this visit. 
VISITS 2-10+ – Six-Month Visits (Months 6, 12, 18, 24, 30, 36, 42, 48, 54, and later visits 
after Month 60 visits as required) 
• Review of any signs or symptoms or changes in your medications. 
• Questions about your HIV viral load and CD4 count from routine medical care. 
• Physical exam, including vital signs. 


 
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• Anal swab for anal cytology and research. 
• Two additional anal swabs for research. 
• An anal exam with a gloved finger and a groin exam for lumps or tenderness. 
• Blood sample for research studies. 
• If you are a woman who could become pregnant, you will have a urine pregnancy test before 
getting any treatments. 
• High resolution anoscopy. The study doctor will collect biopsies of any lesions that look like 
HSIL. A part of your biopsies may also be used for research studies. 
• You will receive your next study treatment if needed or discuss treatment changes with the 
study doctor. 
• If you agreed to take the A-HRSI questionnaire, you will take it at month 12, 24, 36, 48, and 
60. 
• You will have additional visits for treatment follow-up after each 6-month visit when treatment 
is given (e.g., Visit 2A, 2B for imiquimod treatment), as described above under Visit 1. 
• If the provider finds anal cancer at any visit, we will ask you to return to the clinic for an extra 
anal biopsy for future research studies before you stop the study. 
Final Study Visit (Month 60 or later) 
• You will have all of the evaluations listed above. 
• High resolution anoscopy. You will have biopsies of all anal HSIL lesions. If no lesions are 
seen, the study doctor will take biopsies of areas that were treated to be sure that there is no 
more anal HSIL. If this totals less than 4 biopsies, normal-appearing tissue will be biopsied so 
that at least 4 areas are sampled. 
• If you still have anal HSIL after the final study visit is complete, the researchers will contact 
you. You will be referred for appropriate medical care outside of this study. 


 
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Attachment 2: Diagram of the Anus 
 
 
 


 
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Attachment 3: ANCHOR Study Comprehension Assessment 
Instructions: We would like to ask you some questions about the ANCHOR Study to be sure that 
the information we provided about study was clear. Please read each statement about the 
ANCHOR Study below. Please circle the best response or “I don’t know” if you do not know the 
answer. 
 
Name: __________________________________ 
 
Date: _________________________ 
 
1. Once I sign the consent form, I… 
a. can never leave the study. 
b. must provide a compelling reason for leaving the study. 
c. can only leave the study after consulting with the study investigators. 
d. can leave the study at anytime for any reason. 
e. I don’t know. 
 
2. What is HPV? 
a. cancerous tissue. 
b. a procedure where a clinician will insert a scope into the anus. 
c. a virus that can potentially cause anal cancer. 
d. a wart. 
e. I don’t know. 
 
3. What are high grade squamous intraepithelial lesions (HSIL)? 
a. areas of skin damaged by HPV. 
b. cancerous tumors. 
c. anal warts that turn white with vinegar. 
d. a sexually transmitted infection. 
e. I don’t know 
 
4. Why is this study being done? 
a. To find a cure for HPV. 
b. To find a cure for HIV. 
c. To find out if anal cancer is caused by HIV and HPV infection. 
d. To find out if treating HSIL prevents anal cancer. 
e. I don’t know 


 
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5. How long will I be followed if I enroll in the study? 
a. 1-2 years 
b. 3-4 years 
c. 5-8 years 
d. for as long as I have HSIL 
e. I don’t know 
 
6. If I am in the active monitoring group, what is expected of me? 
a. I will not have to come in for visits. 
b. I will be given a cream, surgery, or heat/laser to remove my HSIL. 
c. I will come in for visits every 6 months but will not have any anal biopsies. 
d. I will come in for visits every 6 months, will have biopsies at least every year, and my 
HSIL will be closely monitored by the clinician. 
e. I don’t know. 
 
7. If I am in the treatment arm, what is expected of me: 
a. I will not have to come in for visits. 
b. I will come in for visits every 6 months but will not have any anal biopsies. 
c. All of the treatments will take place in the operating room. 
d. I may undergo several courses of treatment, and possibly different kinds of treatment, 
over the course of the study. 
e. I don’t know. 
 
8. Can I switch from the study group to which I have been assigned to the other study group: 
a. No, I can’t switch to the other study group at any time during the study. 
b. Yes, I can switch to the other study group within the first year after I join the study. 
c. Yes, I can switch to the other study group  at any time during the study. 
d. Yes I can switch to the other group and then switch back if I want to. 
e. I don’t know. 


 
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Informed Consent Instructions for Study Staff (omit from local ICF document given to study 
participants): 
Administer the questionnaire to the participant after the informed consent discussion has occurred 
before the participant signs the informed consent form. After the participant provides responses, 
review the answers to the questionnaire with the participant, and re-review or provide additional 
information as relevant to all incorrect responses to answer any questions. If the participant 
declines to answer any questions, the correct responses may be reviewed with the participant. 
There is no minimum score requirement for the assessment; follow-up to the questionnaire is 
important to reinforce key information about the study and assist comprehension before the 
participant provides consent to participate. The completed Attachment 3 and documentation of the 
research staff’s review of this assessment with the participant must be maintained in the site’s 
source documents with the participant’s signed informed consent form for all screened 
participants. 
Answer Key 
1. D 
2. C 
3. A 
4. D 
5. C 
6. D 
7. D 
8. A 
 
 


 
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Attachment 4: ANCHOR Informed Consent Frequently Asked Questions 
 
What is HPV? 
• 
“Human papillomavirus” is a virus passed 
between people during sex 
• 
Some of the 80 different types of HPV cause 
warts, while others can cause cancer, including 
cervical or anal cancer 
• 
HIV weakens the immune system and reduces the 
body's ability to fight HPV, increasing the risk of 
warts and cancer 
 
What is HSIL? 
• 
“High-grade squamous intraepithelial lesions” 
• 
HSIL is tissue damaged by 1 or more types of 
HPV 
• 
This tissue is at risk of turning into cancer 
• 
HSIL is common among people living with HIV 
 
Is HSIL anal cancer? 
• 
No, HSIL is pre-cancer, HSIL rarely becomes 
cancer 
• 
We estimate that 1 in 100 HIV+ people with 
HSIL will develop anal cancer after 5 years 
 
How is HSIL treated and what are the risks of 
treatment? 
• 
HSIL can be removed with one or more laser, heat 
or acid treatments, a daily cream, or surgery 
• 
Treatment side effects can be severe, but for laser, 
heat or acid treatments only last a short time, 
usually less than a week; Side effects due to the 
cream may last longer 
 
Why is this study being done? 
We don’t know if treating HSIL prevents anal cancer 
 
Who can be in the study? 
• 
We need 5,085 people to volunteer for 5-8 years 
• 
35 years of age or older 
• 
Living with HIV (we’ll need to see which meds 
you’re on) 
• 
Have not been treated for anal HSIL in the last 6 
months 
• 
Never had anal, vulvar, vaginal, or cervical cancer 
• 
Must have anal HSIL found during screening 
 
What possible benefits can I expect in this study? 
• 
You may not benefit from the study, because we 
don’t know what’s best: treatment or monitoring 
• 
This study will hopefully benefit future 
generations 
 
 
What happens in the study? 
• Volunteers receive either treatment or monitoring 
• After 5-8 years, we will compare cancer rates by 
group 
 
Can I choose which study group I am in? 
• No – a computer randomly assigns you 
• Once you’re in your group, that’s your group 
 
Other than this study, what are my choices? 
Manage HSIL outside of this study 
 
What are the advantages of being in each group? 
• Active Monitoring: Treating anal HSIL may not be 
better than active monitoring to reduce the risk of 
anal cancer. If so, you would have avoided 
uncomfortable treatments that provide little benefit to 
you. 
• Treatment: If you are in the treatment arm your HSIL 
may be reduced in size or removed entirely. 
 
What tests and procedures happen in both groups? 
• Physical/genital exam: 1st visit & maybe more often 
• 3 anal swabs: every 6 months 
• Finger exam: every 6 months 
• Anoscopy (also called HRA): every 6 months, maybe 
more often 
• Biopsies: once per year, maybe more often 
• Blood draw: every 6 months 
• Pregnancy test: at screening and before treatments 
• Follow-up phone call: every 6 months 
 
What are the general risks in this study? 
• Screening and treatment procedures may cause 
discomfort; please see full consent for details 
• You may lose work time while recuperating 
 
Are all of these tests and procedures necessary? 
• Two anal swabs, blood samples, and some anal 
biopsies are extra 
• These will be kept for future research 
 
What happens to my leftover tissue after the study? 
• You can donate these for future studies 
• Some samples may be used for genetic testing 
• You can say no to donating your samples and still be 
in the ANCHOR study 
 
What’s expected of me in “active monitoring”? 
• Study visits every 6 months 
• More frequent visits if cancer is suspected 


 
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How long will I be in the study? 
5 to 8 years or longer, with your permission 
 
What’s expected of me in “treatment”? 
• 
HSIL treatment in consultation with a study 
doctor 
• 
Possibly extra visits & biopsies to check for side 
effects and to see if treatment has removed HSIL 
• 
If HSIL doesn’t go away/returns, more treatment 
• 
Women should not get pregnant while they are 
in the study 
• 
Men who could father a baby should use birth 
control (condoms) while using topical creams 
to treat HSIL 
 
What are the treatment side effects? 
• 
Each treatment has common side effects 
• 
Some treatment side effects can be severe 
• 
Please see the full consent for details 
• 
Always tell our doctor about your side effects; we 
can usually help you feel more comfortable 
 
What are the specific risks of treatment? 
The risks of your specific treatment will be discussed 
with you by your health provider. 
 
Can I still have anal sex? 
• 
Yes! – but you will need time to heal for 2 weeks 
or more after procedures 
• 
While using creams, sex partners should use a 
condom to avoid exposure to the chemicals 
 
What happens if I develop anal cancer? 
The study stops for you and we refer you to cancer 
care 
 
Could the study end early? 
Yes – if we learn that one group is better or that 
neither group is better at preventing anal cancer 
 
Can I stop being in the study? 
• 
Yes – you may leave, at any time for any reason 
• 
Always let our doctor know, so you can stop 
safely 
• 
We’d like to stay in touch even if you stop 
visiting us 
 
Could my participation end earlier for other 
reasons? 
• Yes – if you cannot follow the study procedures 
• Yes – if the doctor takes you out of the study 
• Yes – if the study is stopped for administrative 
reasons 
• Treat anal HPV/warts only with your study doctor 
 
• Biopsies (every year, possibly more often) 
What are my rights in this study? 
• There’s no penalty if you choose not to be in the 
study; you will not lose medical care or legal rights 
• Questions? Call the IRB at [Phone] 
• Email the IRB at [IRB email] 
 
What are my costs in this study? 
• A co-pay or deductible, based on insurance coverage 
• If you have no insurance, the study covers the costs 
 
Will I be paid for this study? 
• No – but we’ll reimburse for your time and expenses 
as listed below 
• When HRA is performed, we give you $100 
• When only swabs are taken, we give you $25 
 
What if I’m hurt because of this study? 
• If you’re hurt or injured, please tell the study doctor 
• You’re responsible for medical treatment for injury 
• You keep your legal rights to seek payment 
 
Who will see my medical information? 
• We make every effort to protect your information 
• Names and contact information are stored by local 
site and separately from ANCHOR clinical database 
• Some organizations may inspect your records; they 
are required to keep your information private 
 
Where can I get more information about this study? 
• 1-800-4-CANCER or http://cancer.gov 
• http://www.clinicaltrials.gov/NCT02135419 
• https://anchorstudy.org/ 
 
Who can answer my questions about this study? 
Contact [Site PI's name and phone number] 
 
Will the study interfere with my other healthcare? 
• Generally speaking, your healthcare won’t change 
• But please treat HPV, including warts, only with us 
 
What should I do if I feel a change in my body? 
• You know how your body feels, better than anybody 
• A change in your body may be important 
• Please tell our doctor, so we can pay attention too! 
 


 
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Attachment 5: Certificate of Confidentiality Statement 
 
ANCHOR: Anal Cancer Prevention Study 
Anal Cancer/HSIL Outcomes Research Study 
Conducted by the AIDS Malignancy Consortium (AMC) 
 
The NIH has given the AMC and the ANCHOR Study researchers a Certificate of Confidentiality. 
The Certificate does not mean that the NIH or the U.S. Government recommend that you take part 
in this study. This Certificate helps us keep your health information private. 
Your records for this study will have information that may identify you. This Certificate lets us 
turn down legal demands for your study records. We can use the Certificate to turn down demands 
for records from a U.S. court. The Certificate can be used in any federal, state, or local legal 
matters. We will use the Certificate to turn down any demands for your study records. The cases 
where we cannot use the Certificate are explained below. 
We cannot use the Certificate to turn down a demand from the U.S. Government for study records. 
This applies to audits or reviews of the ANCHOR Study, or records that we have to report to the 
NIH or FDA. 
The Certificate does not stop you or your family members from sharing your health information. 
It does not stop you from talking about taking part in this study. You may give written permission 
for an insurer, employer, or other person to get copies of your study records. If you give 
permission, we cannot use the Certificate to say no to a request for your study records.