Treatment of High-Grade Pre-Neoplastic Cervical Lesions (CIN 2/3) Using a Novel "Prime and Pull" Strategy
Phase: PHASE2
Conditions: Cervical Intraepithelial Neoplasia, Cervical Dysplasia
Sponsor: Yale University

ClinicalTrials.gov ID: NCT02864147 
Oﬃcial Title: Treatment of High-Grade Pre-NeoplasƟc Cervical Lesions (CIN 2/3) Using a Novel "Prime 
and Pull" Strategy   
Document Type: Protocol w SAP 
Date: 2/24/2023 

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Protocol version 11.0
Version date: December 20, 2022
Treatment of High-Grade Pre-Neoplastic Cervical Lesions (CIN 2/3) Using a 
Novel “Prime and Pull” Strategy
STUDY CONTACTS:
Reference the Study Contact List
STUDY PI:
Alessandro D Santin MD 
Yale University School of Medicine
Department of Obstetrics, Gynecology & Reproductive Sciences, 
Division of Gynecologic Oncology
333 Cedar Street, LSOG Bld. Room 305, PO Box 208063, 
New Haven, CT, 06520-8063 
Phone: 203-737-4450
Fax: 203-737-4339
E-mail: alessandro.santin@yale.edu
Co-PI:
Sangini S. Sheth, MD, MPH
Yale University School of Medicine
Department of Obstetrics, Gynecology & Reproductive Sciences, 
Division of Gynecologic Specialties
330 Cedar Street, FMB 339E
New Haven, CT, 06520
Phone: 203-785-5545
Fax: 203-737-7675
E-mail: sangini.sheth@yale.edu
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SCHEMA
Women with high-grade (CIN 2/3) cervical dysplasia 
Subjects must have histologically-confirmed pre-invasive HPV lesion(s), cervical intraepithelial 
neoplasia grades 2 or 3 (CIN 2/3), as determined by colposcopy-guided cervical biopsies
 
Primary Outcome 
Assessment 
(Weeks 20-24)
Randomized
Control 
(observation)
Imiquimod 
(16 week course)
HPV vaccine + 
Imiquimod 
(16 week course) 
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TABLE OF CONTENTS
PAGE
 1.0
OBJECTIVES
1
 2.0
BACKGROUND AND RATIONALE
2
 3.0
SUBJECT ELIGIBILITY AND EXCLUSIONS; INCLUSION OF MINORITIES   
4
 4.0
STUDY MODALITIES
6
 5.0
CORRELATIVE STUDIES                                                                                                   8
 6.0     TREATMENT PLAN AND ENTRY/REGISTRATION PROCEDURE
            10
 7.0
TREATMENT MODIFICATIONS: IMIQUIMOD
13
 8.0
TREATMENT MODIFICATIONS: 9-VALENT HPV VACCINE
20
 9.0
STUDY PARAMETERS
            25
10.0
EVALUATION CRITERIA
26
11.0
DURATION OF STUDY
27
12.0
STUDY MONITORING AND REPORTING PROCEDURES
                                    27
13.0     YALE PRINCIPAL INVESTIGATOR SAE REPORTING REQUIREMENT                    34
14.0    SITE MANAGEMENT AND COORDINATION                                                    
36
15.0
STATISTICAL CONSIDERATIONS                                                                                   38
     
16.0
REFERENCES
                                                            43
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1.0
OBJECTIVES
1.1
Primary objectives
1.1.1
To determine treatment efficacy defined as histologic regression to CIN 1 or less at 
weeks 20-24 (4 to 8 weeks after the end of imiquimod treatment) in the HPV 
Vaccine + Imiquimod group compared to control
1.1.2
To determine treatment efficacy defined as histologic regression to CIN 1 or less at 
weeks 20-24 (4 to 8 weeks after the end of imiquimod treatment) in the Imiquimod 
group compared to control
1.2
Secondary objectives
1.2.1
To assess complete regression (i.e., histologic remission) at weeks 20-24 (4 to 8 
weeks after the end of imiquimod treatment) in each group 
1.2.2
To assess HPV clearance in each group
1.2.3
To assess treatment tolerability
1.3
Exploratory/correlative objectives
1.3.1
To assess T cell infiltration in post-treatment cervical biopsies and endocervical 
cytobrush samples 
1.3.2
To assess HPV16 E7 immunity in CD4/CD8 T cells
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2.0 
BACKGROUND AND RATIONALE
Human papillomavirus (HPV) infection, which is sexually transmitted, leads to a wide range of 
conditions in women from genital warts and precancerous cervical dysplasia to cancers of the genital tract 
and elsewhere. Globally, 9 million cases of precancerous high-grade “cervical intraepithelial neoplasia 
(CIN 2/3) are detected annually through cervical cancer screening programs; 330,000 in the U.S. alone.1 
Persistent infection with oncogenic (“high-risk”) HPV types is one of the most important risk factors for 
development of CIN 2/3 which poses significant risk of progression to invasive cervical cancer.2-4 In order 
to prevent this progression to invasive cancer, CIN 2 and 3 lesions require frequent surveillance and 
management through serial pelvic exams, cytology, colposcopy-directed biopsies, and various invasive 
procedures, including cervical ablation or excision that can negatively impact future pregnancies. 
Surveillance is often complicated by poor compliance, significant stress and anxiety, and low tolerance 
for serial pelvic exams and procedures. In addition, the direct medical costs in the U.S. associated with 
both screening for and treating HPV-associated diseases is estimated to be $8.0 billion dollars, over 80% 
of which is for routine cervical cancer screening and follow-up.5 
Current FDA-approved prophylactic HPV vaccines have not shown therapeutic effect in patients 
with pre-existing HPV-associated cervical lesions.6-8 Treatment options for CIN 2-3 involve a variety of 
destructive or excisional procedures, although in the U.S. excision is generally favored in these women at 
higher risk of invasive cervical cancer as it allows for pathologic evaluation of the specimen for diagnosis 
and for evaluation of surgical margins. Ablation techniques typically utilize cryotherapy or laser while 
excision techniques include scalpel, electrosurgery or laser. 
A major concern with the destructive or excisional treatment modalities currently available for 
CIN 2-3 include future obstetric risk (pregnancy loss, preterm premature rupture of membranes, and 
preterm delivery) and cervical stenosis, or narrowing of the cervical canal, which can result from scarring 
after treatment. The risk of cervical stenosis after an excision procedure was shown to be 7% in one study 
and this can have several negative outcomes including difficulty with normal cervical dilation during 
labor, difficulty with common office procedures (e.g., endometrial biopsy), and might impede normal 
menstrual flow.9 One large population-based study found that women who gave birth after a cervical 
excision procedure were significantly more likely to have a preterm delivery and had a significantly higher 
risk of a late abortion (< 24 weeks gestation) compared with women who gave birth prior to the surgical 
excision.10 
Given these circumstances, precancerous cervical dysplasia is an ideal candidate for therapeutic 
vaccination to clear CIN 2-3 without destroying cervical tissue. Previous attempts at developing vaccines 
that mount systemic cellular immunity responses against other viral sexually transmitted infections have 
been unsuccessful.11,12 We believe that one of the problems in current therapeutic vaccine approaches is 
the lack of effector T cell migration to the sites of infection. Although systemic memory T cells can gain 
entry to certain organs, other anatomic locations including the vagina are immunologically restrictive and 
prevent entry of memory T cells.13 Inflammation or infection is often needed to permit entry of 
systemically circulating activated T cells into restrictive tissues to establish tissue-resident memory T 
cells.14-17 
Our group has recently reported a highly effective vaccination strategy (“prime and pull”) to 
establish tissue-resident memory T cells in the genital tract.18 An initial systemic T cell response is 
triggered (“prime”) by parenteral vaccination. Activated T cells are then recruited into the genital 
mucosa (“pull”) through the application of specific chemokines to the restrictive genital tract and then 
establish a long-term tissue-resident memory T cell (TRM) pool and mediate protective immunity.14 In 
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Version date: December 20, 2022
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mice, this strategy reduces spread of infectious viruses and prevents clinical disease. These results strongly 
suggest the possibility of inducing T cell responses against oncogenic HPV genotypes through 
immunization with a prophylactic HPV vaccine, followed by recruitment of HPV-activated T cells into 
cervical lesions using a local immune-response modulator. 
Imiquimod, a topical immune response modulator, has antiviral and antitumor activity, induces 
cytokines including interferon, tumor necrosis factor, and interleukins 1, 6, and 8, and activates T cells 
triggering an immune response associated with HPV clearance.19,20 Imiquimod is approved for treatment 
of genital warts, 90% of which are caused by non-oncogenic HPV types 6 and 11.21,22 One group has 
studied the use of imiquimod in the weeks prior to a planned surgical cervical excision procedure as a 
potential method for reducing recurrence of disease.23 They found application of imiquimod to the cervix 
to be safe and tolerable. Only one prior study has evaluated the efficacy of imiquimod for therapeutic 
purposes in high grade cervical dysplasia. This Austrian study on the efficacy of imiquimod for treatment 
of CIN 2/3, found significantly higher rates of histologic regression (73% vs 39%) and complete remission 
(47% vs 14%) in the imiquimod group.24 We are not aware, however, of any published study evaluating 
a “prime and pull” vaccination strategy for the potential treatment of CIN 2/3 in women. In the case of 
imiquimod treatment alone, this relies on the ability of T cell responses to endogenous antigens to elicit 
clearance of the HPV infected cells. 
We believe that “prime and pull” will provide further protection to imiquimod treatment alone, by 
eliciting robust T cell responses that can be pulled into the genital mucosa using chemokines induced by 
imiquimod application. Currently, a 9-valent prophylactic HPV vaccine (9vHPV) is commercially 
available and provides protection against 7 high-risk HPV types that cause 90% of all cervical cancers 
(and protects against 2 additional HPV types that cause 90% of all genital warts).22 9vHPV is a sub-unit 
protein vaccine; vectors expressing the papillomavirus L1 gene have been used to generate virus like 
particles (VLPs) that mimic the wild type virus capsid morphologically and antigenically but contain no 
DNA and are therefore noninfectious. This vaccine is highly immunogenic, inducing a robust serum 
neutralizing antibody response which prevents incident infection.25,26 Previous studies have demonstrated 
induction of antigen-specific T cell responses by prophylactic HPV vaccines as well.27-30 
Published treatment efficacy with imiquimod24 and the preclinical in vitro data presented above 
and fully reported in Nature18 clearly suggest that a “prime and pull” vaccination strategy may provide 
significant therapeutic effect for HPV-associated CIN 2/3 lesions. Because the development of a novel, 
effective treatment modality without the need of destructive interventions on the cervix in CIN 2/3 remains 
an unmet medical need, in this study we are planning a multi-arm randomized clinical trial to determine 
the therapeutic clinical effect and resulting immune response of a “prime and pull” method of treatment 
in women with CIN 2-3 lesions using imiquimod vaginal suppository in conjunction with the 
commercially available 9-valent HPV vaccine.
3.0
SUBJECT ELIGIBILITY AND EXCLUSIONS
3.1       Eligible Subjects: 
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3.1.1
Subjects must have untreated cervical biopsy-proven, CIN 2/3 ectocervical lesion(s).  
3.1.2
Subjects must have satisfactory colposcopy with visualization of the entire transformation 
zone or a negative endocervical curettage if colposcopy is unsatisfactory.
3.1.3
Subjects must be high-risk HPV+ as determined by commercially available DNA 
hybridization test which tests for 13 high-risk HPV types.
3.1.4
All Subjects must have a histologic diagnosis of CIN 2,3 cervical lesion(s) confirmed by a 
study pathologist. 
3.1.5
Subjects must have signed an approved informed consent.
3.1.6
Subjects of childbearing potential must have a negative urine pregnancy test within 7 days 
prior to the study entry and be practicing an effective form of contraception.      
3.1.7
Subjects must be at least 18 years of age based on previous and current cervical cancer 
screening guidelines. 
3.1.8
Subjects must be fluent in speaking English or Spanish.
3.2
Ineligible Subjects:
3.2.1
Subjects with unsatisfactory colposcopy* (unable to visualize entire transformation zone) 
or evidence of endocervical disease defined as CIN 2/3 diagnosed on endocervical curettage.
*Subjects with unsatisfactory colposcopy but negative endocervical curettage are eligible 
(see 3.1.2).
3.2.2
Subjects with a history of invasive cervical cancer 
3.2.3
Subjects with a history of other invasive malignancies, with the exception of non-melanoma 
skin cancers are excluded if there is any evidence of other malignancy being present within 
the last five years.  Subjects are also excluded if their previous cancer treatment 
contraindicates this protocol therapy.
3.2.4
Subjects with any unstable medical issue (including cardiac issues as above, active 
treatment for pulmonary embolism, CVA, renal or hepatic insufficiency, active 
infection/sepsis requiring IV antibiotics). 
3.2.5
Subjects who have an uncontrolled seizure disorder, or active neurological disease.
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3.2.6
Subjects known to be seropositive for HIV and active hepatitis, even if liver function 
studies are in the normal range. Subjects otherwise immunocompromised will also be 
excluded (chronic steroid use, taking immunosuppressive medications).
3.2.7
Pregnant or breastfeeding subjects.
3.2.8
Subjects who have had a total hysterectomy (removal of uterus and cervix) or 
trachelectomy (removal of cervix).
3.2.9
Subjects with a known hypersensitivity to imiquimod. Subjects with a known 
hypersensitivity to any prophylactic HPV vaccine or severe allergic reactions to yeast 
(vaccine component).
3.2.10 Subjects who have received their first dose of HPV vaccine < 4 weeks ago or their second 
dose < 12 weeks ago.
3.2.11 Known hypersensitivity or prior intravaginal treatment with Imiquimod
3.3
Inclusion of Minorities:
3.3.1
As men do not have uteri, only women will be enrolled in this trial. Women fluent in 
English or Spanish will be eligible for study enrollment based on limited resources in 
providing appropriate informed consent and research follow up to subjects only fluent in 
other non-English or Spanish languages. We will not exclude potential subjects from 
participating in this or any study solely on the basis of socioeconomic status.  Every attempt 
will be made to enter all eligible subjects into this protocol and therefore address the study 
objectives in a broad population of women with high-grade cervical dysplasia.
4.0
 STUDY MODALITIES             
4.1 
Imiquimod
4.1.1
Packaging, labelling, and storage
Medication numbers will be unique to each box and will be used for tracking purposes only. 
4.1.2 Imiquimod
Imiquimod will be supplied as vaginal suppositories. Available dosage strengths will be 6.25mg and 3.125 
mg. Suppositories will be supplied in boxes. 
Handling instructions will include washing hands before and after vaginal suppository is placed. Subjects 
will be advised not to have sexual intercourse during the nights in which they applied vaginal suppositories 
and to perform a vaginal douche in the morning. Subjects will be instructed to suspend the application of 
vaginal suppositories during the first 3 days of their menses. In addition, subjects may use a tampon 
following insertion to keep the suppository in place and apply Vaseline to skin surrounding the insertion 
area to prevent irritation.  A 24-hour phone number is provided should subjects have any issues or 
concerns.
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Boxes will be labelled according to local regulations and will include the following as a minimum:

Study number.

Product name (Imiquimod)

Contents of the boxes (18 and 24 vaginal suppositories)

Suppository strength (mg)

Batch number

Medication number

Use-by date

Storage information

Instructions for use

Sponsor name and address

A statement that the medication is for clinical study use only

A caution statement
A new box of medication will be dispensed on day 1 (day of enrollment). The subject will initially receive 
one box of 6.25 mg tablets and in the event that dose reduction is necessary the subject will return to the 
clinic and new medication will be dispensed.
4.1.3
Storage conditions
Imiquimod must be stored refrigerated in the original package. Suppositories must be stored according to 
label instructions. 
4.1.4
Drug accountability
Drug supplies, which will be purchased from Apex Pharmacy (Hamden, CT), will be kept in a secure, 
limited access storage area under the storage conditions. Where necessary, a temperature log must be 
maintained to make certain that the drug supplies are stored at the correct temperature.
The responsible person must maintain records of the product’s delivery to the study site, the inventory at 
the site, the use by each subject, and the return to Apex Pharmacy or alternative disposition of unused 
product(s). 
These records will include dates, quantities, batch/serial numbers, expiry (‘use by’) dates, and the unique 
code numbers assigned to the investigational product(s) and study subjects. The responsible person will 
maintain records that document adequately that the subjects were provided the doses specified by the CSP 
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and reconcile all investigational product(s) received from Apex Pharmacy. The responsible person must 
verify that all unused or partially used drug supplies have been returned by the clinical study subject.
4.2 
9-valent Human Papillomavirus Vaccine (Gardasil 9)
4.2.1
Packaging, labelling, and storage
Commercially available vaccine (GARDASIL®9- Merck) will be purchased by the Investigational Drug 
Service at Yale New Haven Hospital and stored under appropriate conditions in its original container 
until dispensed to study participants.  
4.2.2 9-valent Human Papillomavirus Vaccine
9-valent HPV vaccine will be administered in the clinic by a nurse. The vaccine will be supplied in single-
dose vials and is administered as 0.5 mL per dose intramuscularly in the deltoid region of the upper arm 
or in the anterolateral area of the thigh. Thorough agitation immediately prior to administration is 
necessary to maintain suspension of the vaccine.
Subjects randomized to the Imiquimod + HPV vaccine arm of the study will receive a dose of the vaccine 
on day 1 (day of enrollment). Subjects meeting criteria for a booster dose will return to the clinic to receive 
a second dose of the vaccine in week 8. 
4.2.3
Storage conditions
HPV vaccine must be stored in the original package to protect from light and refrigerated at 2oC to 8oC 
(not to be frozen) and administered as soon as possible after being removed from refrigeration.
4.2.4
Drug accountability
9-valent HPV vaccine will be supplied, stored, and maintained by Yale-New Haven Hospital’s 
Investigational Pharmacy (oversight by Sam Abdelghany).  Per their protocol, the vaccine supply will be 
kept in a secure, limited access refrigerated storage area under the storage conditions. A temperature log 
must be maintained to make certain that the drug supplies are stored at the correct temperature.
The responsible person must maintain records of the product’s delivery to the study site, the inventory at 
the site, the use by each subject, and the return to YNHH Investigational Pharmacy or alternative 
disposition of unused product(s). 
These records will include dates, quantities, and batch/serial numbers, expiry (‘use by’) dates. The 
responsible person will maintain records that document adequately that the subjects were provided the 
doses specified by the CSP and reconcile all product(s) received from YNHH Investigational Pharmacy. 
The responsible person must verify that all unused or partially used drug supplies have been returned by 
the clinical study subject.
5.0 CORRELATIVE STUDIES 
5.1
Histopathology: Cervical biopsies will be collected at the time of initial colposcopic evaluation 
based on clinical guidelines for management of abnormal pap tests. Cervical biopsies will be processed 
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in the surgical pathology department. Cervical biopsies with high-grade (CIN 2/3) dysplasia will be 
centrally reviewed and diagnosis confirmed by study pathologists, in the Pathology Department at Yale 
University. Subjects with confirmed CIN 2/3 are eligible for enrollment. Colposcopy will be performed 
and cervical biopsies collected during weeks 20-24 (4-8 weeks after completion of Imiquimod therapy) 
will be centrally reviewed in the Pathology Department at Yale University to assess for the primary 
outcome. Pathologists will be blinded to study randomization.
5.2
HPV DNA Testing & HPV 16/18 Genotyping: Confirmatory high-risk HPV DNA testing will be 
performed under supervision of cytopathologist Dr. Angelique Levi in the Pathology Department at Yale 
University from cervical cytobrush sample obtained on day of enrollment. HPV 16/18 genotyping will 
be performed via PCR from cervical cytobrush sample by Santin Lab to establish baseline HPV status. 
This testing will be repeated between weeks 16-18 from cervical cytobrush samples and at final study 
visit during weeks 20-24 or following at least 3 weeks after completion of imiquimod therapy. 
5.3
Exploratory Objectives 
5.3.1  In the translational research component of this study we are planning to systematically evaluate 
and compare T cell infiltration into the cervical dysplasia and correlate with clinical response. The 
ultimate goal being to determine if the prime and pull strategy of Imiquimod + HPV vaccine leads to the 
establishment of TRM cells and if this correlates to clinical regression of high-grade cervical dysplasia. 
The pre- and post-treatment cervical biopsies will be examined to determine the frequencies and 
anatomical distributions of CD4 and CD8 T cells within the CIN. We will stain paraffin embedded tissue 
sections with antibodies to CD4 and CD8. These sections will be costained with well-established markers 
of human TRM (CD69, CD103).31 The comparison of TRM frequencies and distributions will be conducted 
both within the individual (pre and post prime and pull) as well as amongst the groups at the relevant 
time points. We will assess possible correlation between TRM and viral clearance and clinical outcomes. 
Because the biopsy sample is quite limited in size, we will also collect cells from the endocervical 
cytobrush to analyze the local T cell populations. The protocol for isolating and analyzing T cells from 
cytobrush is well established.32  We will stain the collected CD4 and CD8 T cells with respect to their 
TRM (CD69, CD103) phenotype, as well as ex vivo stimulation with HPV peptide pool (the same one used 
in the CFSE experiments briefly described below) and measuring TNF- and IFN- secretion by 
intracellular staining.   
5.3.2 In addition, we will explore the changes of pre- and post-immune therapy responses to 
HPV16/18 E7 antigens in subject’s peripheral blood lymphocytes (PBL) and serum. We will utilize CFSE 
flow cytometry-based proliferative assays after in vitro stimulation with HPV16 or 18 E7 peptides (i.e., a 
set of 15-mer peptides overlapping by the central ten amino acids and a set of 9-mer peptides overlapping 
by the central eight amino acids for the HPV 16 or 18 E7 protein) and a streptavidin-biotin capture ELISA 
method to investigate anti-HPV E7 antibody prevalence in serum as described by our group (references 
below).33-37 These explorative assays have been previously validated in studies using reagents obtained 
from both healthy women as well as cervical cancer subjects vaccinated within Phase I clinical studies 
with HPV type 16 and 18 E7 antigen-loaded autologous dendritic cells (DC) as a therapeutic cellular 
vaccine.33-37
6.0
TREATMENT PLAN AND ENTRY PROCEDURE/REGISTRATION PROCEDURE
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6.1
Study Entry: Subjects will be screened and consented by the P.I. or a member of the study staff.  
Consent may be obtained remotely.  The consent form can be mailed or emailed to the research participant 
who will sign a printed version of the consent form and fax/scan/email/send a picture of the signed consent 
form or mail the signed consent form back to the researcher.
 
FIGURE 1: Study Timeline
6.2 
Treatment Plan This is a randomized Phase II, three arm control trial in subjects with CIN 2/3 high 
grade cervical dysplasia. Subjects with CIN 2/3 meeting eligibility criteria will have cervical biopsy 
specimens centrally reviewed by study pathologist to confirm diagnosis. HPV DNA test and HPV 16/18 
genotyping will be performed from endocervical cytobrush samples to determine HPV status associated 
with the dysplasia.
Subjects who have CIN 2/3 with HPV+ disease will be enrolled in this study. Subjects will be randomized 
to one of three arms: observation only (control), imiquimod only, imiquimod + 9-valent HPV vaccine. All 
cytology and cervical biopsy specimens related to the study will be centrally reviewed by study 
pathologists who will be blinded to treatment assignment. 
6.2.1 Pre-Treatment Visit/Study Enrollment: After the index cervical biopsy demonstrating CIN 2-3, all 
subjects will be brought back to clinic to discuss results and management. After obtaining informed written 
consent the following pre-treatment tests will be performed: medical history, physical exam with pelvic 
exam, vital signs, urine pregnancy test (must be performed within 7 days of treatment), translational blood 
-10
 .
.
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 weeks 
.
.
    3 mo. 6 mo.
Vaccine
Booster (if applicable)
Colposcopy
Blood draw
Cytobrush
(Biopsy if necessary)
Urine pregnancy test
Colposcopy
Biopsy
Pap
Blood draw
High risk HPV test
HPV 16/18 genotype
Cytobrush
Urine pregnancy test
*Week 22 visit will 
occur at least 3 weeks 
after last dose of 
therapy 
Colposcopy
Blood draw
HPV 16/18 genotype
Cytobrush
(Biopsy if necessary)
Urine pregnancy test
Imiquimod self application
Baseline colpo
Baseline biopsy
Blood draw
High risk HPV test
HPV 16/18 
genotype
Cytobrush 
Urine pregnancy 
test
Pap
High risk HPV 
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samples (may be drawn after randomization but must be drawn prior to treatment), endocervical specimen 
to be sent to Yale Pathology for high-risk HPV DNA testing (if high-risk HPV DNA results are already 
available, this testing may not have to be repeated) and a second endocervical cytobrush specimen for 
translational research purposes. Blood samples will be collected in sodium heparin tubes with about 28 cc 
collected each time as follows: 7 cc in each of 3 green top tubes and 7 cc in 1 EDTA/purple top tube (see 
follow up visits below). Endocervical specimen for high-risk HPV DNA test to be performed by Yale 
Pathology will be submitted in usual fashion as a SurePath specimen. Endocervical cytobrush sample for 
study purposes will be collected into a 50ml conical tubes containing PBS using sterile techniques and 
will be stored on ice until picked up by a laboratory personnel. Once all eligibility has been met the subject 
will be randomized to one of three arms.
Additional components of visit outline by study group:
(1) Control group: Observation at specified study timepoints. 
(2) Imiquimod only: At the baseline visit, this group will receive instruction on imiquimod self 
application (16 week course) using a 6.25 mg vaginal suppository, storage of medication, possible 
side effects. Subjects will be provided a diary and case report form to record vaginal suppository 
application and side effects will be recorded per NCI Common Terminology Criteria for Adverse 
Events (CTCAE 4.0). Imiquimod self-application regimen will be similar to one previously 
described24 and is as follows:
Treatment weeks 1 and 2:  Subjects apply one vaginal suppository per week
Treatment weeks 3 and 4: Subjects apply two vaginal suppositories per week
Treatment weeks 5 to 16: Subjects apply three vaginal suppositories per week
Vaginal suppositories will be self-applied by the subjects in the evening right after going to bed. 
Subjects will be advised not to have sexual intercourse during the nights in which they applied 
vaginal suppositories and to perform a vaginal douche in the morning. Subjects will be instructed 
to suspend the application of vaginal suppositories during the first 3 days of their menses. Subjects 
will be advised to keep the study medication refrigerated. In case of persistent side effects in 
subjects of the imiquimod group, the dose of the study medication will be modified and participants 
will receive vaginal suppositories containing only 50% of the original imiquimod dose (vaginal 
suppositories containing 3.125 mg imiquimod).
Missed doses of imiquimod can be made up the following day. Otherwise, the dose must be 
skipped and subjects should take the next scheduled dose at the usual time. 
Imiquimod will be dispensed in boxes containing 18 vaginal suppositories (treatment weeks 1-8) 
at the beginning of the treatment course. During follow up visit at week 8 (see below), subjects 
will be provided a new box of 24 vaginal suppositories to complete treatment course for weeks 9-
16. Study drug will be prescribed by the investigator and may be dispensed either by the 
investigator or study staff. 
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(3) Imiquimod + HPV vaccine: At the baseline visit, this group will be instructed about the correct 
method of self-application of imiquimod 6.25mg as a vaginal suppository as described above, 
storage of medication, possible side effects. Subjects will be provided a diary and case report form 
to record vaginal suppository application and side effects will be recorded per NCI Common 
Terminology Criteria for Adverse Events (CTCAE 4.0). In addition, all women (regardless of age) 
will be administered a dose of the HPV vaccine on day of enrollment (regardless of previous HPV 
vaccination history). Women previously unvaccinated will receive an additional booster dose at 8 
weeks.  Local pain will be assessed with a visual analog scale immediately after vaccine 
administration and 5 minutes later. Subjects will be provided a separate case report form to record 
vaccine side effects per CTCAE 4.0.
Missed doses of imiquimod can be made up the following day. Subjects are encouraged to 
complete taking all prescribed doses. If additional time is required toward this goal of treatment, 
the scheduled dosing period may be extended.
Imiquimod will be dispensed in boxes containing 18 vaginal suppositories (treatment weeks 1-8) 
at the beginning of the treatment course. During follow up visit at week 8 (see below), subjects 
will be provided a new box of 24 vaginal suppositories to complete treatment course for weeks 9-
16. Study drug will be prescribed by the investigator and may be dispensed either by the 
investigator or study staff. 
6.2.2 Follow-up Visits: Follow-up visits will occur at week 8 (between weeks 7-9), at week 16 (between 
weeks 16-18) and once at week 22 (between weeks 20-24).   For subjects randomized to treatment arms 
(2) Imiquimod only and (3) Imiquimod + HPV vaccine, the week 22 visit will occur at least 3 weeks after 
last dose of therapy. Urine pregnancy test will be performed at all follow-up visits. Subjects with exam 
findings concerning for worsening disease at any point in the study will be taken out of the study and 
treated with surgical excision procedure.
Week 8 (7-9 weeks) Visit: This visit will be timed to occur part way through the 16 week 
imiquimod treatment course. For women using imiquimod (Imiquimod only and Imiquimod + 
HPV vaccine arms), a review of medication diary, monitoring of correct use of study medication, 
and report of local and systemic side effects using a standardized case report form will occur at all 
follow up visits. For women who received HPV vaccine as part of study protocol (Imiquimod + 
HPV vaccine arm), a review of vaccination diary and report of local and systemic side effects 
using a standardized case report form will occur at all follow up visits. A pelvic exam with 
colposcopy will be performed to objectively assess for gross cervical lesions in all subjects and 
also for local side effects from imiquimod use in the Imiquimod only and Imiquimod + HPV 
vaccine arms. Endocervical cytobrush samples will be obtained at this time for study purposes. 
Cervical biopsies will be performed as clinically indicated. Blood sample collections will also 
occur. In addition, women in the Imiquimod + HPV vaccine groups who were previously 
unvaccinated will receive their booster dose of the HPV vaccine.
Week 16 (16-18 weeks) Visit: This visit will be timed to occur at completion of imiquimod 
treatment course. For women using imiquimod (Imiquimod only and Imiquimod + HPV vaccine 
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arms), a review of medication diary, monitoring of correct use of study medication, and report of 
local and systemic side effects using a standardized case report form will occur at all follow up 
visits. For women who received HPV vaccine as part of study protocol (Imiquimod + HPV vaccine 
arm), a review of vaccination diary and report of local and systemic side effects using a 
standardized case report form will occur at all follow up visits. A pelvic exam will be performed 
to objectively assess for gross cervical lesions in all subjects and also for local side effects from 
imiquimod use in the Imiquimod only and Imiquimod + HPV vaccine arms. Endocervical 
cytobrush samples will be obtained at this time. HPV 16/18 genotyping will be performed using 
endocervical cytobrush samples at this time point. Colposcopy and cervical biopsies will be 
performed as clinically indicated. Blood sample collections will also occur.
Week 22 (20-24 weeks): This visit will be timed to occur at least 3 weeks after completion of 
imiquimod treatment course. For women using imiquimod (Imiquimod only and Imiquimod + 
HPV vaccine arms), a review of medication diary, monitoring of correct use of study medication, 
and report of local and systemic side effects using a standardized case report form will occur at all 
follow up visits. For women who received HPV vaccine as part of study protocol (Imiquimod + 
HPV vaccine arm), a review of vaccination diary and report of local and systemic side effects 
using a standardized case report form will occur at all follow up visits. A pelvic exam will be 
performed to objectively assess for gross cervical lesions in all subjects and also for local side 
effects from imiquimod use in the Imiquimod only and Imiquimod + HPV vaccine arms. 
Endocervical cytobrush samples will be obtained at this time. High risk HPV test and HPV 16/18 
genotyping will be performed using endocervical cytobrush samples at this time point. Colposcopy 
and cervical biopsies will be performed on all subjects at this final study visit to assess primary 
endpoints. Blood sample collections will also occur. 
Persistence of CIN 2-3 or worsening cervical dysplasia diagnosed at the 20-24 weeks visit will be 
recommended for treatment with surgical conization.
At approximately 3 months and 6 months after the last study visit, as per standard of care, it is 
recommended that subjects will have a pap smear with high-risk HPV testing.  The results of this 
testing will be recorded in the subject’s study record. 
7.0 TREATMENT  MODIFICATIONS: IMIQUIMOD
7.1       Dose reduction levels
7.1.1
Dose reduction for imiquimod
Treatment related toxicities will be managed by treatment interruptions and possible dose reductions of 
imiquimod according to the schedule described in the below Tables. Dose reductions will apply to 
individual subjects only. Once the dose has been reduced, it cannot be increased later.
To prevent the development of more severe adverse events, treatment related rash/skin irritation should 
be managed early and proactive as described in the below Tables. Table 1: Dose reduction scheme for 
imiquimod
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AE type and CTCAE Grade
Action
Dose reduction scheme
Events related to study drug:
                                                  Grade 2
Dermatologic or local inflammatory 
reactions                 which responds 
promptly to symptomatic treatment 
(e.g., 
antihistamines, 
NSAIDS, 
narcotics); 
prophylactic 
medications indicated for ≤ 24 hrs
Vulvar edema with readily apparent 
obscuration 
of 
anatomic 
architecture, obliteration of skin 
folds, 
deviation 
from 
normal 
anatomic contour
Any drug related AE Grade ≥3
Pause 
treatment 
until 
subject has recovered to 
Grade ≤1 or baseline1.
Resume 
treatment. 
May 
reduce dose according to 
schedule opposite.
If subject has not recovered 
to Grade ≤1 or baseline1 
within 
21 
days 
study 
treatment 
must 
be 
permanently discontinued2.
If subject was receiving      6.25 
mg, resume treatment at a dose 
of 3.125.
If subject was receiving      3.125 
mg, discontinue imiquimod.
1
Baseline is defined as the CTCAE (version 4) Grade at the start of treatment
2
In the event that the subject is deriving obvious clinical benefit according to the investigator’s judgement, 
further treatment with imiquimod will be decided by the investigator.
In the event of any unrelated adverse events, the investigator may choose to interrupt the medication for 
up to 21 days, but no dose reduction should occur.  If the medication is interrupted for more than 21 days, 
the decision to continue with imiquimod will be made by the investigator.
7.2
Management of adverse events, concomitant therapy, restrictions and rescue treatment
7.2.1 Rescue medication, emergency procedures, and additional treatment(s)
7.2.1.1 
Rescue medication
Rescue medications to reverse the actions of imiquimod are not available. There is no specific antidote 
for overdosage. Potential adverse events should be treated symptomatically. Common adverse events of 
treatment with imiquimod with specified management recommendations and/or requirements include 
local inflammatory reactions with site itching or burning or localized edema and occasionally 
accompanied by dermatologic, respiratory, or infectious symptoms. To improve tolerability and the 
probability of clinical benefit, subjects should receive prompt and appropriate supportive care at the first 
signs of symptoms. Suggested treatments for AEs are described below.
7.2.1.2 
Concomitant treatment(s)
Concomitant medications or therapy to provide adequate supportive care may be given as clinically 
necessary. 
All concomitant therapy, including anaesthetic agents, vitamins, homeopathic/herbal remedies, nutritional 
supplements, must be recorded in the (e)CRF during the screening and treatment period, starting from the 
date of signature of informed consent, and ending at the EOT visit. After the EOT visit, only concomitant 
therapy indicated for treatment of an AE has to be reported. 
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In case of major surgery (as judged by the investigator), it is recommended to stop treatment with 
imiquimod around one week prior to the surgery, and to restart treatment after surgery (unless surgery 
involves genitourinary or anogenital tracts in which case treatment should be suspended until complete 
wound healing achieved). If imiquimod is interrupted for more than 21 days, the decision to continue will 
be made by the sponsor in agreement with the investigator.
7.2.2       Management of expected adverse events
Dermatologic and local inflammatory adverse events are the most common side-effects associated with 
treatment with imiquimod. Respiratory symptoms are less commonly reported but can include sinusitis, 
upper respiratory tract infection or flu-like symptoms. Treatment of these side-effects should be proactive 
and should be started as early as possible after onset of symptoms.
7.2.2.1 
Management of dermatologic and local inflammatory adverse events following 
treatment with imiquimod
Dermatologic adverse events including localized erythema, xeroderma, crusted skin, erosion or ulceration, 
edema, vulvar pain or pruritus can occur after a few applications of imiquimod. Although usually mild to 
moderate, dermatologic reactions can cause significant discomfort (Table 7.2.2.1: 2). 
Table 2: Grade specific treatment recommendations for imiquimod-related dermatologic/ local 
inflammatory reactions:
Severity 
(CTCAE Grading)
Description 
Intervention 
concerning imiquimod 
treatment
Specific intervention
Pain of skin
Mild (Grade 1)
Mild pain
Continue same dose
Consider oral acetaminophen or 
ibuprofen
Moderate (Grade 2)
Moderate pain; limiting 
instrumental ADL
Continue 
same 
dose 
unless Grade 2 skin pain 
continues for ≥ 2 days 
(48 hours) in which case 
treatment 
must 
be 
interrupted 
until 
recovered to ≤ Grade 1 
followed 
by 
dose 
reduction
See 
Grade 
1; 
Continue 
acetaminophen or ibuprofen; 
assess 
for 
signs 
of 
local 
infection 
and 
additional 
dermatologic  findings 
Severe (Grade 3) 
Severe pain; limiting self 
care ADL
Dose interruption until 
recovered to ≤Grade 1 
followed 
by 
dose 
reduction*
See Grade 2; plus:
an infectious process should be 
ruled out with pelvic exam and 
cultures.
 
Pruritis
Mild (Grade 1)
Mild or localized; topical 
intervention indicated
Continue same dose
Topical polidocanol cream.
Consider oral antihistamines, 
e.g. 
diphenhydramine, 
dimethindene, 
cetirizine, 
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levocetirizine, 
desloratidine, 
fexofenadine or clemastine)
Moderate (Grade 2)
Intense or widespread; 
intermittent; skin changes 
from 
scratching 
(e.g., 
edema, 
papulation, 
excoriations, 
lichenification, 
oozing/crusts); 
oral 
intervention 
indicated; 
limiting instrumental ADL
Continue 
same 
dose 
unless Grade 2 skin pain 
continues for ≥ 2 days 
(48 hours) in which case 
treatment 
must 
be 
interrupted 
until 
recovered to ≤ Grade 1 
followed 
by 
dose 
reduction
See 
Grade 
1 
plus 
oral 
histamines; 
Consider topical steroids (e.g., 
topical hydrocortisone)
Severe (Grade 3)
Intense or widespread; 
constant; limiting self care 
ADL 
or 
sleep; 
oral 
corticosteroid 
or 
immunosuppressive 
therapy indicated
Dose interruption until 
recovered to ≤Grade 1 
followed 
by 
dose 
reduction*
See Grade 2; plus: an infectious 
process should be ruled out with 
pelvic exam and cultures
 
Skin Ulceration
Mild (Grade 1)
Combined area of ulcers < 
1cm; 
nonblanchable 
erythema of intact skin 
with associated warmth or 
edema
Continue same dose
Consider oral acetaminophen or 
ibuprofen; 
consider 
topical 
polidocanol cream
Moderate (Grade 2)
Combined area of ulcers 1 
- 2 cm; partial thickness 
skin loss involving skin or 
subcutaneous fat
Continue 
same 
dose 
unless Grade 2 skin 
ulcers 
continue 
to 
worsen after ≥ 2 days 
(48 hours) in which case 
treatment 
must 
be 
interrupted 
until 
recovered to ≤ Grade 1 
followed 
by 
dose 
reduction
See Grade 1; consider topical 
antibiotics 
and 
additional 
topical analgesic treatments
Severe (Grade 3)
Combined area of ulcers 
>2 cm; full-thickness skin 
loss involving damage to 
or 
necrosis 
of 
subcutaneous tissue that 
may extend down to fascia
Dose interruption until 
recovered to ≤Grade 1 
followed 
by 
dose 
reduction*
See Grade 2;
Consider oral antibiotics and  
institute 
additional 
symptomatic therapy (topical or 
systemic) 
as 
clinically 
indicated. 
Consider 
dermatology consultation
Life 
threatening 
(Grade 4)
Any 
size 
ulcer 
with 
extensive 
destruction, 
tissue necrosis, or damage 
to 
muscle, 
bone, 
or 
supporting structures with 
or without full thickness 
skin loss
Dose interruption until 
recovered to ≤Grade 1 
followed 
by 
dose 
reduction*
See Grade 3. 
Consider oral antibiotics
Localized (Vulvar) Edema
Mild (Grade 1)
Localized to dependent 
areas, no disability or 
functional impairment
Continue same dose
Avoid prolonged dependent 
positions
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Moderate (Grade 2)
Moderate localized edema 
and intervention indicated; 
limiting instrumental ADL
Continue 
same 
dose 
unless Grade 2edema 
continue to worsen after 
≥ 2 days (48 hours) in 
which case treatment 
must be interrupted until 
recovered to ≤ Grade 1 
followed 
by 
dose 
reduction
See Grade 1
Severe (Grade 3)
Severe localized edema 
and intervention indicated; 
limiting self care ADL
Dose interruption until 
recovered to ≤Grade 1 
followed 
by 
dose 
reduction*
See Grade 1; Consider Foley 
catheter placement if urethral 
obstructed
* If despite optimal supportive care and a treatment interruption, dermatologic reactions does not resolve to CTC AE Grade ≤ 
1 within 21 days, treatment with imiquimod must be permanently discontinued. In the event that the subject is deriving obvious 
clinical benefit according to the investigator’s judgement, further treatment with imiquimod will be decided by the investigator.
7.2.2.2 Management recommendations for respiratory AEs following treatment with Imiquimod
Respiratory adverse events including sinusitis, flu-like symptoms, upper respiratory tract infection can 
occur, although usually mild to moderate (Table 7.2.2.2: 3). 
Table 3: Grade specific treatment recommendations for imiquimod-related respiratory reactions:
Severity 
(CTCAE Grading)
Description 
Specific intervention
Upper respiratory infection
Mild (Grade 1)
-
-
Moderate (Grade 2)
Moderate 
symptoms; 
oral 
intervention 
indicated 
(e.g., 
antibiotic, antifungal, antiviral)
Consider oral antibiotic, antifungal or antiviral; 
Consider anti-pyretic 
Severe (Grade 3) 
IV 
antibiotic, 
antifungal, 
or 
antiviral intervention indicated; 
radiologic, 
endoscopic, 
or 
operative intervention indicated
See Grade 2; if not improving within 48 hours of 
initiation 
of 
oral 
medication, 
consider 
IV 
formulation and also radiologic imaging
Life-threatening 
(Grade 4)
Life-threatening 
consequences; 
urgent intervention indicated
IV antibiotic therapy, radiologic imaging, evaluate 
for other systemic infections, hospitalization to 
monitor 
progress 
and 
if 
urgent 
respiratory 
interventions required
Sinusitis
Mild (Grade 1)
-
Moderate (Grade 2)
Localized; 
local 
intervention 
indicated (e.g., topical antibiotic, 
antifungal, or antiviral)
Consider oral antibiotic, antifungal or antiviral; 
Consider anti-pyretic
Severe (Grade 3)
IV 
antibiotic, 
antifungal, 
or 
antiviral intervention indicated; 
See Grade 2; if not improving within 48 hours of 
initiation 
of 
oral 
medication, 
consider 
IV 
formulation and also radiologic imaging
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radiologic, 
endoscopic, 
or 
operative intervention indicated
Life 
threatening 
(Grade 4)
Life-threatening 
consequences; 
urgent intervention indicated
See Grade 3
Death (Grade 5)
Death
Flu like symptoms
Mild (Grade 1)
Mild flu-like symptoms present
Consider anti-pyretic
Moderate (Grade 2)
Moderate 
symptoms; 
limiting 
instrumental ADL
See Grade 1
Severe (Grade 3)
Severe symptoms; limiting self 
care ADL
See Grade 1
* If despite optimal supportive care and a treatment interruption, respiratory reactions does not resolve to CTC AE Grade ≤ 1 
within 21 days, treatment with imiquimod must be permanently discontinued. In the event that the subject is deriving obvious 
clinical benefit according to the investigator’s judgement, further treatment with imiquimod will be decided by the investigator.
7.2.3
Restrictions
7.2.3.1 Restrictions regarding concomitant treatment
Concomitant medications, or therapy to provide adequate supportive care, may be given as clinically 
necessary.
Additional experimental immunosuppressant treatment and/or standard immunotherapy is not allowed 
concomitantly with the administration of study treatment. 
7.2.3.2 Restrictions on diet and life style
Subjects should be advised there are no dietary restrictions.
To prevent skin related adverse events it is recommended to avoid intense irradiation with UV light and 
harsh detergents. In addition, subjects will be advised not to have sexual intercourse during the nights in 
which they applied vaginal suppositories and to perform a vaginal douche in the morning. Subjects will 
be instructed to suspend the application of vaginal suppositories during the first 3 days of their menses
7.2.3.3
Women of Child-Bearing Potential and Pregnancy Prevention
Subjects who are not of childbearing potential due to being postmenopausal (1 year without menstruation) 
or surgical sterilisation (bilateral oophorectomy and/or tubal ligation) do not need to use contraception to 
be eligible for the trial.
All other subjects are considered to have childbearing potential and must use adequate contraception 
throughout the trial (from screening until end of trial participation).
Acceptable methods of contraception include surgical sterilisation and double barrier method, and must 
be in accordance with local regulations where applicable. Double barrier method of contraception is 
defined as two barrier methods used simultaneously each time the subject has intercourse. Accepted 
barrier methods include diaphragm, female condom, cervical cap, male condom and intrauterine device 
(IUD) (the diaphragm and cervical cap must be used in conjunction with spermicidal jelly/cream). Those 
using hormonal contraceptives, or with partners using hormonal contraceptives, must also be using an 
additional approved method of contraception (as described above).  Partner vasectomy, natural "rhythm" 
and spermicidal jelly/cream are not acceptable methods of contraception.
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Women who become pregnant while participating in the study must discontinue study medication 
immediately.  The pregnancy must be reported following procedures detailed in section 12.10.2.
7.3
Treatment compliance
The study medication will be given in accordance with the protocol and the instructions of the investigator. 
The appropriate number of imiquimod suppositories for the first 8 weeks of imiquimod treatment course 
will be provided to subjects to be self-administered at home. At week 8 follow up visit, the appropriate 
number of imiquimod suppositories for weeks 9-16 imiquimod treatment course will be provided to 
subjects. Subjects will be asked to bring the remaining study medication at each follow-up visit (weeks 8, 
16) to the investigator site for a compliance check. The remaining suppositories will be counted by the 
investigator/site staff and recorded at the investigator site. Discrepancies between the number of 
suppositories remaining and the calculated number the subjects should have used must be documented 
and explained. At the end of the 16-week period, any remaining medication will be collected. 
The investigator can withdraw a subject from the study in the event of serious and persistent non-
compliance which jeopardizes the subject’s safety or render study results for this subject uninterpretable.  
8.0 TREATMENT  MODIFICATIONS: 9-valent HPV Vaccine
8.1       Dose reduction levels
8.1.1
Dose reduction for 9-valent HPV Vaccine
As the 9-valent HPV vaccine is not a continuous use medication, dose reduction will not be applied in the 
setting of treatment related toxicities. Subjects experiencing allergic reactions as described in table below 
will not receive additional doses of the HPV vaccine per FDA and CDC guidelines. Subjects experiencing 
local, self-limited site reactions will be eligible to receive additional doses of the vaccine.
Table 4: Discontinuation of HPV vaccine
AE type and CTCAE Grade
Action
Dose reduction scheme
Events related to study drug:
Grade 3:
Anaphylaxis: 
Symptomatic 
bronchospasm, with or without 
urticaria; parenteral intervention 
indicated; 
allergy-related 
edema/angioedema; hypotension
Any drug related AE Grade ≥3
Treat adverse event or 
provide symptomatic relief 
if treatment not available.
Discontinue or resume HPV 
vaccination 
protocol 
as 
noted in next column. 
 
For 
anaphylactoid 
reaction, 
discontinue 
additional 
HPV 
vaccine administration.
For all other Grade 3 AE, if 
subject has not recovered to 
Grade ≤2 or baseline1 within 21 
days, 
discontinue 
additional 
HPV vaccine administration. 
The decision to continue with 
additional HPV vaccine doses 
following an AE Grade ≤2 or a 
Grade 3 AE that resolves within 
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21 days will be made by the 
investigator.
3
Baseline is defined as the CTCAE (version 4) Grade at the start of treatment
In the event of any unrelated adverse events, the investigator may choose to delay HPV vaccine 
administration for up to 21 days, but no dose reduction should occur.  If vaccine administration is delayed 
for more than 21 days, the decision to continue with vaccination and possibly with study will be made by 
the investigator.
8.2
Management of adverse events, concomitant therapy, restrictions and rescue treatment
8.2.1 Rescue medication, emergency procedures, and additional treatment(s)
8.2.1.1 
Rescue medication
Rescue medications to reverse the actions of 9-valent HPV vaccine are not available.  Potential adverse 
events should be treated symptomatically. Common adverse events of vaccination with 9-valent HPV 
vaccine with specified management recommendations and/or requirements include local reactions with 
pain, swelling or erythema at injection site and occasionally headaches or syncope. Rare events include 
anaphylactoid/hypersensitivity reactions which should serve as contraindication to administration of 
additional doses of 9-valent HPV vaccine as described above. To improve tolerability and the probability 
of clinical benefit, subjects should receive prompt and appropriate supportive care at the first signs of 
symptoms. Suggested treatments for AEs are described below.
8.2.1.2 
Concomitant treatment(s)
Concomitant medications or therapy to provide adequate supportive care may be given as clinically 
necessary. 
All concomitant therapy, including anaesthetic agents, vitamins, homeopathic/herbal remedies, nutritional 
supplements, must be recorded in the (e)CRF during the screening and treatment period, starting from the 
date of signature of informed consent, and ending at the EOT visit. After the EOT visit, only concomitant 
therapy indicated for treatment of an AE has to be reported. 
If vaccination is delayed by more than 21 days, the decision to continue will be made by the investigator.
8.2.1.3 
Emergency procedures
Careful management and assessment of all subjects with syncope should be performed to prevent serious 
secondary injuries (skull fracture, cerebral haemorrhage). Procedures will be in place to avoid injuries 
from falling and to restore cerebral perfusion if syncope occurs. 
Subjects who develop anaphylactoid/hypersensitivity symptoms (symptomatic bronchospasm with or 
without urticarial, angioedema, hypotension) should receive immediate treatment including epinephrine 
1:1000. If anaphylactoid/hypersensitivity reaction to 9-valent HPV is suspected, additional doses of the 
vaccine should be discontinued. 
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8.2.2       Management of expected adverse events
Local injection site and headache adverse events are the most common side-effects associated with 
administration of 9-valent HPV vaccine. Syncope and anaphylactoid/hypersensitivity reactions are less 
commonly reported. Treatment of these side-effects should be proactive and should be started as early as 
possible after onset of symptoms.
8.2.2.1 
Management of local injection site adverse events following administration of 9-
valent HPV vaccine
Local injection site adverse events including pain, erythema, or swelling can occur shortly after injection. 
Swelling and erythema have been noted to be increased in some subjects with successive doses. Although 
usually mild to moderate, local site reactions can cause significant discomfort (Table 8.2.2.1: 5). 
Table 5: Grade specific treatment recommendations for local injection site reactions:
Severity 
(CTCAE Grading)
Description 
Specific intervention
Injection site reaction
Mild (Grade 1)
Tenderness with or without associated 
symptoms 
(e.g., 
warmth, 
erythema, 
itching)
Advise 
symptomatic 
treatment 
with 
acetaminophen or ibuprofen and local ice 
application
Moderate (Grade 2)
Pain; lipodystrophy; edema; phlebitis
Continue acetaminophen or ibuprofen; 
assess for signs of local infection and additional 
dermatologic  findings. Addition of topical 
antibiotic or corticosteroid if clinically indicated
Severe (Grade 3) 
Ulceration or necrosis; severe tissue 
damage; operative intervention indicated
See Grade 2; Consider oral or IV antibiotic as 
clinically indicated and institute wound care, 
including surgical consultation if needed
Life 
threatening 
(Grade 4)
Life-threatening 
consequences; 
urgent 
intervention indicated
See Grade 3
Localized edema
Mild (Grade 1)
Localized to dependent areas, no disability 
or functional impairment
Avoid prolonged dependent positions
Moderate (Grade 2)
Moderate localized edema and intervention 
indicated; limiting instrumental ADL
See Grade 1
Severe (Grade 3)
Severe localized edema and intervention 
indicated; limiting self care ADL
See Grade 1
* If despite optimal supportive care, reactions do not resolve to CTC AE Grade ≤ 2 within 21 days, administration of additional 
HPV vaccine doses must be permanently discontinued. The decision to continue with additional HPV vaccine doses following 
an AE Grade ≤2 or a Grade 3 AE that resolves within 14 days will be made by the investigator.
8.2.2.2 Management recommendations for headache AEs following administration of 9-valent 
HPV vaccine: Headaches can occasionally develop, although usually mild to moderate (Table 8.2.2.2: 6). 
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Table 6: Grade specific treatment recommendations for 9-valent HPV vaccine-related headache 
symptoms:
Severity 
(CTCAE Grading)
Description 
Specific intervention
Headaches
Mild (Grade 1)
Mild pain
Consider 
symptomatic 
treatment 
with 
acetaminophen or ibuprofen
Moderate (Grade 2)
Moderate pain; limiting instrumental ADL
See Grade 1
Severe (Grade 3) 
Severe pain; limiting self care ADL
See Grade 1
* If despite optimal supportive care, reactions do not resolve to CTC AE Grade ≤ 2 within 21 days, administration of additional 
HPV vaccine doses must be permanently discontinued. The decision to continue with additional HPV vaccine doses following 
an AE Grade ≤2 or a Grade 3 AE that resolves within 21 days will be made by the investigator.
8.2.2.3 Management recommendations for syncopal AEs following administration of 9-valent 
HPV vaccine: Syncopal events occur rarely with injectable vaccines and are typically reported in 
adolescents and young adults, occurring within 15 minutes of vaccination. Avoiding falls is imperative.
Table 7: Grade specific treatment recommendations for 9-valent HPV vaccine related syncopal events:
Severity 
(CTCAE Grading)
Description 
Specific intervention
Presyncope / Syncope
Mild (Grade 1)
-
Moderate (Grade 2)
Presyncope present (e.g., near fainting)
Advise subject to sit down, check vitals, 
consider oral hydration
Severe (Grade 3) 
Fainting; orthostatic collapse
 Help subject to lie down and avoid fall, check 
vitals and EKG, consider IV hydration
* If despite optimal supportive care, reactions does not resolve to CTC AE Grade ≤ 2 within 21 days, administration 
of additional HPV vaccine doses must be permanently discontinued. The decision to continue with additional HPV vaccine 
doses following an AE Grade ≤2 or a Grade 3 AE that resolves within 21 days will be made by the investigator.
8.2.2.4 Management recommendations for anaphylactoid/hypersensitivity reactions following 
administration of 9-valent HPV vaccine: A rare AE seen with 9-valent HPV vaccine that clinically may 
present with breathing difficulty, dizziness, hypotension, cyanosis and loss of consciousness. 
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Table 
8: 
Grade 
specific 
treatment 
recommendations 
for 
9-valent 
HPV 
vaccine-related 
anaphylactoid/hypersensitivity reactions
Severity 
(CTCAE Grading)
Description 
Specific intervention
Anaphylaxis
Mild (Grade 1)
-
-
Moderate (Grade 2)
-
 -
Severe (Grade 3) 
Symptomatic 
bronchospasm, 
with 
or 
without urticaria; parenteral intervention 
indicated; 
allergy-related 
edema/angioedema; hypotension
 Administer 
epinephrine 
(1:1000) 
intramuscularly 
immediately; 
additional 
supportive therapy as indicated including IV 
fluids, hospitalization for observation and 
airway management
Life 
threatening 
(Grade 4)
Life-threatening 
consequences; 
urgent 
intervention indicated
See Grade 3
8.2.3
Restrictions
8.2.3.1 Restrictions regarding concomitant treatment
Concomitant medications, or therapy to provide adequate supportive care, may be given as clinically 
necessary.
Additional experimental immunosuppressant treatment and/or standard immunotherapy is not allowed 
concomitantly with the administration of study treatment. 
8.2.3.2 Restrictions on diet and life style
Subjects should be advised there are no dietary or lifestyle restrictions.
8.2.3.3
Women of Child-Bearing Potential and Pregnancy Prevention
Subjects who are not of childbearing potential due to being postmenopausal (1 year without menstruation) 
or surgical sterilisation (oophorectomy, hysterectomy and/or tubal ligation) do not need to use 
contraception to be eligible for the trial.
All other subjects are considered to have childbearing potential and must use adequate contraception 
throughout the trial (from screening until end of trial participation).
Acceptable methods of contraception include surgical sterilisation and double barrier method, and must 
be in accordance with local regulations where applicable. Double barrier method of contraception is 
defined as two barrier methods used simultaneously each time the subject has intercourse. Accepted 
barrier methods include diaphragm, female condom, cervical cap, male condom and intrauterine device 
(IUD) (the diaphragm and cervical cap must be used in conjunction with spermicidal jelly/cream). Those 
using hormonal contraceptives, or with partners using hormonal contraceptives, must also be using an 
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additional approved method of contraception (as described above).  Partner vasectomy, natural "rhythm" 
and spermicidal jelly/cream are not acceptable methods of contraception.
Women who become pregnant while participating in the study may not receive any additional doses of 
the HPV vaccine.  The pregnancy must be reported following procedures detailed in section 12.10.2.
8.3
Vaccine compliance
The vaccine will be administered in accordance with the protocol and the instructions of a site investigator. 
The 9-valent HPV vaccine will be administered in the clinic by a clinic or study nurse based on study 
protocol. 
The investigator can withdraw a subject from the study in the event of serious and persistent non-
compliance with receiving the vaccine dose(s) which jeopardizes the subject’s safety or render study 
results for this subject unacceptable. 
9.0
STUDY PARAMETERS 
9.1
Observations and Tests
The following observations and tests are to be performed and recorded before, during, and 
after treatment:
Pre-Treatment
Treatment 
Period
Post-Treatment 
Period
End of 
Treatment 
****
3 and 6 Month 
Post End of 
Treatment
Study week
8
16
22
Study Procedures Window (weeks)
-10
 1
  2
 2
 2
History, PE w/ pelvic exam
X
X
X
X
Vital signs
X
X
X
X
Urine pregnancy test
X**
X
X
X
Pap
X
High risk HPV test
X***
X
X
HPV 16/18 genotype
X
X
X
Cytobrush collection*
X
X
X
X
Colposcopy, cervical biopsy
X
X 
(biopsy if 
clinically 
indicated)
X
(biopsy if 
clinically 
indicated)
X
Peripheral blood sample collection*^
X****
X
X
X
Toxicity assessment 
X
X
X
X
Randomization
X*****
*
For translational studies. Translational blood may be drawn after randomization but must be drawn prior to treatment.
^          See Section 6.2.1 for collection information.
**        Urine pregnancy test must be performed within 7 days of treatment
***      If high-risk HPV DNA results are already available, this testing may not have to be repeated
****    Week 22 visit will occur at least 3 weeks after last dose of therapy
*****  Randomization to occur following confirmation of eligibility
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10.0 EVALUATION CRITERIA
10.1
 Objective Response
The major parameters of response to be assessed include treatment efficacy defined as 
histologic regression of cervical dysplasia to CIN 1 or less 12 to 16 weeks after the end of 
imiquimod treatment, HPV clearance and treatment tolerance.
10.11 Histologic regression: Only subjects with histologically confirmed CIN 2,3 as 
reviewed by a central study pathologist will be enrolled in the study.  All index biopsy sites 
noted to have CIN 2,3 at baseline must be assessed via colposcopy. Cervical biopsy at the 
final colposcopy will be collected from the worst areas of residual disease. Included in the 
evaluations are the following criteria:  

Histologic regression (HR): Histologic regression of all index lesions to CIN 1 
or less at 12 to 16 weeks after end of imiquimod treatment period.

Histologic remission (HM):  Complete regression of cervical dysplasia at all 
index biopsy sites at 12 to 16 weeks after end of imiquimod treatment period.
 

Persistent Disease (PR):  One or more index lesions persists with CIN 
2,3 high grade dysplasia or new lesions are identified colposcopically 
and histologically confirmed to be CIN 2,3.

Progressive Disease (PD): Worsening histology of an index lesion.
10.12 HPV Clearance: HPV clearance will be measured by both the Roche cobas HPV  
Test utilized by pathology concomitant with the pap test at final study visit which 
assesses for presence of 14 high risk HPV types as well as HPV 16/18 genotyping 
performed by Santin Lab. 
10.13 Treatment Tolerance: The grade level of the various toxicities will be classified using 
the Common Toxicity Criteria, version 4 (CTC, v.4) guidelines. Acute toxicities 
will be scored if occurring </= 30 days from treatment completion, and chronic if 
> 30 days. Frequency and duration of treatment interruptions due to the treatment 
toxicity will be assessed.  
11.0
     DURATION OF STUDY
11.1 This study will continue as long as treatment protocols remain activated.
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11.2
The subject will be followed with routine clinical care with a 6 month colposcopy and 
cytology if histologic remission or regression noted at final study visit and in 12 months if 
cytology remains negative with normal colposcopy. Any abnormal cytology or colposcopy 
at 6 months should prompt cervical biopsy and management per guidelines of American 
Society for Colpsocpy and Cervical Pathology (ASCCP).  
Those subjects with persistent CIN 2,3 or progression of disease at end of study period will 
be offered surgical excisional procedure per routine clinical care and should be followed 
by ASCCP guidelines.
12.0
STUDY MONITORING AND REPORTING PROCEDURES 
12.1 Personnel responsible for the safety review and its frequency:
The principal investigator will be responsible for monitoring the data, assuring protocol compliance, and 
conducting the safety reviews at the specified frequency which must be conducted at a minimum of every 
6 months (including when re-approval of the protocol is sought).  During the review process, the principal 
investigator will evaluate whether the study should continue unchanged, require modification/amendment, 
or close to enrollment. Either the principal investigator, the IRB or the Yale Cancer Center Data and Safety 
Monitoring Committee (DSMC) have the authority to stop or suspend the study or require modifications.
12.2 The risks associated with the current study are deemed moderate for the following reasons: 
1. The risk of progression of CIN 2,3 to invasive cervical cancer over 6-8 months from time of initial 
diagnosis is low and observation of CIN 2 diagnosed in young women for up to 12 to 24 months 
is already recommended by the ASCCP.
2. The risks associated with the FDA-approved drugs imiquimod and 9-valent HPV vaccine are 
minimal.
Although we have assessed the proposed study as one of moderate risk, the potential exists for anticipated 
and/or unanticipated adverse events, serious or otherwise, to occur since it is not possible to predict with 
certainty the absolute risk in any given individual or in advance of first-hand experience with the proposed 
study methods. Therefore, we provide a plan for monitoring the data and safety of the proposed study as 
follows:
12.3 Attribution of Adverse Events:
Adverse events will be monitored for each subject participating in the study and attributed to the study 
procedures / design by the principal investigator Dr. Alessandro Santin, according to the following 
categories:
a.) Definite: Adverse event is clearly related to investigational procedures(s)/agent(s).
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b.) Probable: Adverse event is likely related to investigational procedures(s)/agent(s).
c.) Possible: Adverse event may be related to investigational procedures(s)/agent(s).
d.) Unlikely: Adverse event is likely not to be related to the investigational procedures(s)/agent(s).
e.) Unrelated: Adverse event is clearly not related to investigational procedures(s)/agent(s).
12.4 Plan for Grading Adverse Events:
The following scale will be used in grading the severity of adverse events noted during the study:
1. Mild adverse event
2. Moderate adverse event
3. Severe 
12.5 Plan for Determining Seriousness of Adverse Events:
An adverse event may be graded as severe but still not meet the criteria for a Serious Adverse Event.  
Similarly, an adverse event may be graded as moderate but still meet the criteria for an SAE.  It is 
important for the PI to consider the grade of the event as well as its “seriousness” when determining 
whether reporting to the IRB is necessary.
Refer to Section 13.0 of the protocol for guidelines
12.6 Plan for reporting Reportable Adverse Events and other unanticipated problems involving 
risks to subjects or others to the IRB
The principal investigator will report the following types of events to the IRB: a) adverse events that are 
serious or life-threatening AND unanticipated (or anticipated but occurring with a greater frequency than 
expected) AND possibly, probably or definitely related to the drug/device/intervention; and b) other 
unanticipated problems involving risks to subjects or others.
These adverse events or unanticipated problems involving risks to subjects or others will be reported to 
the IRB in accordance with IRB Policy 710, using the appropriate forms found on the website. This 
information is discussed in Section 13.0 of the protocol.
12.7 Plan for reporting adverse events to co-investigators on the study, as appropriate the protocol’s 
research monitor(s), e.g., Yale Cancer Center Data and Safety Monitoring Committee (DSMC), 
Protocol Review Committee (PRC), DSMBs, study sponsors, funding and regulatory agencies, and 
regulatory and decision-making bodies.
For the current study, the following individuals, funding, and/or regulatory
agencies will be notified (choose those that apply):
X All Co-Investigators listed on the protocol.
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X Yale Cancer Center Data and Safety Monitoring Committee (DSMC)
The principal investigator Alessandro Santin will conduct a review of all adverse events upon completion 
of every study subject. The principal investigator will evaluate the frequency and severity of the adverse 
events and determine if modifications to the protocol or consent form are required.
12.8
ADVERSE EVENT REPORTING 
This study will utilize the CTC version 4.0 for toxicity and Adverse Event Reporting.  A copy of the CTC 
version 4.0 can be downloaded from the CTEP home page.
Toxicity Grade
Typea
Local IRB
Study 
Coordinators
Via 
email/fax/phone
4,5
Unknown
Yes
Yes
5
Known
No
Yes*
2,3
Unknown
No
No
4 
Known
No
No
*  If clearly related to the commercial agent(s)
a  Type (Known or unknown) is based on toxicities included in the package insert or literature of known 
toxicities associated with the study drug(s).
12.9
ASSESSMENT OF SAFETY
12.9.1 SPECIFICATION OF SAFETY VARIABLES 
Safety assessments will consist of monitoring all adverse events and reporting adverse events 
(AEs) and serious adverse events (SAEs) that are considered related to Imiquimod or 9-valent 
HPV vaccine, all events of death, and any study specific issue of concern.
12.9.1.1 Adverse Events
An AE is any unfavorable and unintended sign, symptom, or disease temporally associated with 
the use of an investigational (medicinal) product or other protocol-imposed intervention, 
regardless of attribution.
This includes the following:
 AEs not previously observed in the subject that emerge during the protocol-specified AE 
reporting period, including signs or symptoms associated with high-grade cervical dysplasia 
CIN 2,3 that were not present prior to the AE reporting period. See Section 12.10.
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Complications that occur as a result of protocol-mandated interventions (e.g., cervical biopsy).
If applicable, AEs that occur prior to assignment of study treatment associated with medication 
washout, no treatment run-in, or other protocol-mandated intervention.
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.
12.9.2 Serious Adverse Events
Refer to Section 13.0 of the study protocol.
12.9.3 Methods and Timing for Assessing and Recording Safety Variables
The investigator is responsible for ensuring that all AEs and SAEs that are observed or reported 
during the study, as outlined in Section 12.10 and 13.0 respectively, are collected and reported to 
the appropriate IRB(s).
12.9.4 Adverse Event Reporting Period
The study period during which all AEs and SAEs must be reported begins after informed consent 
is obtained and initiation of study treatment and ends 30 days following the last administration of 
study treatment or study discontinuation/termination, whichever is earlier.  After this period, 
investigators should only report SAEs that are attributed to prior study treatment.
12.9.5 Assessment of Adverse Events
All AEs and SAEs whether volunteered by the subject, discovered by study personnel during 
questioning, or detected through physical examination, laboratory test, or other means will be 
reported appropriately.
Each reported AE or SAE will be described by its duration (i.e., start and end dates), regulatory 
seriousness criteria if applicable, suspected relationship to imiquimod or 9-valent HPV vaccine  
(see following guidance), and actions taken.
To ensure consistency of AE and SAE causality assessments, investigators should apply the 
following general guideline:
Yes
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There is a plausible temporal relationship between the onset of the AE and administration 
of imiquimod or 9-valent HPV vaccine and the AE cannot be readily explained by the 
subject’s clinical state, intercurrent illness, or concomitant therapies; and/or the AE follows 
a known pattern of response to imiquimod or 9-valent HPV vaccine and/or the AE abates 
or resolves upon discontinuation of imiquimod or dose reduction and, if applicable such as 
in case of 9-valent HPV vaccine, reappears upon re-challenge.
No
Evidence exists that the AE has an etiology other than imiquimod or 9-vvalent HPV 
vaccine (e.g., preexisting medical condition, underlying disease, intercurrent illness, or 
concomitant medication); and/or the AE has no plausible temporal relationship to 
imiquimod or 9-valent HPV vaccine administration (e.g., cancer diagnosed 2 days after 
first dose of study drug).
Expected adverse events are those adverse events that are listed or characterized in the Package Insert or 
current Investigator Brochure. 
Unexpected adverse events are those not listed in the Package Insert (P.I.) or current Investigator 
Brochure (I.B.) or not identified.  This includes adverse events for which the specificity or severity is not 
consistent with the description in the P.I. or I.B.  For example, under this definition, hepatic necrosis 
would be unexpected if the P.I. or I.B. only referred to elevated hepatic enzymes or hepatitis. Refer to 
Section 13.0 for further guidelines.
12.10 PROCEDURES FOR ELICITING AND RECORDING ADVERSE EVENTS
12.10.1
Eliciting Adverse Events
A consistent methodology for eliciting AEs at all subject evaluation time points should be adopted.  
Examples of non-directive questions include: 
“How have you felt since your last clinical visit?
“Have you had any new or changed health problems since you were last here?” 
12.10.2
Specific Instructions for Recording Adverse Events
Investigators should use correct medical terminology/concepts when reporting AEs or SAEs.  
Avoid colloquialisms and abbreviations.  All information will be recording using Oncore Clinical 
Trials Management System.
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a. Diagnosis vs. Signs and Symptoms
If known at the time of reporting, a diagnosis should be reported rather than individual signs and 
symptoms (e.g., record only liver failure or hepatitis rather than jaundice, asterixis, and elevated 
transaminases).  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 ok to report the 
information that is currently available.  If a diagnosis is subsequently established, it should be 
reported as follow-up information.
b. Deaths
All deaths that occur during the conduct of the research must be reported to the Yale Principal 
Investigator and the Remote Monitoring Coordinator immediately (if possible) to ensure that all 
over site committees are properly informed.  All information will be entered into Oncore Clinical 
Trials Management System and updated appropriately.  Further determination regarding the need 
to submit to the subjects local IRB, Yale University HHRP following IRB Policy 710 for reporting 
Unanticipated Problems Involving Risk to Subjects will be reviewed. Specific information is 
outlined in section 13.0 of the protocol. 
When recording a death, the event or condition that caused or contributed to the fatal outcome 
should be reported as the single medical concept.  If the cause of death is unknown and cannot be 
ascertained at the time of reporting, report “Unexplained Death”.
c.
Preexisting Medical Conditions
A preexisting medical condition is one that is present at the start of the study.  Such conditions 
should be reported as medical and surgical history.
A preexisting medical condition should be re-assessed throughout the trial and reported as an AE 
or SAE only if the frequency, severity, or character of the condition worsens during the study.  
When reporting such events, it is important to convey the concept that the preexisting condition 
has changed by including applicable descriptors (e.g., “more frequent headaches”).
d. Hospitalizations for Medical or Surgical Procedures
Any AE that results in hospitalization or prolonged hospitalization should be documented and 
reported as an SAE.
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If a subject is hospitalized to undergo a medical or surgical procedure as a result of an AE, the 
event responsible for the procedure, not the procedure itself, should be reported as the SAE.  For 
example, if a subject is hospitalized to undergo coronary bypass surgery, record the heart condition 
that necessitated the bypass as the SAE.
Hospitalizations for the following reasons do not require reporting:
Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures for 
preexisting conditions
Hospitalization or prolonged hospitalization required to allow efficacy measurement for the study 
or
Hospitalization or prolonged hospitalization for scheduled therapy of the target disease of the 
study.
e.
Pregnancy
If a female subject becomes pregnant while receiving imiquimod or 9-valent HPV vaccine series 
or within 30 days of awareness after the last dose of study drug, a report should be completed and 
expeditiously submitted to Yale HIC. Follow-up to obtain the outcome of the pregnancy should 
also occur. 
Abortion of a pregnancy in a study subject receiving Imiquimod, whether accidental, therapeutic, 
or spontaneous, should always be classified as serious, and expeditiously reported as an SAE.  
Similarly, any congenital anomaly/birth defect in a child born to a female subject exposed to 
imiquimod or 9-valent HPV vaccine should be reported as an SAE.
f.
Post-Study Adverse Events
The investigator should expeditiously report any SAE occurring after a subject has completed or 
discontinued study participation if attributed to prior Imiquimod or 9-valent HPV vaccine 
exposure.
If the investigator should become aware of the development of cancer or a congenital anomaly in 
a subsequently conceived offspring of a female subject who participated in the study, this should 
be reported as an SAE. 
13.0 PROCEDURES FOR REPORTING UNANTICIPATED PROBLEMS INVOLVING RISKS 
TO SUBJECTS OR OTHERS, INCLUDING ADVERSE EVENTS (UPIRSOs)
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13.1 Expedited Reporting of UPISROs Occurring at Yale
AEs classified as “serious” and “unexpected” that are possibly, probably, or definitely attributed to drug 
administration, or SAEs whose frequency exceeds expectations, require expeditious handling and 
reporting.
Serious Adverse Event (SAE)
Any adverse event that results in any of the following outcomes:  

death, 

a life-threatening experience, 

inpatient hospitalization or prolongation of existing hospitalization, 

a persistent or significant disability/incapacity, 

a congenital anomaly/birth defect, or

any other adverse event that, based upon appropriate medical judgment, may jeopardize the 
subject’s health and may require medical or surgical intervention to prevent one of the other 
outcomes listed in this definition. 
The PI will promptly investigate all safety information related to an adverse experience.  If the results of 
the PI’s investigation show an adverse drug experience not initially determined to be reportable (based on 
whether the event is serious, unexpected, and associated with drug administration) is so reportable, the PI 
will report such experience.  Follow-up information to a safety report shall be submitted as soon as the 
relevant information is available.
Reporting to the Yale Human Investigation Committee
Timeframe for Reporting 
1. Events that may require a temporary or permanent interruption of study activities by the Principal 
Investigator or sponsor to avoid potential harm to subjects should be reported to the IRB 
immediately (if possible), followed by a written report to the IRB using the UPIRSO Reporting 
Form (710 FR 4) no more than 5 calendar days after the Yale Principal Investigator becomes 
aware of the event. 
                                          
2. Internal Events (defined above) should be reported to the IRB using the UPIRSO Reporting Form 
(710 FR 4) within 5 calendar days of the Principal Investigator becoming aware of the event. 
3. External Events (defined above) should be reported to the IRB using the UPIRSO Reporting Form 
(710 FR 4) within 15 calendar days of the Yale University Principal Investigator (PI) becoming 
aware of the event ONLY IF either of the following are true:
(a) The Yale PI has concluded that an immediate change to the protocol is necessary to address 
the risks raised by the event, and BI agreed to the changes OR 
(b) A monitoring entity (e.g., an external IRB at the site where the problem or event occurred, the 
sponsor, or the Data Safety Monitoring Board) has required modifications/amendments to the 
research protocol or consent documents as a result of the event. 
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For all reports of external events, the UPIRSO Reporting Form (710 FR 4) must include the 
following information: 
(a) a clear explanation of why the event or series of events has been determined to meet criteria 
for reporting; 
(b) a description of the proposed protocol changes and  any corrective actions to be taken by the 
PI in response to the external event; and 
(c) any aggregated data and an analysis or summary from the sponsor or DSMB, when applicable 
and available, sufficient to explain the significance of the event or series of events in order to 
ensure the information is interpretable and relevant to the IRB’s task of protecting the rights 
and welfare of human participants. 
MedWatch 3500A Reporting Guidelines:
In addition to completing appropriate subject demographic and suspect medication information, the report 
should include the following information within the Event Description (section 5) of the MedWatch 
3500A form:

Protocol description (and number, if assigned)

Description of event, severity, treatment, and outcome if known

Supportive laboratory results and diagnostics
Investigator’s assessment of the relationship of the adverse event to each investigational product and 
suspect medication
Follow-up information:  
Additional information may be added to a previously submitted report by any of the following methods:

Adding to the original MedWatch 3500A report and submitting it as follow-up

Adding supplemental summary information and submitting it as follow-up with the original 
MedWatch 3500A form 
Summarizing new information and faxing it with a cover letter including subject identifiers (i.e. D.O.B. 
initial, subject number), protocol description and number, if assigned, brief adverse event description, 
and notation that additional or follow-up information is being submitted  (The subject identifiers are 
important so that the new information is added to the correct initial report)
MedWatch 
3500A 
(Mandatory 
Reporting) 
form 
is 
available 
at
http://www.fda.gov/medwatch/getforms.html 
At all times, the Remote Monitoring Coordinator is available to facilitate submissions and answer any 
questions regarding the process for all sub site staff.
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14.0  STUDY MANAGEMENT AND COORDINATION
14.1
Overview
This is a single site trial where Dr. Alessandro D. Santin is the Principal Investigator. The research team 
at Yale University consisting of a Site Research Coordinator, Data Manager, Clinical Research Nursing 
Coordinator, Specimen Procurement Coordinator and an Administrative Assistant will provide research 
support.  The YCCI clinical trials management support staff will provide clinical database systems support 
and training through the use of Oncore Clinical Trials Management System. 
14.2.    Data Collection
Study will use Oncore Clinical Trials Management System for all data associated with the protocol. Data 
manager will receive Oncore access, complete an Oncore access request and submit HIPAA training 
certification. Individuals will receive Oncore training by a member of the YCCI clinical trials management 
support staff.  Subjects entered into the system will be identified by a subject number assigned after 
randomization. The confidentiality of records that could identify subjects will be protected, respecting the 
privacy and confidentiality rules in accordance with the applicable regulatory requirement(s).  
14.3
Study Enrollment Procedures
Any modification of the original consent document must be approved in advance by the IRB. Rationale 
will be provided with any request for a change. A copy of the IRB and approved informed consent 
document must be on file at the Regulatory office before subjects may be enrolled.  
To register eligible subjects on this study, contact the Site Coordinator; Lisa Baker, RN (203-785-6398) 
or Martha Luther, RN (203-737-2781) and provide the signed and dated eligibility checklist, completed 
signature page of the consent form and additional source documents if requested by the Principal 
Investigator or Site Coordinator. Once the Principal Investigator or Site Coordinator verifies eligibility, a 
unique subject study number will be issued.  The subject will not be identified by name. 
14.4
Responsibilities of the Principal Investigator Dr. Alessandro D. Santin: 
The principal investigator is responsible for the overall conduct of the study and for monitoring the safety 
and quality of the data as well as compliance to the protocol and with applicable federal regulations and 
Good Clinical Practice (GCP). 
The principal investigator will monitor accrual rates for adequate progress.   
The principal investigator will ensure appropriate coordination, submission and approval of the protocol 
as well as the consent documents and any subsequent amendments. There will be only one version of these 
documents and the principal investigator is responsible for assuring that the correct versions are used.
In addition, this study is subject to YCC Discovery Fund Grant Oversight.  The principal investigator will 
provide periodic reports to YCC leadership and attend meetings in accordance with the YCC Discovery 
Fund Grant Monitoring Plan to ensure study progress toward milestones and timelines are being achieved.
14.5   IRB Approvals
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No subjects are to be entered on study without full IRB approval. IRB re-approval must be appropriately 
maintained. Documentation of reapproval must be obtained in a timely manner or registration will be 
halted.
14.6
Amendments and Consents
The Study Coordinator will maintain a copy of all amendments, consent forms, and approvals. Consent 
forms will be reviewed and approved by the Yale IRB. Should changes to the protocol or consent become 
necessary, protocol amendments will be issued by the Yale IRB. 
15.0   DATA ANALYSIS / STATISTICAL METHODS 
The analyses and methods described in this section, for this randomized, Phase II, single-blinded,  3-arm 
clinical trial, apply to the primary, secondary, and exploratory/correlative objectives provided in Sections 
1.1 to 1.3.  
15.1 General Statistical Considerations

Type I error for hypothesis testing will be α = 0.05, and 95% confidence intervals will be used for 
parameter estimation, unless otherwise specified.  Type II error will be based on β = 0.10, unless 
otherwise specified.  Only the primary analysis will use an adjustment for multiple comparisons.

Summary statistics for continuous variables will include (but may not be limited to) the number of 
observations, mean, standard deviation, minimum, median and maximum. For categorical 
variables, summary statistics will include (but may not be limited to) counts, percentages, and 
cross-tabulation tables.

Standard diagnostics will be performed to assess whether the assumptions for specific parametric 
statistical methods are adequately supported by the data.  Should parametric methods be 
inappropriate, the analogous non-parametric methods will be used and only these results will be 
reported.

Imputation of missing values will not be applied.

No interim analyses for efficacy or futility are planned, although the trial may be stopped early 
based on observations from continual safety monitoring.

Data analysis will be conducted using SAS® v9.4 (or later).  
15.2 Analysis Set Definitions

full analysis set—all randomized subjects

safety analysis set—all subjects who have received at least one dose of any study drug, and/or 
have had any assessments at baseline or later that could be associated with an AE; analyzed by 
treatment received

ITT analysis set—all randomized subjects with valid baseline measurements; analyzed by 
treatment assigned

modified ITT analysis set—all randomized subjects with valid baseline measurements,
receiving at least one dose of any study drug, and have at least one valid post-baseline 
measurement of outcome; analyzed by treatment assigned
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
modified ITT completers analysis set—all randomized subjects who receive at least one dose of 
any study drug and have complete and valid post-baseline assessments of outcome; analyzed by 
treatment assigned

per protocol analysis set—all randomized subjects who receive all doses of their assigned study 
drug, have complete and valid post-baseline assessments of outcome, and have no protocol 
deviations (e.g. dose modification, assessment outside of time window, non-compliance)
15.3 Subject Disposition
Using a flowchart with counts and percentages, a breakdown of the study dispositions for all screened 
subjects will be provided. ‘Disposition’ refers to eligibility, consent, enrollment, randomization 
assignment, dosing, drop-out prior to completion of full protocol, completion of protocol, follow-up.  
Reasons for drop-out will also be summarized.
15.4 Demographics, clinical measures, and safety
Summary statistics will be used to describe demographics and clinical measures collected at baseline 
using ITT analysis set, and will be presented by assigned treatment group and overall. Baseline 
characteristics will be tested for homogeneity across treatment arms using a 2-sided Fisher’s Exact Test 
for categorical measures and ANOVA or Wicoxon Rank-Sum Test for continuous variables.  Standard 
tables and listings will be provided for ongoing assessments, including (but not limited to) study drug dose 
received, treatment modifications, concomitant medications, labs, vital signs, and clinical tests, and will 
be reported by subject, by time point, and by treatment group, as appropriate.  AEs/SAEs will be reported 
by subject, by time point, and by treatment group, using the safety analysis set.
15.5 Analysis for the Primary Objective
The co-primary objectives of this study are to test superior treatment efficacy of Imiquimod + HPV 
vaccine (Trt1) compared to control (Trt0), and likewise Imiquimod only (Trt2) compared to control (Trt0). 
The primary outcome is histologic regression (yes/no) after completion of the 12 to 16 weeks imiquimod 
course. The full definition of histologic regression and how it is determined is described in Section 5.1. 
The co-primary hypothesis tests are: 
{H01: p1 - p0 = 0 versus H11: p1 - p0 > 0} 
and
{H02: p2 - p0 = 0 versus H12: p2 - p0 > 0}
p0, p1, and p2 are the proportion of subjects on Trt0, Trt1, and Trt2, respectively, who are determined to 
have histologically regressed.  Fisher’s Exact Test will be used to assess if the data support rejecting the 
null hypothesis H01, and likewise for H02.  To control Type I error, we will use a conservative Bonferroni 
adjustment for multiple comparisons with one-sided α = (0.05)/2 = 0.025 as the significance level for each 
test.  Rejection of a particular null hypothesis suggests superior efficacy of that specific treatment 
compared to control. Exact 95% confidence intervals will be calculated for p1 - p0 and for  p2 - p0.  
Confidence intervals for ‘risk ratios’ (p1/p0, p2/p0) in addition to ‘risk differences’ (p1 - p0, p2 - p0)  may 
also be estimated.  The main analysis will use the modified ITT analysis set.  Sensitivity analyses for the 
same statistical method using the modified ITT completers and/or per protocol analysis sets may also 
be provided.
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15.6 Analysis for the Secondary Objectives
The secondary objectives are:

to estimate treatment efficacy of Imiquimod + HPV vaccine (Trt1) compared to control (Trt0), and 
likewise Imiquimod only (Trt2) compared to control (Trt0), for the secondary outcome histologic 
remission (yes/no) after completion of the 12 to 16 weeks imiquimod course. Exact 95% 
confidence intervals for p1 - p0 and for p2 - p0 will be calculated.  The main analysis will use the 
modified ITT analysis set.  Sensitivity analyses for the same statistical method using the modified 
ITT completers and/or per protocol analysis sets may also be provided.

to estimate treatment efficacy of Imiquimod + HPV vaccine (Trt1) compared to control (Trt0), and 
likewise Imiquimod only (Trt2) compared to control (Trt0), for the secondary outcome HPV 
clearance (yes/no) after completion of the 12 to 16 weeks imiquimod course. Exact 95% 
confidence intervals for p1 - p0 and for p2 - p0 will be calculated. The main analysis will use the 
modified ITT analysis set.  Sensitivity analyses for the same statistical method using the modified 
ITT completers and/or per protocol analysis sets may also be provided.

to characterize treatment safety and tolerability using descriptive statistics (safety analysis set)
15.7
 Exploratory/Correlative Objectives
To characterize the association of  T cell infiltration into the cervical dysplasia (i.e. frequencies and 
anatomical distributions of CD4 and CD8 T cells within the CIN)  and pre- and post-immune therapy 
responses to  HPV16/18 E7 antigens in subject peripheral blood lymphocytes (PBL) and serum with 
clinical outcomes (histologic regression/remission) and viral clearance. TRM frequencies ,distributions 
and responses will be measured within subjects (pre and post ‘prime and pull’), and changes from baseline 
will be compared between the treatment groups at relevant time points. Descriptive summary statistics 
will be used to evaluate associations and ANOVA will be the used to test the statistical significance of 
associations. The main analysis will use the modified ITT analysis set.  Sensitivity analyses for the same 
statistical method using the modified ITT completers and/or per protocol analysis sets may also be 
provided.
15.8 Sample Size Justification
Anticipated annual accrual is approximately 40-45 subjects. 
Our general philosophy regarding Phase II trials of new drug combinations is to facilitate the prompt 
discovery of inactive agents, yet allow for a reasonable estimate of the response rates for those regimens 
demonstrating some degree of activity. However, excessive accrual to inactive study regimens to 
document precise response rates is to be avoided. The purpose of this study is to evaluate the activity and 
potential toxicity of the combination therapy of Imiquimod + HPV vaccine in subjects with untreated 
high-grade CIN 2,3 cervical dysplasia. This study will implement a 3 arm randomized trial design that 
will allow us to estimate the treatment effect of Imiquimod + HPV vaccine compared to observation as 
well as Imiquimod alone compared to observation. 
Based on published literature for the primary outcome,38 we assume a 28% spontaneous histological 
regression rate in CIN 2-3 lesions with observation alone (control group). From Grimm et al.’s efficacy 
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trial of imiquimod for treatment of CIN 2-3, we would like to have sufficient power to detect a 35% 
absolute increase in rate of histologic regression in the imiquimod only arm compared to control. Based 
on this data, we consider a conservative effect size estimate also of an absolute 35% higher rate of 
histologic regression in the imiquimod + HPV vaccine intervention arm compared with the control arm. 
We therefore aim to recruit a total sample size of 114 subjects (38 in each arm). Calculations based on 
Fisher’s Exact Test indicate we will have ≥ 90% power to detect a treatment effect of ≥ 29% in both 
treatment arms independently compared to the observation arm (α = 0.025 for each comparison to control). 
In addition, with n = 38 subjects per arm, we would have 80% power to detect a treatment effect size of ≥ 
25% in one treatment arm compared to placebo (while maintaining 90% power to detect the treatment 
effect size of ≥ 29% in the other arm). Assuming a subject dropout rate of 20%, a total sample size of 138 
(n = 46 per arm) subjects is calculated which will be recruited over 5 years.
Some operating characteristics of the study design are as follows:
Exact Operating Characteristics for the Treatment Effect Size Δ
given control group rate=0.28, n=38 subjects per arm, one-sided α
Exact binomial probabilities for α given minimum required power = (1-β)
Δ =
0.00
0.10
0.20
0.30
0.32
0.35
0.37
0.40
(1-β ) ≥ 0.80
0.8080
0.8008
0.8101
0.8126
0.8011
0.8168
0.8119
0.8113
α =
0.5321
0.7745
0.1537
0.0366
0.0220
0.0118
0.0058
0.0027
(1-β) ≥ 0.90
0.9033
0.9009
0.9049
0.9060
0.9068
0.9062
0.9012
0.9009
α =
0.4835
0.6574
0.2876
0.0863
0.0616
0.0363
0.0203
0.0103
Exact binomial probabilities for power = (1-β) given maximum required α
Δ = 
0.00
0.10
0.20
0.30
0.32
0.35
0.37
0.40
α ≤ 0.025
0.0147
0.0147
0.0143
0.0140
0.0140
0.0143
0.0146
0.0149
1-β =
0.0147
0.1050
0.3472
0.6860
0.7472
0.8269
0.8706
0.9215
α ≤ 0.05
0.0305
0.0327
0.0272
0.0304
0.0317
0.0330
0.0331
0.0323
1-β =
0.0305
0.1795
0.4847
0.7870
0.8328
0.8898
0.9197
0.9535
Software: G*Power version 3.1.9.2, ©1992-2014, Franz Faul, Universität Kiel, Germany
At 7-8 months from time of initial CIN 2,3 diagnosis, persistent or progressing dysplasia will be surgically 
excised per current standard of care. Risk of progression of high-grade cervical dysplasia during the study 
period in the observation group is very low and observation periods of 6-9 months for CIN 2,3 are 
commonly described in the literature.24,39 In this regard, studies estimating the risk of clinical progression 
from CIN2/3 lesions to cervical cancer found that the median time was 23.5 years with only 1.6% of these 
lesions becoming invasive within 10 years40. In addition, any clinical concern for disease progression 
based on history, physical exam or colposcopy at an interval follow up visit will trigger cervical biopsies 
to objectively evaluate histopathology and, should disease progression be noted, the subject will be 
removed from study and offered surgical excision. We will also employ early stopping rules as follows: 
Evaluability for Efficacy and Toxicity:
Only those subjects who are deemed “ineligible” will be eliminated from the analysis. All subjects who 
receive any therapy will be evaluated for both treatment efficacy and toxicity. While on occasion, 
circumstances may prevent the determination of treatment efficacy, such subjects will be included in the 
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analysis and labeled as “unknown”. This category will be listed and be reflected in the calculation of the 
response rate.
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