protocol synopsis
2.3.10 rationale for health-related quality

The ANCHOR study presents a unique opportunity for the assessment of healthrelated 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 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 AHRSI 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 userfriendly 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 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.
protocol synopsis
final stage of a-hrsi development

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 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 71112 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 AHRSI subscales as compared to those who changed for the better or had no change.  
| in PGIC and ECOG PS, mean (SD) PGIC   | ECOG PS      |                            |                         |              |              |             |
|---------------------------------------|--------------|----------------------------|-------------------------|--------------|--------------|-------------|
| A-HRSI                                | Change for   |                            |                         |              |              |             |
| Subscale                              | better N=34  | Change for  Worse N=24     |                         |              |              |             |
| Physical                              | -0.12 (0.86) | 0.05 (0.74)                | 0.27 (0.65)             | -0.23 (0.61) | -0.06 (0.80) | 0.37 (0.78) |
| Symptoms                              | No Change    | Change for  the worse N=12 | Change for  better N=18 | No Change    |              |             |
| N=38                                  | N=43         |                            |                         |              |              |             |
| Physical                              | 0.01 (1.14)  | -0.06 (0.99)               | 0.65 (1.25)             | -0.76 (1.18) | 0.10 (0.74)  | 0.63 (1.22) |
| Impacts Psychological                 | -0.22 (1.39) | 0.05 (1.19)                | 0.90 (1.50)             | -0.34 (1.42) | -0.08 (1.11) | 0.64 (1.55) |
| Symptoms                              |              |                            |                         |              |              |             | | confidence interval)                                                                                                                                                                                                                    | PGIC                 | ECOG PS              |                      |                      |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|----------------------|----------------------|----------------------|
| A-HRSI                                                                                                                                                                                                                                  | Change for worse vs. | Change for worse     | Change for worse vs. | Change for worse vs. |
| Subscale                                                                                                                                                                                                                                | no change            | vs. better           | no change            | better               |
| Physical                                                                                                                                                                                                                                | 0.22 (-0.30 to 0.73) | 0.39 (-0.13 to 0.90) | 0.43 (0.05 to 0.82)  | 0.60 (0.13 to 1.07)  |
| Symptoms                                                                                                                                                                                                                                | P=0.401              | P=0.144              | P=0.027              | P=0.013              |
| Physical                                                                                                                                                                                                                                | 0.71 (-0.01 to 1.43) | 0.65 (-0.08 to 1.38) | 0.53 (0.02 to 1.03)  | 1.39 (0.77 to 2.01)  |
| Impacts                                                                                                                                                                                                                                 | P=0.052              | P=0.081              | P=0.041              | P<0.001              |
| Psychological                                                                                                                                                                                                                           | 0.86 (-0.02 to 1.73) | 1.12 (0.23 to 2.01)  | 0.72 (0.04 to 1.39)  | 0.98 (0.14 to 1.82)  |
| Symptoms                                                                                                                                                                                                                                | P=0.055              | P=0.014              | P=0.037              | 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 |                      |                      |                      |                      |  
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 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.
protocol synopsis
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),
protocol synopsis
2.4 overview of 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, 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 costeffective (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.
protocol synopsis
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 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. 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:  
| 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 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.
protocol synopsis
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 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.  
  
  
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 (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 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 review.
protocol synopsis
2.6 ancillary study sars-cov-2 and

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.
protocol synopsis
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 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 SARSCoV-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 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.
protocol synopsis
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-CoV2 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 SARSCoV-2, evidence for intestinal ACE-2 expression and intestinal SARS-CoV-2 infection and replication, and our preliminary evidence of ACE-2 and TMPRSS2 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 SARSCoV-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.
protocol synopsis
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 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 COVID19 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 SARSCoV-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 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.
protocol synopsis
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.
protocol synopsis
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)
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 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.
protocol synopsis
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
protocol synopsis
3.3 inclusion criteria for the

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
protocol synopsis
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
protocol synopsis
3.4 inclusion criteria for the

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
protocol synopsis
3.5 inclusion criteria for the

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
protocol synopsis
3.6 number of participants to

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.
protocol synopsis
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.
protocol synopsis
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).
protocol synopsis
registration for optional a-hrsi substudy

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 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.
protocol synopsis
registration for optional sars-cov-2 ancillary

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.
protocol synopsis
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.
protocol synopsis
4.0 treatment plan 4.1 group

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.
protocol synopsis
4.1.1 definition and management of

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.
protocol synopsis
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 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 normalappearing areas that were previously treated. If this totals less than 4 biopsies, normalappearing 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 6month 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 for pre-existing HSIL. The following treatment modalities may be used in the treatment arm of the ANCHOR study:
protocol synopsis
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% 5fluororacil 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 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.
protocol synopsis
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.
protocol synopsis
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 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 6month 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.  
Figure 4-A: Participant initiates imiquimod/5-FU at visit 1 (randomization visit)
Eight 1-week imiquimod/Four  2-week 5-FU cycles of treatment Visit 1 + 8 weeks  
  
Tolerating therapy  
  
If no lesions are seen, participant will return for HRA at the next 6 month visit.  If HSIL is  found, alternative treatment is initiated per guidelines Figure 4-B: IRC, hyfrecation, or electrocautery performed at visit 1 (randomization visit)  
  
If no lesions are seen, participant will return for HRA at the next 6 month visit.  If
protocol synopsis
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.
protocol synopsis
4.3 general concomitant medication and

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.
protocol synopsis
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
protocol synopsis
- 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 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.
protocol synopsis
4.4 concurrent hpv-related disease treatment

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- 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.
protocol synopsis
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 diagnosed. Participants removed from study for unacceptable adverse event(s) will be followed until resolution or stabilization of the adverse event.
protocol synopsis
4.6 criteria for removal from

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.
protocol synopsis
5.0 dosing delays/dose modifications 5.1

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.  
| Topical 5-fluorouracil  5%   | Topical Imiquimod 5%                                       |                                                           |
|------------------------------|------------------------------------------------------------|-----------------------------------------------------------|
| Standard dose                | Twice daily for 5 days                                     | 1 single use sachet applied 3 times per week prior to     |
| followed by 9 days off       | normal sleeping hours and left on the skin for 6–10 hours. |                                                           |
| Reduced dose                 | Twice daily for 3 days                                     | 1 single use sachet applied twice per week prior to       |
| followed by 11 days off      | normal sleeping hours and left on the skin for 6–10 hours. |                                                           |
| Minimal dose                 | Once daily for 3 days                                      | 1 single use sachet applied once per week prior to normal |
| followed by 11 days off      | sleeping hours and left on the skin for 6–10 hours.        |                                                           |  
Table 5-A:1. Dose modifications
protocol synopsis
6.0 adverse events list and

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.
protocol synopsis
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%.
protocol synopsis
6.1.2 adverse event list for

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 5fluorouracil 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.
protocol synopsis
6.1.3 adverse event list for

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.  
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 postprocedure 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.
protocol synopsis
6.1.5 adverse events for surgical

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.
protocol synopsis
6.2 adverse event characteristics and

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 protocolspecified 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 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 investigator must assign an attribution to the AE using the following attribution categories:
protocol synopsis
table 6-a mapping adverse event

| RELATIONSHIP                 | ATTRIBUTION                                   | DESCRIPTION                                  |    |            |         |    |     |
|------------------------------|-----------------------------------------------|----------------------------------------------|----|------------|---------|----|-----|
| Unrelated to investigational | Unrelated                                     | The AE is clearly NOT related to the         |    |            |         |    |     |
| agent/intervention           | intervention                                  |                                              |    |            |         |    |     |
| Unlikely                     | The                                           | AE                                           | is | doubtfully | related | to | the |
| intervention                 |                                               |                                              |    |            |         |    |     |
| Related to investigational   | Possible                                      | The AE may be related to the intervention    |    |            |         |    |     |
| agent/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.
protocol synopsis
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 301251-1161, 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 DMC is responsible for distribution of SAE reports to other parties on behalf of the sponsor.
protocol synopsis
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).
protocol synopsis
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.
protocol synopsis
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
- 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.
protocol synopsis
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.
protocol synopsis
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.
protocol synopsis
7.0 pharmaceutical and study device

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.
protocol synopsis
7.1 5-fluorouracil 5% cream

NOTE: Please refer to the commercial package insert for more information.  
7.1.1 Other names Efudex Cream
protocol synopsis
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 5fluorouracil, 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.
protocol synopsis
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.
protocol synopsis
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 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).
protocol synopsis
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.
protocol synopsis
7.1.7 side effects

See Section 6.1.2. Refer to the approved package insert for complete prescribing and toxicity information.
protocol synopsis
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.
protocol synopsis
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 8week course of perianal treatment (dose doubled to treat the perianus).
protocol synopsis
7.2.6 preparation

Chemically, imiquimod is 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4amine. 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.
protocol synopsis
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.
protocol synopsis
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.
protocol synopsis
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. 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 studysupplied 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.
protocol synopsis
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.
protocol synopsis
8.1 special instructions and definitions

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.
protocol synopsis
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.
protocol synopsis
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.
protocol synopsis
8.1.6 complete physical exam (at

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).
protocol synopsis
8.1.7 targeted physical exam (subsequent

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 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).
protocol synopsis
8.1.11 blood samples for correlative

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 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.
protocol synopsis
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.
protocol synopsis
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 8.2.7 Digital anorectal examination and examination of inguinal nodes 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 13, as outlined in Section 8.1.12.
protocol synopsis
8.3 baseline visit with randomization

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 checklist)
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.
protocol synopsis
8.4 evaluations between six month

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).
protocol synopsis
8.5 evaluations at visit 2

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)
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 6month 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.
protocol synopsis
8.6 evaluations at follow-up (visits

Evaluations are to be conducted ± 4 weeks of the 6-month follow-up visit. 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 6month 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.  
8.6.11 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.  
8.7.2 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 8.7.8 High resolution anoscopy
protocol synopsis
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 8.7.4 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 8.7.9 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.  
8.7.10 *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).
protocol synopsis
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
I).
protocol synopsis
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                   |
protocol synopsis
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.
protocol synopsis
9.1 viral factors in hsil

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 Spf10 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). 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.
protocol synopsis
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 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.
protocol synopsis
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.
protocol synopsis
9.2 identification of host factors

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. 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 nonprogressing 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).
protocol synopsis
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).
protocol synopsis
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 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 wellmatched 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.
protocol synopsis
9.3 viral and host biomarker

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.
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 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 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.
protocol synopsis
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 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), 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 betacatenin (114).
protocol synopsis
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 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 preprogression time points. Similar comparisons will include location of staining
(superficial, basal, diffuse) and weak, moderate or strong) among these tissues.
protocol synopsis
9.4 medical history and behavioral

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