A Randomized Controlled Trial Comparing Cure Rates of Cervical Intraepithelial Neoplasia Grade 2 and Higher (CIN2+) Treated With CO2-based Cryotherapy, CropPen, and Thermoablation (UH3)
Phase: NA
Conditions: Cervical Intraepithelial Neoplasia
Sponsor: The Cleveland Clinic

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
v. 04 Date: April 20, 2020 
 
 
 
Research Protocol  
A randomized controlled trial comparing cure rates of cervical 
intraepithelial neoplasia grade 2 and higher (CIN2+) treated 
with CO2-based cryotherapy, CryoPen, and thermoablation 
 
 
 
 
Sponsor: National Cancer Institute- UH3CA189883 
 
 
 
 
 
 
 
 
 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
v. 04 Date: April 20, 2020 
Table of Contents  
 List of Abbreviations  
1.0  
Background and rationale 
 
2.0  
Study objectives 
 
3.0  
Study design and statistical analysis  
 3.1  
Study design  
 3.2  
Sample size and statistical analyses  
4.0  
Research participants  
 4.1  
Characteristics of research participants 
 
 4.2  
Inclusion and exclusion criteria 
 
5.0  
Study procedures 
 
 5.1       Initial Exam 
 5.2  
Study procedures for cervical ablations 
 
 5.3  
Laboratory evaluation  
6.0  
Recruitment 
 
7.0  
Consent process 
 
8.0  
Study instruments 
 
9.0  
Risks  
10.0 Mitigation of risks 
 
11.0 Benefits 
 
12.0 Study and safety monitoring  
13.0 Managing and reporting adverse events and unanticipated problems  
 13.1  Adverse Events 
 
 13.2  Serious Adverse Events 
 
 13.3  Reporting procedures for AEs, SAEs, and unanticipated problems  
14.0 Confidentiality and data management  
15.0 Study costs 
 
16.0 Care for injury  
17.0 Compensation  
18.0 Investigator responsibilities 
 
 Appendices 
 
 
 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
v. 04 Date: April 20, 2020 
List of Abbreviations 
AE  
 
Adverse Event 
C  
 
Celsius 
FDA  
Food and Drug Administration, United States 
HPV  
Human papillomavirus 
IRB 
 
Institutional Review Board 
kg   
 
kilograms 
mm  
 
millimeters 
PI  
 
Principal Investigator 
PID 
 
Pelvic inflammatory disease 
REC 
 
Research Ethics Committee 
SAE 
 
Serious Adverse Event 
SCJ 
 
Squamo-Columnar Junction 
 
 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
v. 04 Date: April 20, 2020 
1.0 Background and rationale  
Cancer of the cervix is preventable but continues to cause the deaths of an estimated 
275,000 women worldwide each year, more than 88 percent of them in developing 
countries.1 Vaccination against human papillomavirus (HPV), a means of primary 
prevention, has been shown to be over 90 percent effective in protecting against the 
lesions that lead to the majority of cervical cancer cases, but only if administered before 
viral infection,2,3 which occurs soon after initiation of sexual activity.4 Secondary 
prevention is accomplished with cervical screening followed by treatment of precancer 
and is essential for protecting women who have been sexually active. The World Health 
Organization recently declared that “screening for cervical cancer once, at age 40, 
followed by removal of any discerned precancerous lesion” is a “best buy.”5 Best buys 
are defined as actions that should be undertaken immediately to produce accelerated 
results in terms of lives saved, diseases prevented, and heavy costs avoided. 
Screening, with treatment when necessary, has been the key to the 70 percent or more 
reduction in cervical cancer cases in industrialized countries over the past 50 years, and it 
is critical to reducing the huge burden of cervical cancer in low-resource countries. 
Screening technologies appropriate to low-resource settings have advanced considerably 
in the past decade, but a key component of any effective screening strategy is treatment—
and treatment technologies have lagged behind. Treatment methods for developing 
countries must be low-cost, effective, and adaptable for areas with limited resources in 
terms of infrastructure and health care providers.  
 
Cryotherapy, the current gold standard for treatment of precancerous cervical lesions in 
low resource settings,6-9 has many appealing features; however, in recent years a number 
of issues—particularly the use of compressed gas, which requires storage and 
replenishment of large and potentially dangerous tanks—have emerged as clear 
limitations.  
 
Two technologies—the CryoPen®, manufactured and sold by CryoPen®, Inc. (TX, USA), 
and the cold coagulator commercialized by WISAP Medical Technology (Germany) are 
treatment options for precancerous cervical lesions that run on electricity and avoid the 
difficulties associated with compressed gas. The CryoPen® is a treatment option for 
precancerous cervical lesions that are able to meet core criteria of cost, safety, and 
potential for use in remote campaign locations as well as in clinic settings. The CryoPen® 
differs from previous cryotherapy methods in that it does not require an external source 
of gas (a tank), since the gas for cooling is built into the system. The original device has 
been redesigned to be portable, durable, low-cost and low-maintenance for use in low- 
and middle-income countries (LMICs). 
 
Cold coagulation (“thermocoagulation”) has been used since the 1970s for treatment of 
cervical lesions. In spite of its name, this is treatment with a probe heated to temperatures 
of 100 to 120ºC. Henceforth this treatment method will be referred to as 
“thermoablation.” It is also an effective, inexpensive method that does not require high-
level healthcare providers.  
 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
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The purpose of this study is to conduct a randomized non-inferiority clinical trial to 
compare the LMIC-adapted CryoPen® and WISAP C3 thermoablator to CO2-based 
cryotherapy for the treatment of CIN2 or more severe diagnoses (“CIN2+”) but excluding 
microinvasive or invasive cervical cancer. 
 
2.0 Study objectives 
 
The primary objective of the study is to compare CIN2+ cure rates at twelve-months 
post-treatment with the LMIC-adapted CryoPen®, the WISAP thermoablator, and CO2-
based cryotherapy. 
 
Secondary objectives of the study are:  
● To measure patient acceptability of the CryoPen® and thermoablator with pain scores 
and surveys  
● To actively assess side effects of cryotherapy and thermoablation including infection, 
pain, bleeding, discharge, and stenosis at a six-week post-treatment visit 
● To evaluate the sensitivity of high-risk HPV DNA testing post-ablation  
 
3.0 Study design and statistical analysis 
3.1 Study design 
 
Women will be recruited from the Salvadorian Social Security Hospital (ISSS) in San 
Salvador, El Salvador, Hospital Universitario San Ignacio (HUSI) and Ginesalud SAS in 
Bogota, Colombia and  Shanxi Bethune Hospital – Shanxi Academy of Medical Sciences 
(SBH) in Taiyuan, China.  Women attending a regional screening program will also be 
recruited at three hospitals in Xinxiang, Henan Province, China: Yanjin County Women 
and Children Hospital, Fengqiu County Women and Children Hospital, and Weihui 
Municipal Hospital for Women and Children Healthcare.  
 
All hospitals are referral sites for women with abnormal cytology.  
 
Eligible women will be those over the age of 18 with biopsy results of CIN2+ (high grade 
CIN not otherwise specified) who consent to participate after being informed about the 
study and are willing and able to provide a permanent and reliable address. Women will 
be excluded if they are pregnant or plan to become pregnant during the study, have had 
previous surgery destructive to the cervix in the last 5 years, or have a history of total 
hysterectomy. 
 
Women who meet the eligibility criteria listed above, will be invited to participate in the 
study.  The diagnosis of CIN2+ will be based on the local pathology diagnosis. 
 Women will be randomly assigned to one of three study arms: 
A. CO2-based cryotherapy (double freeze) 
B. LMIC-adapted CryoPen® (single freeze)  
C. Thermoablation for 40 seconds at 100ºC followed by additional 20-second 
applications if necessary according to clinician’s criteria in order to fully 
ablate the squamo-columnar junction (SCJ).  

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
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Recruitment, enrollment, and patient flow are summarized in Figure 1. 
 
Figure 1 Consort diagram of study patient flow 
The single or double freeze modalities were chosen given the results of the UH2 trial 
where single-freeze CryoPen® achieved a greater depth of necrosis than double freeze, 
and the WHO cryotherapy guidelines which endorse CO2 double-freeze as the standard 
cryotherapy treatment. While these are the preferred modalities, in a few cases clinicians 
may decide to repeat the CryoPen® modality to correct placement of the first application 
and maximize treatment efficacy and patient safety.  
 
We will approach approximately 1,602 women with CIN2+ during a 2.5 year enrollment 
phase (30 months). Assuming an approximately 80% participation rate,10 approximately 
1,281 women will be enrolled— 1,152 (90%) will be eligible for cryotherapy (i.e. no 
contraindications) and 129 will be ineligible and undergo alternative treatments. We 
estimate that approximately 20% will be lost to follow-up at the twelve-month visit, so 
that we will have complete data on a total of 922 women (57.5% of 1,602) treated with 
either CO2-based cryotherapy, LMIC-adapted CryoPen®, or thermoablation. We will 
monitor the percentages of ineligibles, contraindications, and loss to follow-up in real 
time and adjust recruitment accordingly to achieve the target sample size. In addition, we 
will conduct a non-essential 6-week follow-up visit to evaluate adverse effects from the 
ablative procedures. The primary endpoint of the study will be to determine residual 
disease at a 12-month follow up visit as determined by colposcopy and four-quadrant 
biopsies. 
 
Enrollment and Treatment 
Enrollment diagnosis will be based on the local pathology. These slides will be read by 
an expert gynecologic pathologist for quality assurance. At the pre-treatment visit, 
consented women will be administered a short questionnaire to collect demographic 
Women with 
CIN2+ (1,602)
Ineligible/No 
Consent
Local Standard 
of Care
Eligible and 
Consenting 
(1,281)
Cryo Eligible?
Yes (1,152)
Randomizatio
n
WISAP 
thermoablator
LMIC-adapted 
CryoPen
CO2-based 
cryotherapy
12-month follow-up
(930)
VIA, cytology, HPV 
testing, 4-quadrant 
biopsy
CIN2/3
No (129)
LEEP/CKC

 
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information and health history. Women will receive a pregnancy test as part of the 
eligibility criteria for enrollment in the study. Vaginal samples will be collected to be 
analyzed for bacterial vaginosis, yeast, gonorrhea, trichomonas, and chlamydia. Samples 
will be collected again at the 6-week follow-up visit to conduct the same analyses. 
Pathology samples from Latin America will be sent to the Pathology Department at the 
Medical College of Wisconsin (Milwaukee, WI) for molecular analyses. The China 
samples will be analyzed in-country at both SDH and Hybribrio labs using molecular 
tests as well. In addition, the ability to pass the cervical swab used for vaginal samples 
into the endocervix will be recorded at both the pre-treatment and the six-week follow-up 
visits to gather information about treatment-induced stenosis. All women will also 
undergo HPV genotyping with next-generation sequencing. The specimen is taken prior 
to treatment to establish a baseline of which HPV types are present. If a different HPV 
type is present in the post-treatment specimen, this will be classified as a new rather than 
persistent infection. Evaluation of the HPV genotyping from Latin America will be 
conducted at the National Cancer Institute in the United States and in China, where these 
samples will be analyzed at local labs. All samples that are sent to the United States for 
analysis will be shipped using a specialized courier service.  
 
All women will also undergo a pelvic exam and visual inspection of the cervix following 
placement of diluted acetic acid on the cervix. Women will be immediately withdrawn 
from the study if any of the following contraindications for cryotherapy are present on 
visual inspection 1) lesion extends to over 75% of the cervix, 2) lesion is too large to be 
covered by the cryotherapy probe, 3) lesion extends into the endocervical canal, 4) the 
cervix is distorted or has severe atrophy; 5) the cervix is not visible, 6) the squamo-
columnar junction is not completely visible, 7) the cervix is hard to reach, and/or 8) 
suspicion of cancer. Women deemed ineligible for cryotherapy will undergo LEEP 
immediately. In the case of suspected cancer, women will be appropriately referred. Once 
a patient is deemed eligible for cryotherapy, the study coordinator at each site will log 
into REDCap (a secure, HIPAA-compliant web application for building and managing 
surveys and databases) to obtain the randomized treatment assignment.  
 
Women will then undergo treatment with the LMIC-adapted CryoPen®, the WISAP 
thermoablator, or CO2-based cryotherapy. Patient pain will be evaluated four times 
during the treatment visit as described in section 5.2 below. 
 
Images of the cervix will be taken with the EVA System Mobile ODT device before and 
after treatment. De-identified cervical images from the initial visit and the follow-ups will 
be made available to Mobile ODT. The company will use these images to help develop 
an Automated Evaluated System (AVE), a software algorithm to detect pre-cancerous 
lesions through images rather than laboratory tests. This algorithm is currently in 
development and the more images it “learns”, the more accurate results it can obtain. If 
successful, AVE can greatly ease diagnosis and triage in low-income settings. 
 
 
 
 

 
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Six-week follow-up 
Six weeks post-treatment, women will return for a visit to evaluate side-effects. Data will 
be collected on women who attend this visit at any time between 4 to 12 weeks post-
treatment. If a woman does not attend this visit, she will still remain in the study and be 
invited to return at 12 months post-treatment. At the 6-week visit, women will complete a 
questionnaire to assess the presence of pain or cramps, bleeding, watery discharge, and 
malodorous discharge. Women will also be asked some questions about their attitudes 
and perceived acceptability of the treatment they received. Finally, women will be asked 
to answer a checklist to assess their compliance with post-treatment recommendations. In 
addition, a pelvic exam will be performed and vaginal samples will be collected and 
analyzed for bacterial vaginosis, yeast, gonorrhea, trichomonas, and chlamydia. If an 
infection is suspected, the provider may prescribe medication as necessary. The provider 
will note whether a vaginal culture swab can be introduced into the endocervix. If it 
cannot, stenosis will be diagnosed.  Women will also be evaluated for acute clinical 
cervicitis at the follow-up visit and this will be recorded.  Another photo will be taken 
with the Mobile ODT device.  
 
12-month follow-up 
One year post-treatment, women will return for a follow-up visit to determine residual 
disease, which is the primary endpoint. Residual disease will be determined by 
colposcopy and four-quadrant biopsies. In addition, VIA, cytology, and high-risk HPV 
DNA testing with careHPV will be performed. Genotyping and next-generation 
sequencing will be repeated to differentiate between persistent and new infections. The 
goal is to evaluate which test has the highest sensitivity after an ablative treatment: VIA, 
cytology, or HPV. 
 
We will use local health systems, including healthcare promoters, to encourage women to 
return for the follow-up visit. Attempts to contact women who do not return for this visit 
will continue until 18 months post-treatment. If a woman does not return within this time 
period, she will be considered lost to follow-up. 
 
At the 12-month visit, the following procedures will be performed:  
1. Pelvic examination 
2. Urine pregnancy test 
3. Unaided VIA 
4. Cytology 
5. High-risk HPV testing with careHPV 
6. Colposcopic evaluation and impression  
7. Four-quadrant microbiopsy protocol using the protocol for sensitive detection 
of any recurrent/untreated CIN2+.11 All visible lesions in each quadrant of the 
cervix (12-3, 3-6, 6-9, and 9-12 o’clock) will be biopsied. Endocervical 
curettage (ECC) will be performed on all patients. If no lesion is visible in a 
given quadrant, the most suspicious appearing area in that quadrant will be 
biopsied. 
8. Cervical swab collection for HPV genotyping using next-generation 
sequencing (NGS) 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
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Treatment acceptability questions will again be administered at the end of this visit. 
Women diagnosed with recurrent/untreated CIN2+ on biopsy will be asked to return to 
undergo LEEP. Women with suspected cancer on biopsy will be referred to the local 
cancer hospital for standard of care. 
 
3.2 Sample size and statistical analyses 
 
Treatment efficacy 
Analytic Approach. The primary analysis will follow an “intent to treat” approach, and 
we will conduct an additional sub-analysis excluding patients referred to excisional 
treatments after randomization. Similarity of the two study arms in terms of follow-up 
rates, distribution of individual and demographic variables, and disease distributions will 
be assessed. We will calculate the cure fraction and the corresponding binomial 95% 
confidence interval (95% CI) of each treatment as the percentage of women diagnosed 
with CIN2+ at the twelve-month visit. The non-inferiority of the LMIC-adapted 
CryoPen® and the WISAP thermoablator relative to CO2-based cryotherapy will be 
assessed by calculating 95% CIs for the differences (LMIC-adapted CryoPen® minus 
CO2-based cryotherapy and WISAP thermoablator minus CO2-based cryotherapy) in cure 
fractions. Non-inferiority will be concluded if the lower confidence bound is greater than 
the non-inferiority margin (-10%). Exploratory analysis of the effectiveness and 
differences between arms will be evaluated within strata defined by quartiles of age and 
by the baseline diagnosis. Both the local and expert pathologists’ diagnoses will be used 
in these analyses. As warranted, a logistic regression model will be used to mutually 
control for all important variables and chance differences between study arms to identify 
the key determinants of treatment failure and differences (if any) in effectiveness between 
treatments.  
 
Statistical Power and Sample Size. With a total of 922 women with a diagnosis of 
CIN2+, 307 per treatment arm, the study will have at least 80% power (alpha = 0.05) to 
detect a non-inferiority margin difference of 10% between the cure fraction of CO2-based 
cryotherapy (reference) and of the LMIC-adapted CryoPen® (experimental) and the 
WISAP thermoablator (experimental), for a range of efficacy (50% to 80%) for the 
reference treatment and 40% efficacy under the null hypothesis of inferiority. 
We chose a range of 50-80% efficacy for cryotherapy because although the literature 
suggests CO2-based cryotherapy is 80% effective, the disease ascertainment by 
colposcopy following treatment in previous studies was before the recognition that 
colposcopy is not perfectly sensitive. Therefore, the disease ascertainment in those 
studies was not as rigorous as proposed in this study. To ensure that the study is 
sufficiently powered for a wide range of efficacies, we are selecting 50% efficacy for the 
control arm as the worst-case scenario for study power. In order to achieve a sample size 
of 922 with complete follow-up, we will enroll 1281 women with CIN2+ under the 
assumption that 10% will have contraindications for cryotherapy and 20% will be lost to 
follow-up at twelve months post-treatment. 
 
 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
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Test of cure 
Analytic Approach. Since all women returning for the twelve-month visit will undergo 
four-quadrant biopsies and LEEP if diagnosed with CIN2+, regardless of the baseline 
characteristics or ablative treatment, we can assess the test of cure performance 
(sensitivity, specificity, positive predictive values (PPV), and negative predictive values 
(NPV)) of each test (VIA, cytology, and high-risk HPV DNA) for recurrent CIN2+ and 
CIN3 diagnosed by local and expert pathology. Performance will be stratified by 
quartiles of age and by the baseline diagnosis. McNemar chi-square will be used to test 
whether differences in sensitivity and specificity are significant (p < 0.05). A method 
developed by Leisenring et al.12 will be used to test for significant differences in PPV and 
NPV. 
 
Statistical Power. The statistical power for comparing sensitivity for the test of cure for 
VIA, cytology, and high-risk HPV testing is dependent on sample size of the efficacy 
study and the actual efficacy. We are enrolling 1281 women to assure that we have 930 
women with a twelve-month follow-up. The larger the number of women who return for 
their follow-up, the larger the sample size for a given level of efficacy will be. The more 
efficacious the treatments, the smaller the sample size will be. Assuming 70% efficacy in 
treatment (30% recurrence), results of tests for cure will be available for 279 women with 
recurrence. This will provide 85% power (0.05 two-sided alpha) to detect a 10% 
difference in sensitivity between tests assuming 20% discordance between tests. 
 
Interim Analysis of Cure Fractions. An interim analysis of cure fractions will be 
performed after approximately 2 years of study opening, when approximately 1/3 of the 
women have been enrolled. A confidence interval will be calculated for the difference 
(LMIC-adapted CryoPen® minus CO2-based cryotherapy and WISAP thermoablator 
minus CO2-based cryotherapy) in cure fractions. If the upper bound of the confidence 
interval is lower than the non-inferiority margin (-10%), then treatment modification or 
termination may be considered. The confidence interval will be calculated using an alpha 
calculated using an alpha time-squared spending function to maintain an overall 5% Type 
I error (one-sided). 
 
4.0 Research participants 
4.1 Characteristics of research participants 
 
The study will enroll eligible, consenting women aged 18 and older. Eligible women will 
be informed about the study and invited to participate. The legal age of consent in El 
Salvador, Colombia and China is 18 years of age.  
 
4.2 Inclusion and exclusion criteria 
Women who present to the health facility for biopsy results and meet the study criteria 
will be invited to participate in the study. We will collect delinked, anonymous, pooled 
data (e.g., age, geographic area of residence, and diagnosis) on all women, including 
those who decline to participate, to characterize them and assess whether our study 
population is representative of the general population. 
 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
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Criteria for Inclusion 
● Women aged 18 and older  
● Biopsy results of CIN2, CIN2-3, CIN3, or high-grade CIN not otherwise specified 
(quality assurance will be performed by an expert gynecologic pathologist) 
● Willing and able to provide informed consent 
● Willing and able to provide permanent or reliable address 
 
Criteria for Exclusion 
● Pregnant or plans to become pregnant during study 
● History of total hysterectomy (verified by medical record or pelvic evaluation) 
● Previous surgery destructive to the cervix within the last 5 years 
● Patient not eligible for cryotherapy (1) lesion extends to over 75% of the cervix, 
2) lesion is too large to be covered by the cryotherapy probe, 3) lesion extends 
into the endocervical canal, 4) the cervix is distorted or has severe atrophy; 5) the 
cervix is not visible, 6) the squamo-columnar junction is not completely visible, 
7) the cervix is hard to reach, and/or 8) suspicion of cancer) 
● Cervix shape disfigured or hard to reach  
 
4.3 Withdrawal from the study 
Women who sign the informed consent to participate in the study have the right to 
withdraw their consent at any time during the study. Physicians performing the ablation 
procedures have the right to remove participants from the study if, upon pelvic 
examination, the condition of the cervix is not conducive to continuing the procedure.  
 
5.0 Study procedures 
5.1 Initial Exam 
 
After a woman consents to participate in the research study and meets other eligibility 
criteria including testing negative on a pregnancy test, she will answer a short survey 
with demographic and life history information relevant to the study (i.e., age, location of 
residence relative to clinic, smoking and sexual history, parity and history of vaginal 
surgeries). Next, the provider will initiate the ablation procedure by performing a pelvic 
exam. If the provider determines during this exam that the condition of the cervix is not 
conducive to an ablation procedure for anatomical reasons, the participant will be 
removed from the study.  
 
5.2 Study procedures for cervical ablations 
For the traditional cryotherapy arm, providers will use a MedGyn cryotherapy unit with a 
19 or 25 mm conical tip supplied with a refillable tank of medical grade CO2. For the 
experimental CryoPen® arm, providers will use LMIC-adapted CryoPen® prototype with 
a 19 mm conical tip. For the thermoablator arm, providers will use a handheld WISAP 
thermoablator (model C3) with a 20 mm conical tip.  
 
Anesthesia is not required for cervical ablation procedures and none will be provided 
routinely in this research study. Analgesics may be prescribed for discomfort after the 
procedure. Study coordinators will verbally ask study participants to assess their pain 

 
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using the Wong Baker Faces Scale (Figure 2),13 a well-validated and widely used self-
assessment tool. This scale was originally developed for use with children but is now 
available in many languages, including Spanish and Mandarin Chinese, and is used to 
improve communication between patients and providers. It utilizes five simple drawings 
of faces depicting different levels of pain, each shown above a number from 0 (no pain) 
to 10 (worst possible pain). Patients can use the expressions on the faces to rate their own 
experience according to the corresponding number. Pain will be assessed at 4 points: 
before speculum insertion (baseline), after speculum insertion but before treatment, 
during treatment, and after speculum removal. 
 
 
Figure 2. FACES and numeric pain rating scale 
Cervical photographs will be taken immediately pre- and post-procedure for participants 
who have consented to photographs. Photographs will provide additional documentation 
of the cervix anatomy to corroborate the clinical evaluation whether the anatomy of the 
cervix could have interfered with a good contact by the treatment probe at cervix 
positions 3, 6, 9 or 12 (as on a clock face).  
 
Each study procedure is estimated to take approximately 10-15 minutes. The remainder 
of the time will be spent preparing the participant and taking cervical photographs. 
The six-week and 12-month follow-up visits (described in section 3.1) are expected to 
take about 15 minutes. All women will undergo liquid-based cytology, VIA, and HPV 
testing. Women suspected of having invasive cancer at the follow-up visit will be referred 
for appropriate management. 
 
5.2.1 CO2-based cryotherapy procedure 
• Study physicians will use MedGyn cryotherapy units supplied with a refillable tank of 
medical grade CO2 and a 19 or 25 mm conical tip. 
• A pressure gauge attached to the compressed gas tank shows the cylinder gas 
pressure. If there is adequate gas pressure in the cylinder (40-74 kg/cm2), the 
indicator moves to the green zone in the gauge when the cryogun releases the gas. 
The pressure must be in the green zone or there will be insufficient freezing for 
cryotherapy.  
• The cervix is exposed after a speculum is placed to separate the vaginal walls.  
• The cryotherapy probe tip is applied with the center of the tip on the external os. The 
probe should adequately cover the lesion and should not come in contact with any 
part of the vagina during the procedure.  
• The gas trigger in the cryogun is squeezed to administer the freeze. An ice ball will 
form on the tip of the cryoprobe and on the cervix throughout the freeze.  

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
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• A double freeze is applied.  
• The cryoprobe is removed by gently rotating on the cervix once complete thawing has 
occurred and the ice ball has cleared. 
• The cryoprobe and tip must be disinfected using a high-level disinfectant. 
 
5.2.2 CryoPen® procedure 
• Study physicians will use the LMIC-adapted CryoPen® with a 19 mm conical tip. 
• The CryoPen unit is plugged into an electrical outlet. The well is filled with ethanol. 
The unit is ready for use when the light on the temperature indicator turns green. 
• The tip is placed on the applicator and twisted in place. 
• The core is removed from the unit and the well is closed to prevent the well from 
freezing. 
• The core is inserted into the applicator and pulled against the core handle to hold the 
device together. 
• The core and the applicator are inserted together into the vagina and the tip is placed 
on the cervix.  
• When the procedure is completed the core is removed first. Once complete thawing 
has occurred, the applicator is then removed by gently rotating the tip. 
• If the entire SCJ is not frozen, providers may repeat the application once more until 
the entire SCJ is ablated. 
• The core is then returned to the CryoPen® unit. Frost must be wiped from the core 
with a towel before the core is inserted into the well. 
• The applicator must be discarded and the core tip must be disinfected using a high-
level disinfectant. 
 
5.2.3 Thermoablator 
• Study physicians will use a handheld thermoablator with a 20 mm conical tip, 
manufactured and sold by WISAP Medical Technology (Germany).  
• The probe and protector slider are connected to the unit and the unit is plugged into 
an electrical outlet. 
• The unit is turned on when the power button is pressed to the “on” position. The 
switch illuminates green and starts blinking as the therapy probe tip begins to heat up. 
• Unit is ready for use when the green temperature indicator light stops blinking. 
• The physician slides the protector forward to cover the probe tip.  
• When the probe makes contact with the cervix, the protector is moved back.  
• The probe tip is applied to the cervix for 40 seconds. 
• The protector is slid back to cover the tip. 
• 
The probe is removed and the physician visually evaluates the cervix to ensure the 
entire SCJ has been ablated. 
• 
In some cases, providers may continue with additional 20 second applications until 
the entire SCJ is ablated. 
• When the procedure is completed the unit is turned off and the probe and protector 
are removed for disinfection with a high-level disinfectant. 
 
 

 
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5.3 Laboratory evaluation 
A local pathologist will evaluate initial and follow-up cervical biopsy specimens. 
Enrollment diagnosis will be based on the local pathology. These slides will also be 
reviewed by an expert gynecological pathologist for quality assurance. At the one year 
follow-up visit, women will have colposcopic-directed biopsies. This pathology will be 
read by both a local and an expert gynecologic pathologist. The primary endpoint of the 
study will be the consensus achieved by the local and expert pathologists on the follow-
up biopsy samples taken from the colposcopic-directed biopsies at the one year follow up 
visit. Discrepant cases will be discussed between the two pathologists and if agreement 
cannot be reached, the case will be reviewed by a third expert pathologist. Consensus will 
be defined as two of the three pathologists agreeing on the diagnosis. We hope that by 
using this consensus method, the true incident of CIN2+ will be able to be evaluated and 
bias in favor of superior cure rates will be avoided. Standard cytology will be taken as per 
local guidelines. Cytology will be read by a local pathologist and interpreted according to 
the Bethesda System for classification.19 Evaluation of the HPV genotyping will be 
conducted at the National Cancer Institute. 
 
6.0 Recruitment 
Hospital clinicians at Salvadorian Social Security Hospital (ISSS) in San Salvador, El 
Salvador, Hospital Universitario San Ignacio (HUSI), Ginesalud SAS in Bogota, 
Colombia and the four hospitals in China (Shanxi Bethune Hospital – Shanxi Academy 
of Medical Sciences (SBH), Yanjin County Women and Children Hospital, Fengqiu 
County Women and Children Hospital, and Weihui Municipal Hospital for Women and 
Children Healthcare) will be informed of study criteria and will alert local study 
coordinators when participants with CIN2+ are scheduled for procedures.  
● Our collaborators will be paid per patient during the course of this study. To help 
ensure that all of the data is obtained, the remainder of the compensation will be 
provided after the follow-up visit is completed.  
● Potential participants will be invited by hospital staff to a private room to learn about 
the study. This conversation is anticipated to last approximately 30 minutes. The 
potential participant’s family members will be invited to join this conversation if the 
woman so chooses.  
● The study coordinator will assess inclusion/exclusion criteria to confirm eligibility 
based on the potential participant’s clinical record of the most recent pelvic exam, 
performed either prior to or after her hospitalization, and invite her to participate.  
● Participants will be informed that only information necessary to confirm eligibility 
will be entered into the study data collection form. This form will have a number code 
for the patient, not the participant’s name. 
● As part of the information provided, women in El Salvador and Colombia will be 
informed that their tissue sample will be analyzed in a laboratory in the United States. 
All women in the study will be informed that results of their analysis and their de-
identified information may be published in a peer-reviewed medical journal. Women 
will be informed that no personal information will be in any analysis of results or 
publication.  

 
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v. 04 Date: April 20, 2020 
● As part of the information provided, women will be informed that photographs may 
be taken of the uterine cervix during their procedures. The photographs will be 
assigned a study number and will not identify the participant. Women may opt out of 
having photographs taken. De-identified images will be made available to Mobile 
ODT to develop an algorithm to screen pre-cancerous lesions. 
● Women will have the opportunity to ask the study coordinator questions about the 
study of the study coordinator. In addition, the study coordinator will ask the potential 
subject to repeat the major points of the study in order to ensure that the woman 
understands to what she is agreeing and what is expected of her. 
● Women who decline to participate will be offered the standard of care treatment for 
CIN2+. 
● The study coordinator will not be the primary doctor for the potential study 
participants and will not be in a relationship of authority over participants. The study 
coordinator will explain that the woman’s decision to participate in the study will not 
influence her care.  
 
7.0 Consent process 
● The study coordinator, the Principal Investigator (PI), and the co-investigators all 
have certificates showing completion of training in human subjects protections within 
the last five years. 
● Consent will occur at the same time as recruitment by the study coordinator, which is 
described above. As stated above, a potential participant will be in a private room. 
The woman’s family members will be invited to join this conversation if the woman 
chooses. 
● Women will be invited to participate in the study at the time they receive their 
diagnosis of CIN2+. 
● It will be made clear to women that the research study is not a necessary part of their 
care. 
● A woman who agrees to participate in the study will be provided an informed consent 
form. All women who agree to participate will sign the form or provide a fingerprint 
if non-literate. A witness will be present. 
● If any non-literate women are invited to participate in the study, they may sign the 
consent form with a thumbprint. The witness will sign the consent form 
acknowledging that the woman understands the study. 
● Women who consent to participate in the study will be randomized to a study arm 
immediately prior to her ablation 
● Women who sign the informed consent to participate in the study have the right to 
withdraw their consent at any time during the study and stop their participation in the 
study without affecting their treatment. 
 
8.0  Study instruments 
At the beginning of the research study, a meeting of all staff involved in the study will be 
held. The protocol and all procedures for the study will be presented and discussed. The 
discussion will include use of the instruments and their safe storage and handling. The 
operating instructions or manuals for all three instruments will be provided at the 
meeting.  

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
v. 04 Date: April 20, 2020 
Providers in this study have been trained previously in all aspects of the handling and use 
of the standard cryotherapy equipment, the adapted CryoPen®, and the WISAP 
thermoablator model C3. Any providers who have not yet used the CryoPen® or 
thermoablator will be trained in safe handling, use, sterilization, and storage by a 
CryoPen, Inc., or WISAP employee or the PI of this study, Dr. Miriam Cremer.  
All instruments used in this study are the property of the Cleveland Clinic.  The devices 
may be donated to the institutions where they are being used at the end of the study.  Safe 
use, storage, and disposal is the responsibility of the institution. Specifically, each 
institution is responsible for ensuring that the use of all instruments conform to regulatory 
approval granted for each, whether for clinical or research use. 
 
The MedGyn cryotherapy instrument is approved and widely used around the world. The 
CryoPen® freezes tissue in the same manner as the MedGyn instrument: a metal probe 
cooled to -50ºC to -80ºC is applied to the cervix. The WiSAP thermoablator (model C3) 
ablates tissue with heat by applying a probe heated to 100ºC to the cervix.  
 
9.0 Risks 
Traditional cryotherapy is a safe and effective treatment for precancerous cervical 
lesions. The CryoPen® is a recently developed instrument and has FDA approval for 
gynecological procedures. It freezes tissue in a manner similar to the traditional method 
and side effects are similar. In undergoing procedures in this study, women may 
experience some discomfort related to the insertion of the speculum. Some pain, even 
severe pain, and cramping may occur during freezing, but these effects are minor and 
short-lived. While pain evaluations are very subjective, we expect patients’ pain levels to 
allow them to leave the hospital or clinic on their own. Some women may experience a 
vasovagal reaction during treatment, but this is expected to resolve quickly after 
treatment is completed. Women infrequently experience discharge, bleeding, and/or 
cramping within one month of traditional treatment that causes them to seek medical 
care, and long-term sequelae, such as stenosis and a negative impact on fertility and 
obstetrical outcomes, appear to be uncommon. Vaginal infections post-treatment may 
occur but are infrequent. Severe bleeding during and/or after traditional cryotherapy 
treatment requiring further medical attention or blood transfusion is an extremely rare 
occurrence. The development of pelvic inflammatory disease (PID) following 
cryotherapy is rare.  Definitions and reporting guidelines of adverse events can be found 
in Section 13. 
 
Since clinicians at the study sites in El Salvador (Salvadorian Social Security Hospital), 
Colombia (Hospital Universitario San Ignacio and Ginesalud SAS), and China (Shanxi 
Bethune Hospital – Shanxi Academy of Medical Sciences (SBH), Yanjin County Women 
and Children Hospital, Fengqiu County Women and Children Hospital, and Weihui 
Municipal Hospital for Women and Children Healthcare) are experienced gynecologists, 
no additional risk than would normally be expected for this procedure is anticipated. No 
additional risks are expected for CryoPen® since the amount of tissue damage is expected 
to be similar to that achieved with conventional cryotherapy. Women receiving a 
procedure will be provided information about managing the immediate minor symptoms. 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
v. 04 Date: April 20, 2020 
Reviews of cold coagulation performed for more than 2500 women in Scotland reported 
that they occasionally experienced cramps but that anesthesia was not required in 95 
percent of patients.11,12 Bleeding occurred rarely, and this was usually from biopsy 
performed before treatment. The heat of the probe causes blood to clot, reducing the risk 
of bleeding from the procedure itself. As with freezing, there may be some pain, 
including severe pain, during the procedure, and cramping, discharge, and/or bleeding 
after the procedure and a risk of infection post-procedure. As with cryotherapy, we 
expect pain levels to subside quickly after treatment and allow women to leave on their 
own. 
 
All women will be evaluated 6 weeks after treatment. The data from this visit will be 
evaluated in the bi-monthly research calls. We expect that the side effects from all 
ablative treatment will be minor, however, if there are any unanticipated adverse events, 
serious adverse events, or other unanticipated problems these will be discussed as a group 
and we will decide whether to continue with that arm of the study.    
 
There is also the risk that the ablative procedure will not treat the underlying CIN2+ 
lesion. According to a Cochrane review, cure rates for cervical intraepithelial neoplasia 
grade 3 (CIN3) following cryotherapy range from 77% to 93%20.  Women who 
participate in the study will all undergo colposcopy and 4-quadrant biopsy at 12 months. 
Thus, residual disease will be identified and women who still have CIN2+ will be 
referred for treatment with LEEP.  
 
10.0 
Mitigation of risks 
If a participant is experiencing significant pain during the procedure she may request that 
the procedure be stopped. After the procedure, participants will be offered analgesics 
such as acetaminophen or other medication as deemed appropriate by her care team. In 
the unlikely event that a complication occurs, the local study staff have the resources 
available at these high-level hospitals to assist the subject and provide appropriate clinical 
care for any condition. To mitigate side-effects and the risk of infection, women will be 
provided with standardized post-treatment recommendations that include avoiding the 
following behaviors: sexual intercourse, vaginal douches, bathing or swimming in any 
body of water, and the insertion of tampons or other objects into the vagina.  
 
All women are scheduled to have two follow-up visits— 6 weeks and 12 months post-
treatment. The risk of an untreated lesion developing into cancer during this time is 
extremely low21-23. Several attempts will be made to contact women to be sure that they 
come in for follow-up.  
 
11.0 
Benefits 
Patients will receive treatment of cervical precancer. They will also have a higher level of 
post-operative surveillance (VIA, cytology, and HPV testing) than women who do not 
participate in the study. Furthermore, women will have quality analysis done on all of 
their pathology specimens.  

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
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12.0 
Study and safety monitoring 
The PI and the study coordinators are staff members of the institutions participating in the 
study; general study supervision will be conducted by the PI. The local study 
coordinators will conduct recruitment, consent, and interviews of potential study 
participants. Sample labeling will be conducted by the study coordinators. The patient 
database will be managed by the research staff secretary and/or study coordinators. The 
PI and study coordinators will conduct adverse events monitoring.  
 
Data and Safety Monitoring Plan. Participants will include the principal investigator and 
study co-investigators, in addition to an epidemiologist, an OB-GYN, a gynecologic 
oncologist, and 2 researchers who are not part of the study.  After enrollment begins, the 
group will meet bi-monthly by telephone conference and additionally if any unanticipated 
Adverse Events (AEs), Serious Adverse Events (SAEs), or Unanticipated Problems (UPs) 
occurs. They will assess enrollment, loss to follow-up, any safety issues or concerns 
including pain due to treatment, and perform an interim analysis. 
 
Interim Analysis of Cure Fractions. An interim analysis of cure fractions will be 
performed after two years from study opening when approximately 1/3 of the women 
have been enrolled. Confidence intervals will be calculated for the differences (LMIC-
adapted CryoPen® minus cryotherapy and WISAP thermoablator minus cryotherapy) in 
cure fractions. If the upper bound of the confidence interval is lower than the non-
inferiority margin (-10%), then treatment modification or termination may be considered. 
The confidence interval will be calculated using an alpha calculated using an alpha time-
squared spending function to maintain an overall 5% Type I error (one-sided). 
 
13.0 Managing and reporting adverse events and unanticipated problems 
13.1 Adverse Events and Unanticipated Problems 
 
An adverse event (AE) is any untoward or undesirable event experienced by a 
participant regardless of whether the event is expected or related to the participant’s 
involvement in the research. 
 
An unanticipated problem (UP) is an unexpected event given the research procedures 
that are described in the protocol, and given the characteristics of the population being 
studied; it is related or possible related to participation in the research; and it suggests 
that the research places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social) than was previously known or recognized. 
 
13.2 Serious Adverse Events 
A serious adverse event (SAE) is any untoward medical occurrence or effect that 
involves 
● Death 
● A life-threatening condition (participant at immediate risk of death) 
● Persistent or significant disability/incapacity 
● In-patient hospitalization or prolongation of existing hospitalization 
● A congenital anomaly or birth defect  

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
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● An important medical event that may jeopardize the participant or may require 
intervention to prevent one of the outcomes listed above. 
 
AEs, SAEs, and unanticipated problems will be reviewed and evaluated by the site PIs 
throughout the study. They will decide how to report these according to the guidelines 
below.  
 
All AEs, SAEs, and unanticipated problems will be followed until satisfactory resolution 
or until the PI deems the event to be chronic or the patient to be stable. Qualified health 
providers will provide treatment of study-related adverse events at the study sites.  
 
Treatments administered are to be recorded on the participant’s data collection form as a 
complication and will be reported as necessary to the local and Cleveland Clinic’s 
Institutional Review Boards (IRBs). 
 
13.3 Reporting procedures for AEs, SAEs, and unanticipated problems 
The study coordinator at each study site will notify the principal investigator of (Dr. 
Miriam Cremer) of every AE that meets the criteria for an unanticipated problem, SAE, 
and/or other unanticipated problem within 24 hours of finding out about the event, even if 
the study coordinator considers the event not related to the study or is unsure about 
relatedness to the study.  
 
The PI will report AEs that meet the criteria for unanticipated problems, SAEs, and other 
unanticipated problems to the Cleveland Clinic IRB according to REC guidelines. Every 
unanticipated AE, SAE, and/or unanticipated problem occurring during the study must 
also be reported to the local IRB in accordance with local reporting requirements.  
Initial notification of the unanticipated AE, SAE or unanticipated problem to the study PI 
either via phone, fax, or email must be made within 24 hours of the site becoming aware 
of the event. The initial notification should be coded as “Initial AE/SAE notification” and 
should include as much of the information on the AE/SAE reporting form as possible 
including a description and date/s of onset.  
 
The PI should not wait for additional information to fully document the event before 
notification to Cleveland Clinic. The initial notification is to be followed by a completed 
adverse report form as soon as possible, but at latest within three calendar days of the 
initial notification. The completed adverse form should be coded as “Follow-up Adverse 
Event report” or “Final Adverse Event report” depending on whether the event is ongoing 
or has been satisfactorily resolved, and should detail relevant aspects of the adverse 
event/s, action/s taken and event outcomes. If applicable, hospital case records and 
autopsy reports should be obtained.  
 
Any relevant information that becomes available after the SAE report form has been sent 
(outcome, precise description of medical history, results of the investigation, copy of the 
hospitalization report) should be forwarded as soon as possible to the study PI. The 
confidentiality of the participants shall be respected when forwarding this information. 

 
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14.0 
Confidentiality and data management 
All study documents will be coded and this information will be kept in a locked storage 
room in the research office in the respective countries. Copies will be electronically sent 
through a secure website to the PI and the study coordinator in the US.  
 
Coded data will be stored in REDCap database. Data that is sent from El Salvador, 
Colombia and China to the United States via email will be sent in encrypted form. 
 
Any linking documents with the study identification number and women’s names will be 
securely stored and kept separately from the other study records. These linking 
documents will also be entered into a separate, password-protected and HIPAA-
compliant electronic storage folder or file. Only essential biological samples and 
photographs of the cervix will be labeled with the patient study code and will not include 
any identifiers to the subject. Following the standard procedures at for any biopsy, tissue 
will be stored in secure facilities for ten years before being destroyed. No further research 
will be performed on this tissue. 
 
Study staff or clinicians who are obligated to follow the participant for clinical care 
reasons will have access to this linking document in paper or electronic form. Linking 
documents will be destroyed as soon as the study is completed. 
 
Research ethics committees with oversight for this study will have the right to review 
research data. A report of study results will be created and shared with the cervical cancer 
community. No individual patient identifiers will be included. Manuscripts for journal 
submission may be prepared. 
 
The data gathered from this project is of great interest in the field of global cervical 
cancer prevention, and it will be important to share the results and implications for 
clinical practice in LMICs with others. Data and results may be shared with collaborating 
institutions (Cleveland Clinic, Shanxi Bethune Hospital [previously Shanxi Dayi 
Hospital], Instituto Salvadoreño del Seguro Social, Hospital Universitario San Ignacio, 
University of Southern California, Albert Einstein College of Medicine, Medical College 
of Wisconsin, WiSAP Medical Technology, National Cancer Institute, and Basic Health 
International) and distributed widely so that other researchers, clinical investigators, and 
organizations (including the World Health Organization, American College of Obstetrics 
and Gynecology, the Association of Reproductive Health Professionals and The 
American Society of Reproductive Health Professionals, American Cancer Society and 
the International Federation of Gynecology and Obstetrics) may build on it and use it as a 
reference for further investigation to promote the development of affordable treatment 
options for cervical cancer in low-income settings.   
 
15.0 
Study costs  
The costs of the study are borne by the sponsor. There is no cost to the patient for 
participating in the study. 

 
Protocol: Cure Rates for CIN2+ by Treatment Device 
 
v. 04 Date: April 20, 2020 
16.0 
Care for injury 
Since cervical ablation procedures are common and are not high risk, and the physicians 
at the study sites are experienced specialists, no injuries are anticipated. In the unlikely 
event of injury appropriate care will be provided at all sites. All subjects are undergoing 
treatment for their high-grade lesion with ablative therapy, which is the standard of care. 
Participants are responsible to follow up at six weeks and one year to ensure that their 
lesions have been treated.  
 
17.0 
Compensation 
In El Salvador, Colombia and China it is unusual to offer compensation or payment for 
participating in a research study because this adds pressure to participate, so no payment 
will be offered.  
 
18.0 
Investigator responsibilities 
Dr. Miriam Cremer, Associate Professor at Cleveland Clinic and Founder & President of 
Basic Health International, is the Principal Investigator.  
 
Site Principal Investigators include: 
Ana Celia de Uriarte, M.D. – El Salvador (ISSS) 
Raul Hernando Murillo Moreno, M.D. – Colombia (HUSI) 
Gustavo Ciendua, M.D. Sonia Salamanca, M.D. and Diana Valderrama, M.D. – 
Colombia (Ginesalud SAS) 
Suhui Wu, M.D. – China (SDH) 
 
All PIs will submit the initial paperwork and IRB protocol the local IRBs. Dr. Cremer 
will submit the IRB to the Cleveland clinic. General study coordinators will be 
responsible for creating and managing the randomization list. The local study 
coordinators will conduct recruitment, consent, and interviews of potential study 
participants. Sample labeling will be conducted by the local study coordinators. 
Specimen processing will be conducted by study pathologists. The patient database will 
be managed by general study coordinators. Adverse events monitoring will be conducted 
by the PI and the study coordinators. Study supervision will be conducted by the PI. 
Reporting of data will be conducted by the PI and the study coordinators. Data analysis, 
writing, and publication will be conducted by all collaborators. 
 
 
 
 
 
 
 
 
 
 
 
 

 
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