A Safety and Efficacy Trial of Circumferential Anal Canal Radiofrequency Ablation for High-Grade Anal Intraepithelial Neoplasia Using the BARRX™ Anorectal Wand
Phase: PHASE2
Conditions: HSIL, High Grade Squamous Intraepithelial Lesions, Anal Cancer, HPV-Related Squamous Cell Carcinoma, Human Papilloma Virus, Human Papillomavirus Infection, Ain III
Sponsor: Stephen E. Goldstone

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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A Safety and Efficacy Trial of Circumferential 
 
Anal Canal Radiofrequency Ablation for 
  
High-Grade Anal Intraepithelial Neoplasia 
Using The BARRX™ Anorectal Wand 
(Clinical Protocol 01-2017) 
AUGUST 31, 2017 
 
 

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PROTOCOL SIGNATURE PAGE 
 
 
I,                            , Principal Investigator at site             , agree to conduct and follow this 
protocol: PROTOCOL 01-2017: A Safety and Efficacy Trial of Circumferential Anal Canal 
Radiofrequency Ablation for Anal Intraepithelial Neoplasia using the Barrx™ Anorectal 
Wand as written according to FDA guidelines. I understand that no deviations from the above 
protocol may be made without written permission from the Protocol Chair(s). 
 
 
_________________________________ 
_____________________ 
 
Signature 
Date (mm/dd/yyyy) 
 
 
 

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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P R O T O C O L   S U M M A R Y  
 
Title 
A Safety and Efficacy Trial of Circumferential Anal Canal 
Radiofrequency Ablation for Anal Intraepithelial Neoplasia 
using the Barrx™ Anorectal Wand 
Design 
Multi-center prospective trial involving up to 70 subjects 
Subject Population 
HIV-positive and HIV-negative subjects with intra-anal 
intraepithelial neoplasia (AIN) containing at least one high- 
grade squamous intraepithelial lesions (HSIL) involving the 
squamocolumnar junction (SCJ). 
Objective 
Assess the safety, and efficacy of circumferential 
radiofrequency ablation (RFA) to the anal canal using the 
FDA cleared Barrx™ Anorectal Wand to eradicate anal 
HSIL lesions 
Hypothesis 
The study device RFA procedure will have an acceptable 
safety profile and demonstrate superior disease free 
survival at 12 months over standard ablative techniques  
Primary 
Endpoint 
• Histologic clearance of HSIL at the SCJ at 12 months 
Secondary Endpoints 
• Persistence of treated HSIL index lesions at 3 months post 
RFA 
• Safety of circumferential ablation of anal canal HSIL using 
the Barrx™ Anorectal Wand 
• Histologic clearance of low-grade squamous intraepithelial 
lesions (LSIL) at 12 months 
Principal Investigator 
Institution 
Laser Surgery Care 
420 W 23rd St, New York, NY 10011 
Investigator 
Stephen E. Goldstone, MD FACS (Laser Surgery 
Care) (212) 242-6500 
Institutional Review 
Board 
Solutions IRB (Little Rock, AR) 
Sponsor Information 
Stephen E. Goldstone, MD FACS (SEGMD PC) 
Laser Surgery Care 
420 West 23rd St 
New York, NY 10011 
(212) 242-6500 

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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G L O S S A R Y 
 
AE: adverse event 
 
AIN: anal intraepithelial neoplasia 
 
ASCH: atypical squamous cells-cannot exclude high-grade lesions (ASCH), 
ASCUS: atypical squamous cells of undetermined significance 
ANC: Absolute neutrophil count 
BQE: baseline qualifying exam 
CO2: carbon dioxide 
CD4: T-cell 
 
CRF: case report form 
 
CR-HSIL: complete response high-grade squamous intraepithelial lesion 
 
CR-HSIL/LSIL: complete response high-grade squamous intraepithelial lesion and low-grade 
squamous intraepithelial lesion 
 
ELISA: enzyme-linked immunosorbent assay 
 
ETZ: eligible treatment zone 
 
FDA: Food & Drug Administration 
HIV: human immunodeficiency virus 
HPV: Human Papillomavirus 
HRA: high-resolution anoscopy 
 
HSIL: high-grade squamous intraepithelial lesion 
 
ICF: informed consent form 
 
IDSA: Infectious Disease Society of America 
 
IRB: institutional review board 
IRC: infrared coagulation 
J/cm2: Joules per square centimeter 

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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LAT: lower anogenital tract 
 
LSIL: low-grade squamous intraepithelial lesion 
 
MSM: men who have sex with men 
 
Nd:YAG: neodymium-doped yttrium aluminum garnet 
 
NSAID: non-steroidal anti-inflammatory drug 
 
PTT: Partial Thromboplastin Time 
 
QOL: Quality of Life 
 
RFA: radiofrequency ablation 
SAE: serious adverse event 
SCC: squamous cell carcinoma 
SCJ: squamocolumnar junction 
W: Watts 
 
5-FU: 5-Fluorouracil 

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T A B L E  O F  C O N T E N T S 
 
 
1. 
Introduction........................................................................................................................7 
 
 
2. 
Materials and Methods....................................................................................................22 
 
 
3. 
Adverse Events .................................................................................................................33 
 
 
4. 
Statistical Methods...........................................................................................................34 
 
 
5. 
Study Management ..........................................................................................................34 
 
 
6. 
Ethical and Regulatory Considerations .........................................................................36 
 
 
7. 
Subject Withdrawal and Study Termination................................................................37 
 
 
8. 
Regulatory Standards ......................................................................................................37 
 
 
9. 
Appendix A. Schedule of visits and tasks ......................................................................38 
 
 
                                               
 
 
 
 
 

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1.   INTRODUCTION 
 
 
Anal Intraepithelial Neoplasia and Ablative Therapy 
 
Anal squamous cell carcinoma (SCC) is a relatively uncommon cancer, however, it is on the 
rise in men and women worldwide.  In the US, it is estimated that approximately 8,080 new 
cases predicted in the United States in 2016.1 Over the past decade, the rate of anal cancer 
cases has increased by approximately 2.2% each year while mortality has increased 3.8% 
each year.2  
 
Like cervical cancer, anal SCC is thought to be preceded by a spectrum of intra-epithelial 
changes of varying cytological and histological abnormality, referred collectively to as anal 
intraepithelial neoplasia (AIN).1,3,4 AIN involves both the perianal skin and the anal canal 
including the anal transition zone.5 
 
AIN is a multifocal disease process strongly associated with human papillomavirus (HPV; 
usually HPV types 6, 11, 16 and 18). A large majority of anal cancers are attributable to 
HPV-16 (~72%).6 Oncogenic HPV infection persistence is thought to be necessary in the 
development of anal SCC and high-grade squamous intraepithelial lesions (HSIL), the 
precursor to anal SCC.7 
 
The exact prevalence of AIN in the general population is unknown, but is thought to range 
from < 1% to 5.25%; notably, the incidence is increasing.8-10 Several higher-risk groups have 
been identified and include subjects with HIV, those who are systemically 
immunocompromised such as transplant recipients and those on long-term steroids (e.g. for 
connective tissue disorders), women with a history of genital intraepithelial neoplasia and 
those with extensive anogenital condylomata.11 In men and women, common risk factors 
include receptive anal sex, increased lifetime number of sexual partners, a history of genital 
warts and cigarette smoking.4 HSIL is common in HIV-positive men who have sex with men 
(MSM), with a prevalence of 43% to 52% and an incidence of 49% over 4 years.12,13 Though 
less pervasive, HSIL among HIV-negative MSM is still substantial, with a prevalence of 
25% and incidence of 17% over 4 years.13,14 
 
Subjects may undergo screening for anal dysplasia due to their inclusion in populations with 
increased risk (e.g. MSM, subjects with HIV or cervical dysplasia), the presence of 
symptoms such as pruritus and anal discharge, or signs of HPV infection such as anal 
condylomata. Anal Pap cytology was proposed as a potential screening methodology based 
on observations by Palefsky et al. in 1997 and Goldstone et al. in 2001, with the investigators 

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noting physical and cytological similarities between anal and cervical carcinomas and 
intraepithelial neoplasia.15,16 In this procedure, a Dacron swab is inserted to a depth of ~3 to 4 
cm and then slowly removed in a circular motion to sample the cells from all areas of the 
anal canal. Samples are immediately preserved on slides or in a liquid medium to prevent 
drying.10 
 
Typically, interpretation of anal Pap smears is based on the 2001 Bethesda System for 
evaluating cervical cytology. In this system, smears are classified as normal, or inclusive of 
atypical squamous cells of undetermined significance (ASCUS), atypical squamous cells- 
cannot exclude high-grade lesions (ASCH), low-grade squamous intraepithelial lesions 
(LSIL) or high-grade squamous intraepithelial lesions (HSIL).17 
 
Upon detection of ASCUS or worse, further diagnostic work-up includes examination of the 
anus in a manner similar to cervical colposcopy (referred to as high-resolution anoscopy or 
HRA). HRA is performed in conjunction with application of 5% aqueous acetic acid ± 
Lugol’s iodine stain. The anoscope is inserted to allow placement of a small swab wrapped 
in a gauze pad that has been soaked in the acetic acid. The anoscope is removed, leaving the 
gauze pad in place for one minute to allow absorption of the acetic acid. After removal of 
the gauze-wrapped swab, the anoscope is reinserted and an examination of the anal canal 
and perianal skin with magnification is performed. Acetic acid produces an epithelial change 
known as aceto-whitening, which is seen in regions of squamous cellular abnormality. 
Vascular abnormalities, ulceration, and friability are signs of dysplasia. During the exam, 
Lugol’s iodine may be applied with a small swab to further identify lesions suspicious for 
HSIL; such areas do not take up the Lugol’s stain and appear yellow or light beige upon 
application of the stain. Upon identification, lesions suspicious of HSIL are biopsied.18 
 
Grading of AIN is performed based on this histopathological examination. AIN 1 is 
characterized by 20 to 25% of the epithelium being replaced by abnormal basaloid cells and 
by the presence of koilocytes, which are indicative of actively replicating HPV infection. 
AIN 2 and 3 are diagnosed when abnormal basaloid cells replace more than 50% of the 
epithelium.13 
 
Because the current understanding of HPV biology does not support a 3-tiered classification 
system of mild, moderate, and severe dysplasia/carcinoma in situ, recent multi-society 
consensus advocates a separation of morphologic designations to two categories that reflect 
transient active HPV replication and persistent HPV-associated pre-cancer. According to this 

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2013 consensus statement from the College of American Pathologists and the American 
Society for Colposcopy and Cervical Pathology, “The recommended terminology for HPV- 
associated squamous lesions of the LAT (lower anogenital tract) is low-grade squamous 
intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL), which 
may be further classified by the applicable –IN (intraepithelial neoplasia) subcategorization.” 
Using this two-category system, AIN 1 is categorized as LSIL, and AIN 2 and AIN 3 are 
categorized as HSIL. While LSIL lesions may regress or progress to HSIL, HSIL lesions 
rarely regress regardless of the subject’s HIV status.19 
 
While the natural history of AIN has not been as well established as that of cervical 
intraepithelial neoplasia (CIN), HSIL is considered to be the immediate precursor to anal 
cancer.20-22 Several studies have demonstrated the oncogenic potential of AIN 2–3.23-25 
Stanley et al. report that, “…even with surveillance and treatment, on average 9-13% AIN 3 
may progress to invasive disease over 5–10 years, comparable to that observed for 
progression for CIN 3 over similar time periods.”26 Rates of anal HSIL histology are high in 
HIV-positive patients of all sexual risk groups with abnormal anal cytology.27 Compared to 
HIV-negative subjects, in HIV-positive subjects, squamous intraepithelial lesions appear to be 
more aggressive; with ~50% of LSIL lesions progressing to HSIL within 2 years for HIV- 
positive MSM. Risk of progression to invasive cancer may be as high as 50% for lesions in 
 
HIV-positive subjects.24,25,28 
 
Despite increasing acknowledgement of AIN amongst medical and subject communities and 
the availability of screening and diagnostic tools, there is no uniform standard management 
for dysplastic lesions. However, due to the high progression rate to anal SCC, experts 
recommend treatment for HSIL.7, 29, 30 
 
In the cervix, treatment of HSIL focuses on removal of the squamous columnar epithelial 
transformation zone where most dysplasia occurs by performing the Loop Electrosurgical 
Excision Procedure (LEEP) or Conization. Unfortunately, this cannot be done in the anal 
canal and current treatment options are limited, and generally consist of local treatment with 
clinician- or subject-applied creams or gels, clinician-applied ablative techniques such as 
electrocautery, carbon dioxide (CO2) laser, infrared coagulation (IRC; Redfield Corporation, 
Rochelle Park, NJ), and surgical excision. Reports of these treatments are plagued by a lack 
of controlled studies and small study sizes. Unfortunately targeted ablation of lesions has 
been associated with frequent persistent or recurrent disease, reflecting the field nature of 

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infection.31-38  Widespread topical treatment with Imiquimod or 5-Fluorouracil (5-FU) has 
been reported with moderate success but treatment occurs over lengthy periods (4 months), 
with a lack of control subjects and limited study sizes precluding meaningful conclusions.36- 
40 
 
 
Park et al. recently further detailed limitations in current AIN therapeutic management, 
stating, “AIN treatment can be challenging because recurrence after treatment and 
development of metachronous lesions (concurrent separate lesions) is common. There are no 
randomized controlled trials of AIN treatments to inform treatment recommendations, thus 
selection of treatment modality is directed not only by the extent and location of disease but 
also by available expertise and resources.”7 
 
 
Radiofrequency Ablation Study Device 
 
The radiofrequency ablation (RFA) or Barrx™ Ablation System used in this protocol 
comprises an RFA generator (Barrx™ FLEX) and the Barrx™ Anorectal Wand. The 
generator and wand are cleared by the FDA for human use and the wand is specifically 
cleared for treatment of anal neoplasia.  More than 100,000 procedures have been performed 
in the US (mostly RFA of esophageal metaplasia and dysplasia). The present FDA indication 
for use statement is: “The Barrx™ catheters are indicated for use in the coagulation of 
bleeding and non-bleeding sites in the gastrointestinal tract including, but not limited to, the 
esophagus.  Indications include esophageal ulcers, Mallory-Weiss tears, arteriovenous 
malformations, angiomata, Barrett’s esophagus, Dieulafoy lesions, angiodysplasia, gastric 
antral vascular ectasia, and radiation proctitis” (a Dieulafoy lesion is defined as a large 
tortuous arteriole in the stomach wall that erodes and bleeds). Thus, in this study, the device 
will be utilized in accordance with the FDA indications for use. 
 
 
Radiofrequency Ablation of Gastrointestinal Tissue 
 
RFA is used for the treatment of a number of disease states throughout the gastrointestinal 
tract, including; Barrett’s esophagus, vascular lesions, bleeding, tumor palliation, dysplasia, 
carcinoma, and others. Historically, RFA has been applied with the assistance of an 
endoscope inserted via the mouth or anus.  Commonly used terms related to the treatment of 
gastrointestinal tissue with RF energy include “coagulation” which may refer to sealing 
bleeding tissue and vessels, and “ablation” which may refer to the thermal removal of tissue 
such as the case for Barrett’s esophagus. 

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Previously available techniques for coagulation and ablation, such as argon plasma 
coagulation, Nd:YAG (neodymium-doped yttrium aluminum garnet) laser photocoagulation 
and bipolar electrocauterization, rely on small focal probes (~2 mm width) to deliver energy 
to tissue. Variability in tissue effect, lack of uniformity, lack of broad surface area coverage, 
lack of depth control, and occasionally subject toxicity, remain limitations in the application 
of these tools for treating gastrointestinal tissue. 
 
In response to the need for a more uniform energy delivery system, a predecessor of the 
present study device was developed in 2000 and subsequently cleared by FDA in 2001 
(K013139).  This device (Barrx™360 Ablation System) utilizes the same electrode design 
and energy delivery mechanism as the study device and has been studied extensively for the 
ablation of Barrett’s esophagus in human subjects. To date, more than 100,000 procedures 
have been performed with the Barrx™ Ablation System. 
 
The present study device (Barrx™ 60 ) has been used to provide RFA in a controlled 
programmed manner to any endoscopically accessible portion of the gastrointestinal tract, 
using treatment settings derived from experience with the predecessor device (Barrx™360 ). 
 
Supporting Studies involving RFA in the GI Tract 
 
The initial reports assessed the ablation depth and effect in the porcine esophageal model, 
demonstrating that application energy using the Barrx™ 360 device could be safely 
performed in the range of 5-12 J/cm2 (energy density) and at high power (350 W). Such 
control of energy and power prevented stricture formation and other injury.44 
 
Subsequent “treat and resect” studies were performed in subjects having their esophagus 
removed. These authors reported that 10 and 12 J/cm2 applied twice (2x) resulted in 
complete removal of squamous epithelium and that the maximum depth of ablation was 
limited to the superficial submucosa at even the highest treatment settings tested (14 J/cm2 
delivered 4x). 45, 46 
 
Since these initial studies, RFA has become an accepted treatment for the management of 
high-grade dysplasia associated with Barrett’s esophagus.39-41  Preliminary studies in porcine 
models and humans demonstrated that depth of ablation was linearly related to energy 
density of treatment and subsequent studies have utilized energy densities to ablate the 
epithelial mucosa with sparing of the muscularis and submucosa.44-46  Clinical trials using 
RFA for the treatment of esophageal dysplasia (Barrett’s dysplasia and squamous dysplasia) 

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have resulted in the complete eradication of dysplasia in ≥ 90% of individuals with a low 
incidence of side-effects or complications.41,47,48 
 
Several additional trials have been conducted in subjects with Barrett’s esophagus with and 
without dysplasia using both the Barrx™ 360 device and the Barrx™ 90 device. Safety has 
been established for both devices at these treatment setting ranges for use in the esophagus 
and upper stomach (cardia). Further, effectiveness for complete removal of the pre- 
malignant Barrett’s epithelium has been established within this treatment setting range as 
well.41, 51 
 
The use of RFA outside the esophagus is growing. RFA has been applied to colonic samples 
at the time of resection. Two applications at a dose of 12 J/cm2 resulted in changes no deeper 
than muscularis propria and was recommended as the dose to guide future studies of the lower 
GI tract.49  RFA use in the stomach has been reported in two studies in which the bleeding 
dilated vessels in patients with gastric antral vascular ectasia (GAVE) were successfully 
treated. 52, 53 In one study comprising 21 patients who received a median of 2 
RFA sessions, two adverse events were reported consisting of minor bleeding and superficial 
ulceration. 52  In the second study, 6 patients underwent 10 RFA sessions with no 
complications. Baldaque-Silva reported treating 3 patients with gastric dysplasia with 2 RFA 
sessions per patient and noted mild to moderate epigastric pain treatable with acetaminophen 
but no major complications.54 The rectum has also been investigated as a site of RFA to treat 
bleeding sites in 4 patients suffering from chronic radiation proctitis with no adverse events 
reported. 55, 56 
 
RFA in the Anal Canal 
 
Data was published on 19 HSIL subjects who had undergone RFA with the Barrx™90 
device in a Phase I safety/tolerability study. Subjects underwent dose escalation from 1 - 3 
ablation applications at a single location (n=13) to 2 - 3 applications hemi- circumferentially 
(n=6). The procedure was well-tolerated with no serious adverse events, and all subjects 
demonstrated healing of the anal mucosa by 28 days post-procedure.50  
 
The Principal Investigator published the results of a single center, 22-patient study assessing 
this device for hemi-circumferential ablation reviewed and approved by Solutions IRB titled, 
“A Trial of Radiofrequency Ablation for Anal Intraepitheilial Neoplasia Using the Barrx™ 
Ablation System” (B-250,, Rev. 3/14/13).57 The trial included only HIV-negative subjects (3 
women) with HSIL involving a maximum or 50% of the SCJ circumference.  Mean number 

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of index lesions was 1.7 (range1-4). The treatment zone was limited to the 50% of the anal 
canal involving the HSIL.  Participants were followed at 3-month intervals with HRA and 
biopsy of treated lesion sites at any visit following RFA and at the 12-month endpoint.  At 12 
months Kaplan-Meier estimates of histologic clearance of HSIL within the treated half of the 
anal canal was 76%.  Of 35 HSIL treated 4 lesions persisted at 3 months in 4 subjects. These 
were retreated with focal RFA to the quadrant involving the lesion and one persisted again at 
12 months.  One-third of subjects were noted to develop HSIL recurrence in the non-RFA 
treated half of the anal canal (metachronous recurrence).   
 
Post treatment patients completed a diary assessing symptoms and tolerability.  Overall, 
patients experience mild discomfort following RFA. The pain was considered tolerable and 
easily controlled with over the counter medication or mild narcotic. These findings are in 
keeping with other trials using targeted ablation of individual lesions. Bleeding with bowel 
movements as reported in the diary was almost universal and resolved within a median of 13 days.  
2 subjects noted SAE’s that were felt to be unrelated to the treatment (Appendicitis and narcotic 
addiction unrelated to this procedure). AE’s were reported in 4 participants (19%) but only 2 
were felt to be possibly device related.  One subject developed a low-grade fever the night of 
the procedure.  The other subject developed asymptomatic granulation tissue at 9 months post 
treatment after he had been judged to be fully healed at prior visits. At 12 months there were a 
small amount of purulent drainage from the area. It was probed and did not track anywhere.  
It was treated with silver nitrate coagulation and it resolved.  
 
Given that risk of developing metachronous recurrence in high in studies of focal ablation of 
HSIL as well as the prior study of hemicircumferential RFA, the Principal Investigator 
recently published the results of  a 10 participant (9 HIV-infected), single center study 
assessing this device for circumferential ablation reviewed and approved by Solutions IRB 
titled, “Radiofrequency ablation therapy for anal intraepithelial neoplasia: results from a single-
center prospective pilot study in HIV+ participants.”58  The results were recently presented at the 
Infectious Disease Society of America annual meeting and at HPV 2017 in Cape Town South 
Africa. While this represented a pilot study with a primary endpoint of safety and tolerability, 
data was analyzed for a secondary endpoint of efficacy. Participants had to have at least one 
HSIL involving the SCJ and the entire SCJ was ablated (3 x 12 J/cm2 per site) to treat 
baseline and occult HSIL(s).  A post-RFA biopsy was taken. Subjects were assessed with 
HRA at 3, 6, 9, and 12 months. Mandatory lesion site biopsies occurred at month 12. 
Recurrence was retreated with focal RFA. At baseline, subjects had a mean 2.7 HSIL’s (range 

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2-8).  Median time to treat was 6.5 (5-13) minutes. Lesion persistence occurred in 4 subjects 
(3 at 3 months, 1 at 6 months). Metachronous recurrence occurred in only 1 subject at 3 
months. No lesion persisted after retreatment. All subjects were dysplasia free at 12 months. 
Immediate post RFA biopsy of a treated lesion showed dysplasia in 50% and 2 of these 
participants developed persistence.   
 
Safety and tolerability analysis showed that eight subjects were healed by 3 months and one 
each had asymptomatic granulation tissue and superficial erosion. All healed by 9 months. 
Median post-ablation anal pain was 5 (range 2-9) but resolved to a minimal level (0 or 1) 
within a median of 20 (0-29) days. During the first week after RFA, seven subjects 
experienced mild or moderate anal bleeding, not requiring any intervention.  Median days to 
bleeding resolution was 1 (range 0-4) days.  Seven subjects experienced mild or moderate 
incontinence (mucous or stool discharge) following treatment. One subjected developed thin 
stool and straining to pass bowel movements at 3 months after significant diarrhea several 
weeks before. He had a soft, circumferential, mid canal scar that was easily dilated. All 
symptoms resolved. Narcotics usage for pain relief was minimal (N= 3 subjects) and median 
days of pain reliever usage was 13 (3-22) for non-narcotics and 8 (4-14) for narcotics. All 
subjects who tried receptive anal sex post-RFA succeeded, including the subject with the 
dilated stricture.  Median subject score for worry about anal canal condition was 4.5 (range 0-
8) falling to 0.5 (range 0-5) 9 to 12 months post-RFA. Two device-related mild adverse 
events (AE) occurred in one subject each (externally thrombosed hemorrhoid and soft anal 
stricture).  No serious AE occurred. 
 
To date RFA has been shown to be effective in treating individual HSIL lesions and has been 
tolerable without undo complications in treating the hemi-circumference and circumference of the 
anal canal. Most importantly, it appears that there is a trend to decreased metachronous 
recurrence when the entire circumference of the anal canal SCJ is ablated without undo 
complications. Circumferential RFA is hypothesized to reduce metachronous recurrence in 
that it not only ablates identified HSIL, but also treats occult disease. Moreover, flat LSIL is 
not usually ablated during focal ablation. Some LSIL could progress to HSIL over time 
creating metachronous recurrence. With circumferential ablation this disease could be ablated 
thereby preventing progression. We now propose a larger, multicenter trial to better 
determine the efficacy and safety of circumferential RFA of the anal canal SCJ for the 
treatment of anal HSIL to determine if our prior results on a very limited pilot trial hold.  

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Figure 1. Post treatment tolerability. 
 
Subject 21-day diary:  self-reported symptoms after initial RFA 
Variable 
Subjects (N=21) 
Duration (days) 
Bleeding (with bowel movement) 
 20 (95%) 
13 (0-23) 
Bleeding (without bowel movement) 
 12 (57%) 
2 (0-16) 
Variable 
At day 1 
At day 7 
At day 14 
Pain score a  
4 (0-8) 
3 (0-6) 
1.5 (0-6) 
Data shown as n (%) or median (range); a Based on a visual analog scale of 0-10. 
 
 
 
 
Non-Significant Risk Device Study 
 
The use of the Barrx™ Ablation System has been determined to present non-significant risk 
in accordance with 21 CFR 812.3(m) for the intended use in this study. The Barrx™ Ablation 
System is not an implant and does not present a potential risk for serious risk to the health, 
safety, or welfare of a subject. Transient side effects that can be expected after treatment with 
the device include pain, fever, bleeding, and discharge. These are temporary 
and will subside with healing. Although not observed, additional adverse events may include 
anal stenosis, incontinence, scarring, disfiguration, infection, abscess, fissure, or fistula 
formation. Potential serious adverse events, also not been observed to date, may include 
ablation-related visceral perforation, major bleeding or death. There is potential for the 
occurrence of adverse events or serious adverse events related to the monitored sedation. The 
Barrx™ Ablation System will be utilized in accordance with the FDA 510k indications for 
use. 

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References 
 
1. American Cancer Society.  http://www.cancer.org/cancer/analcancer/detailedguide/anal-
cancer-what-is-key-statistics. Accessed February 28, 2016.  
 
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Cancer Society; 2013. 
 
3.   Abbas A, Yang G, Fakih M. Management of anal cancer in 2010. Part 1: 
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4.   Palefsky JM, Rubin M. The epidemiology of anal human papillomavirus and 
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5.   Fenger C, Nielsen VT. Intraepithelial neoplasia in the anal canal: The appearance 
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9.   Ogunbiyi OA, Scholefield JH, Raftery AT, et al. Prevalence of anal human 
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11. Scholefield JH, Harris D, Radcliffe A. Guidelines for management of anal 
intraepithelial neoplasia. Colorectal Dis. 2011; Suppl 1:3-10. 

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12. Palefsky JM, Holly EA, Efirdc JT, et al.: Anal intraepithelial neoplasia in the 
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13. Palefsky JM, Holly EA, Ralston ML, et al.: High incidence of anal high-grade 
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14. Chin-Hong PV, Berry JM, Cheng SC, et al. Comparison of patient- and clinician- 
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2008;149:300-306. 
 
15. Palefsky JM, Holly EA, Hogeboom CJ, et al. Anal cytology as a screening tool 
for anal squamous intraepithelial lesions. J Acquir Immune Defic Syndr Hum 
Retrovirol 1997;14:415-422. 
 
16. Goldstone SE, Winkler B, Ufford LJ, et al. High prevalence of anal squamous 
intraepithelial lesions and squamous-cell carcinoma in men who have sex with 
men as seen in a surgical practice. Dis Colon Rectum 2001;44:690-698. 
 
17. Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology 
for reporting results of cervical cytology. JAMA. 2002;287:2140-2141. 
 
18. Jay N, Berry JM, Hogeboom CJ, et al. Colposcopic appearance of anal squamous 
intraepithelial lesions: relationship to histopathology. Dis Colon Rectum. 
1997;40(8):919-928. 
 
19. Darragh TM, Colgan TJ, Cox JT, et al. The lower anogenital squamous 
terminology standardization project for HPV-associated lesions: Background and 
consensus recommendations from the College of American Pathologists and the 
American Society for Colposcopy and Cervical Pathology. Int J Gynecol Pathol. 
2013;32(1):76-115. 
 
20. Zbar AP, Fenger C, Efron J, et al. The pathology and molecular biology of anal 
intraepithelial neoplasia: Comparisons with cervical and vulvar intraepithelial 
carcinoma. Int J Colorectal Dis 2002;17:203–215. 

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21. Fox PA. Human papillomavirus and anal intraepithelial neoplasia. Curr Opinions 
 
Inf Dis 2006;19:62–66. 
 
22. de Sanjosé S, Palefsky J. Cervical and anal HPV infections in HIV positive 
women and men. Virus Res 2002;89:201–211. 
 
23. Rickert RR, Brodkin RH, Hutter RV. Bowen’s disease. CA Cancer J Clin 
 
1977;27:160–166. 
 
24. Watson AJ, Smith BB, Whitehead MR, Sykes PH, Frizelle FA. Malignant 
progression of anal intra-epithelial neoplasia. ANZ J Surg 2006;76(8):715–717. 
 
25. Scholefield JH, Castle MT, Watson NF. Malignant transformation of high-grade 
anal intraepithelial neoplasia. Br J Surg 2005;92(9):1133–1136. 
 
26. Stanley MA, Winder DM, Sterling JC, Goon PK. HPV infection, anal intra- 
epithelial neoplasia (AIN) and anal cancer: current issues. BMC Cancer. 
2012;12:398. 
 
27. Gaisa M, Sigel K, Hand, J, Goldstone, S. High rates of anal dysplasia in HIV- 
infected men who have sex with men, women and heterosexual men. AIDS. 2014 
Jan 14;28(2):215-22. 
 
28. Devaraj B B, Cosman BC. Expectant management of anal squamous dysplasia in 
patients with HIV. Dis Colon Rectum 2006;49(1):36–40. 
 
29. Stier EA, Baranoski AS. Human papillomavirus-related diseases in HIV-infected 
individuals. Curr Opin Oncol. 2008;20(5):541-546. 
 
30. Pineda CE, Welton ML. Management of anal squamous intraepithelial lesions. 
 
Clin Colon Rectal Surg. 2009;22(2):94-101. 
 
31. Goldstone SE, Hundert JS, Huyett JW. Infrared coagulator ablation of high-grade 
anal squamous intraepithelial lesions in HIV-negative males who have sex with 
males. Dis Colon Rectum 2007;50:565-575. 
 
32. Goldstone RN, Goldstone AB, Russ J, Goldstone SE. Long-term follow-up of 
infrared coagulator ablation of anal high-grade dysplasia in men who have sex 
with men. Dis Colon Rectum. 2011;54(10):1284-1292. 
 
33. Cranston RD, Hirschowitz SL, Cortina G, Moe AA. A retrospective clinical study 
of the treatment of high-grade anal dysplasia by infrared coagulation in a 

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population of HIV-positive men who have sex with men. Int J STD AIDS 
 
2008;19(2):118–120. 
 
34. Marks DK, Goldstone SE. Electrocautery ablation of high-grade anal squamous 
intraepithelial lesions in HIV-negative and HIV-positive men who have sex with 
men. J Acquir Immune Defic Syndr. 2012;59(3):259-265. 
 
35. Singh JC, Kuohung V, Palefsky JM. Efficacy of trichloroacetic acid in the 
treatment of anal intraepithelial neoplasia in HIV-positive and HIV-negative men 
who have sex with men. J Acquir Immune Defic Syndr. 2009;52(4):474-479. 
 
36. Berry JM, Palefsky JM, Welton ML. Anal cancer and its precursors in HIV- 
positive patients: perspectives and management. Surg Oncol Clin N Am 
2004;13:355-373. 
 
37. Goldstone S, Johnstone, AA, Moshier, EL. Long-term outcome of ablation of anal 
high-grade squamous intraepithelial lesions: recurrence and incidence of cancer. 
Dis Colon rectum 2014;57:316-323. 
 
38. Wieland U, Brockmeyer NH, Weissenborn SJ, et al. Imiquimod treatment of anal 
intraepithelial neoplasia in HIV-positive men. Arch Dermatol 2006;142:1438- 
1444. 
 
39. Richel O, Wieland U, de Vries HJ et al. Topical 5-fluorouracil treatment of anal 
intraepithelial neoplasia in HIV-positive men. Br J Dermatol. 2010;163(6):1301- 
1307. 
 
40. Fox PA, Nathan M, Francis N, et al. A double-blind, randomized controlled trial 
of the use of imiquimod cream for the treatment of anal canal high-grade anal 
intraepithelial neoplasia in HIV-positive MSM on HAART, with long-term 
follow-up data including the use of open-label imiquimod. AIDS. 
2010;24(15):2331-2335. 
 
41. Shaheen NJ, Sharma P, Overholt BF, et al. Radiofrequency ablation in Barrett’s 
esophagus with dysplasia. N Engl J Med 2009;360:2277-2288. 
 
42. American Gastroenterological Association, Spechler SJ, Sharma P, et al. 
 
American Gastroenterological Association medical position statement on the 
management of Barrett's esophagus. Gastroenterology. 2011;140(3):1084-1091. 

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43. ASGE Standards of Practice Committee, Evans JA, Early DS, et al. The role of 
endoscopy in Barrett's esophagus and other premalignant conditions of the 
esophagus. Gastrointest Endosc. 2012;76(6):1087-1094. 
 
44. Ganz RA, Utley DS, Stern RA, et al. Complete ablation of esophageal epithelium 
with a balloon-based bipolar electrode: a phased evaluation in the porcine and in 
the human esophagus. Gastrointest Endosc 2004;60:1002-1010. 
 
45. Dunkin BJ, Martinez J, Bejarano PA, et al. Thin-layer ablation of human 
esophageal epithelium using a bipolar radiofrequency balloon device. Surg 
Endosc 2006;20:125-130. 
 
46. Smith CD, Bejarano PA, Melvin WS, et al. Endoscopic ablation of intestinal 
metaplasia containing high-grade dysplasia in esophagectomy patients using a 
balloon-based ablation system. Surg Endosc 2007;21:560-569. 
 
47. Pouw RE, Gondrie JJ, Sondermeijer CM, et al. Eradication of Barrett esophagus 
with early neoplasia by radiofrequency ablation, with or without endoscopic 
resection. J Gastrointest Surg 2008;12:1627-1636. 
 
48. Bergman JJ, Zhang YM, He S, et al. Outcomes from a prospective trial of 
endoscopic radiofrequency ablation of early squamous cell neoplasia of the 
esophagus. Gastrointest Endosc 2011;74:1181-1190. 
 
49. Trunzo JA, McGee MF, Poulose BK, et al. A feasibility and dosimetric 
evaluation of endoscopic radiofrequency ablation for human colonic and rectal 
epithelium in a treat and resect trial. Surg Endosc 2011;25:491-496. 
 
50. Smulian AG, Moore DM, Robertson JC et al. Phase I study demonstrates safety 
and tolerability of radiofrequency ablation (RFA) of the anal mucosa. HIV Clin 
Trials. 2014;15(1):36-44 
 
51. Fleischer DE, Overholt BF, Sharma VK, et al. Endoscopic ablation of Barrett’s 
esophagus: a multicenter study with 2.5-year follow-up. Gastrointest Endosc 
2008;68:867-876. 
 
52. McGorisk T, Krishnan K, Keefer L, Komanduri S. Radiofrequency ablation for 
refractory gastric antral vascular ectasia. Gastrointest Endosc. 2013;78(4):584-8 

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53. Gross SA, Al-Haddad M, Gill KR, et al. Endoscopic mucosal ablation for the 
treatment of gastric antral vascular ectasia with the HALO90 system: a pilot 
study. Gastrointest Endosc.2008; 67(2) 324-7 
 
54. Baldaque-Silva F, Cardoso H, Lopes J, et al. Radiofrequency ablation for the 
treatment of gastric dysplasia: a pilot experience in three patients. Eur J 
Gastroenterol Hepatol 2013;25(7) 863 
 
55. Zhou C, Adler DC, Becker L, et al. Effective treatment of chronic radiation 
proctitis using radiofrequency ablation. Therap Adv Gastroenterol 2009;2(3) 149- 
156 
 
56. Nikfarjam M, Faulx A, Laughinghouse M, et al. Feasibility of radiofrequency 
ablation for the treatment of chronic radiation proctitis. Surg Innov 2010;17(2) 
92-4 
 
57. Goldstone RN, Hasan SR, Drury S, Darragh TM, van Zante A, Goldstone SE. 
A trial of radiofrequency ablation for anal intraepithelial neoplasia. Int J 
Colorectal Dis 2017;32(3) 357-365   
58. Goldstone RN, Hasan SR, Goldstone SE. (in press). Radiofrequency ablation 
therapy for anal intraepithelial neoplasia: results from a single-center 
prospective pilot study in HIV+ participants. Journal of Acquired Immune 
Deficiency Syndromes. Forthcoming 2017. 
 
 

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2.   MATERIALS AND METHODS 
 
 
Subject Selection 
 
The study will include female and male adult subjects who have biopsy-proven HSIL and are 
 
HIV-negative or HIV-positive. 
 
 
Inclusion Criteria 
 
Candidates for this study must meet all of the following criteria: 
 
1.   Age 18-75 years 
 
2.   HRA 2 to 8 weeks prior to the 0 month RFA visit yielding one or more flat, non- 
condylomatous biopsy-proven HSILs that are 
• Located entirely within the eligible treatment zone 
 
AND 
 
• Contiguous with the squamocolumnar junction 
 
3.   Eligible treatment zone (ETZ) is defined as 
 
• 3 cm above the dentate line to the anocutaneous line 
 
AND 
 
• Full anorectal circumference 
 
4.   If female of child-bearing age, negative pregnancy test within 8 weeks of the 0 month 
RFA visit and declared intent to remain on birth control throughout the trial, or 
declaration of infertility defined as subject report of status as post-menopausal or 
surgically sterile (status post hysterectomy or tubal ligation). 
5.   If HIV positive 
 
• HIV positive on antiretroviral therapy for at least 3 months with laboratory 
blood work within 12 weeks prior to the 0 month visit demonstrating viral 
load < 50 
• CD4 count ≥ 250/mm3 
 
• ANC > 750/mm3 
 
• Platelet count ≥ 75,000/mm3 
 
• Hemoglobin ≥ 9.0 g/dl 

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Exclusion Criteria 
 
Candidates will be ineligible for enrollment in the study if any of the following conditions 
apply: 
 
1.   Any biopsy-proven HSIL partially outside of the ETZ (for example, an HSIL lesion 
with extension to the perianal skin) 
 
2.   Any condylomas in the eligible treatment zone > 1/2 cm diameter 
 
3.   Any anal or rectal pathology requiring treatment including ulcer, fistula, fissure, or 
proctitis 
 
4.   Any anal stricture or stenosis in patient history or upon examination. 
 
5.   Symptomatic scarring in anal canal (i.e. not pliable, hyperkeratosis) 
 
6.   History of or present anal or rectal cancer 
 
7.   History of pelvic radiation therapy 
 
8.   History of HPV vaccination or plans to initiate HPV vaccination during the trial 
 
9.   History of ablation or resection therapy within the ETZ within 3 months prior to the 0 
month RFA visit (other than cauterization or excision of condylomata) 
 
10. History of topical therapy (e.g. Imiquimod, 5-FU, Trichloroacetic acid) within the 
 
ETZ within 3 months prior to the 0 month RFA visit 
 
11. Hemorrhoids > grade III 
 
12. Fecal incontinence 
 
13. Concurrent disease requiring systemic immunosuppression therapy 
 
14. Concurrent malignancy requiring systemic therapy 
 
15. Life expectancy < 2 years 

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16. Subject is on a platelet inhibiting agent (i.e. Clopidogrel) and/or anti-thrombotic 
agent (Heparin, Warfarin) and unable to discontinue 7 days before and after 
treatment for 14 days total. 
 
• Exception: Aspirin 81 mg PO daily does not need to be discontinued 
 
 
Subject Enrollment 
 
Upon approval of the protocol and informed consent form (ICF) by the institutional review 
board (IRB), subject enrollment may begin. The goal is to enroll a total of 70 subjects. 
 
Informed Consent 
 
Once the subject’s eligibility has been determined, the Investigator or a member of the 
Research Team will discuss the background of the proposed study and explain the benefits 
and risks of the procedures and follow-up. If this is of interest to the subject, the ICF will be 
discussed and presented. The subject must sign the consent form prior to enrollment. This 
form must have prior approval of the study site's IRB. Failure to obtain a signed ICF renders 
the subject ineligible for the study. 
 
Eligibility Process and Informed Consent 
 
All potential subjects are screened for study eligibility and interest in participation. The 
study is described in detail, all questions answered, and an IRB approved ICF is provided to 
the subject for review and signature. 
 
Study Procedure 
 
The subject is required to attend a minimum of 5 visits to complete the study. This will 
consist of three HRAs with possible biopsy, and one HRAs with RFA with possibility of 
biopsy.   If HSIL is found during follow-up then the subject will be asked to return for additional 
visits with HRA and RFA with possibility of biopsy.  See Figure 2 for study flow diagram. 
 
Preparatory instructions are given consistent with the Investigator’s standard practice for 
HRA.  Topical anesthesia is often sufficient for HRA with or without biopsy. Occasionally 
subjects might require addition of local anesthetic for biopsy. Conscious sedation or 
monitored anesthesia care, along with the administration of topical and local anesthesia, are 
typically adequate for performance of HRA with RFA.  In certain cases due to airway issues, 
sleep apnea, body habitus, or co-morbidities, general anesthesia may be required. 

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HRA entails specific subject positioning, such as jackknife prone or lateral decubitus, and the 
use of a high-resolution video camera to assess the anal canal with the aid of an anoscope or 
tissue retractor. Tissue staining with acetic acid ± Lugol’s solution is used to better identify 
lesions consistent with AIN. 
 
At the baseline qualifying exam, lesion(s) within the ETZ suspicious for HSIL are biopsied 
using standard forceps (one lesion per jar). At the initial RFA visit and prior to RFA, any 
new lesions suspicious for HSIL are biopsied. If a quadrant(s) is void of a lesion and has not 
been biopsied at the BQE a random biopsy will be collected. 
 
At 3, 6, and 9 months, an HRA will be performed and any lesions suspicious for HSIL will 
be biopsied using standard forceps. Patients positive for HSIL at 3, 6 or 9 months will be 
treated with focal RFA.  
 
At 12 months an HRA will be performed and any lesions suspicious for HSIL will be 
biopsied. In the absence of lesions suspicious for HSIL at the site of a prior HSIL lesion, a 
biopsy will be taken from that site. Additionally at 12 months, any quadrant in the anal 
canal without a prior HSIL or new lesion suspicious for HSIL will be biopsied randomly at 
the SCJ. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Figure 2. Study Procedure Flow Diagram 
 
 
 
RFA Procedure 
At visit 1, anal canal RFA is performed to the entire ETZ (the ETZ comprises full anal 
canal, 3 cm above the dentate line to the anocutaneous line proximal to the verge). The 
procedure entails Barrx Anorectal Wand, introducing the device into the anus through the 
anoscope, placing the electrode surface in contact with the target tissue and delivering 3 
bursts of energy in rapid succession at an energy density setting of 12 J/cm2/burst.  
Biopsy proven HSIL identified at the 3, 6, or 9 month visits are subsequently treated with 
focal RFA (3 ablations per site at 12 J/cm2/burst). One cm of normal tissue is ablated on all 
sides of the lesion (the width of the RFA device), unless this would cross the anocutaneous 
line. Field ablation is applied only to the ETZ quadrant(s) from which HSIL-positive 
BQE 
HRA with biopsy or biopsies
Visit 1 (Initial RFA) 
HRA with RFA and possible biopsy or biopsies
Visit 2 (3 Month) 
HRA with biopsies
Visit 2.1 (Follow-up RFA within 4 weeks of Visit 2)
If HSIL at visit 2, HRA with RFA and possible biopsy or biopsies
Visit 3 (6 Month)
HRA with biopsies
V3.1 (Follow-Upwitn 4 weeks of Visit 3) If HSIL at visit 3, HRA with RFA and possible biopsy or biopsies
Visit 4 (9 Month) HRA with Biopsies
Visit 4.1 (Follow-up RFA within 4 weeks of Visit 4)
Visit 5 (12 Month) HRA with biopsies of lesions suspicous for HSIL and previously treated sites or random biopsies of quadrants without 
biopsy 

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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biopsies were obtained at the previous visit without visible lesions identified at treatment. 
HSIL lesions that appear following the 0-month RFA visit outside of the ETZ are ablated in 
a manner consistent with the Investigator’s standard technique. 
 
Subjects are discharged to home using instructions consistent with the Investigator’s standard 
practice for HRA-directed HSIL ablation, which includes pain medication, stool softeners 
and dietary suggestions. 
 
 
Discharge Instructions 
 
Discharge instructions are given consistent with the Investigator’s standard practice for HRA 
with and without ablation, depending on the procedure(s) performed during the visit. After 
undergoing RFA, these instructions may include: 
 
• Pain medication [for example, Vicodin® (hydrocodone 
bitatrate/acetaminophen/hydrocodone 5 mg-300mg), 1 tablet by mouth every 6 
hours as needed for pain]. OTC pain medication such as ibuprofen or 
acetaminophen are acceptable when pain is mild to moderate.  
• Metamucil or Citrucel (over the counter) by mouth, taken twice daily 
 
• Colace 100 mg (docusate sodium) by mouth, take three times a day 
 
• High fiber diet 
 
• Drink 6 to 10 glasses of water every day 
 
• Sitz baths as needed for pain 
 
• Subject instructed to contact treating physician immediately for fever, anal 
bleeding, or other warning signs provided by the physician 
 
Study Pathology 
 
Each individual biopsy specimen is placed in its own jar (i.e. one specimen per jar). If more 
than one specimen is obtained from a single lesion, those specimens may go into the same jar 
(i.e.one lesion per jar). The location from which each specimen was obtained is recorded on a 
CRF with inclusion of octant descriptors. Biopsy specimens are fixed with formalin and 
processed in accordance with the local laboratory protocol for histology. The slides are 
examined by a local pathologist for diagnosis.  
 
 
 

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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Pathology Adjudication 
There is no pathology adjudication. 
 
Baseline Qualifying Exam (BQE) 
 
HRA and assessment of eligibility criteria will largely be completed with the BQE. After 
signing the informed consent approved by the IRB, subjects undergo a medical history and a 
physical exam. Inclusion and exclusion criteria are reviewed to ensure eligibility (a breast and 
pelvic exam will not be done). HRA does not need to be repeated if one has been done at the 
study site within 8 weeks prior to enrollment and biopsy reports and lesion documentation 
are available for study records. 
 
Mapping of the anal canal will be complete based on the BQE and will be used throughout 
the study. The mapping consists of 8 octants starting with the octant pointing to the subject’s 
posterior. Moving clockwise, the octants are labeled Posterior, Right Posterior (RP), Right 
Lateral (RL), Right Anterior (RA), Anterior (A), Left Anterior (LA), Left Lateral (LL), and 
Left Posterior (LP) (See Figure 3).  In addition, the anal canal should be divided into 
quadrants to help with random collection of biopsies at final visit. (See Figure 4); these 
designated quadrants will be kept consistent through the duration of the trial as depicted in 
Figure 3. Lesions will be identified and followed throughout the study. Should a lesion be 
eradicated, the location will still be followed to monitor recurrence. 
 
If female of child bearing age, patient will undergo a pregnancy test (urine test or 
equivalent). 
Figure 3. Mapping of the anal canal by fixed octant designation 
 
Posterior (P) 
 
L Posterior (LP) 
R Posterior (RP) 
 
 
 
 
L Lateral (LL)                                                        R Lateral (RL) 
 
 
 
 
 
L Anterior (LA)                                      R Anterior (RA) 
 
Anterior (A) 
 
 
 
 
 

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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Figure 4. Mapping of the anal canal by a possible quadrant designation 
 
Posterior (P) 
 
L Posterior (LP) 
R Posterior (RP) 
 
A 
 
L Lateral (LL) 
D 
 
 
C 
 
L Anterior (LA) 
 
 
         
 
                R Lateral (RL) 
B 
 
 
 
R Anterior (RA)
   Anterior (A)

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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Visit 1 Month 0 
 
Subjects who have had an HRA and are deemed to meet eligibility requirements return for 
enrollment within 2-8 weeks. All subjects must have at least one lesion containing HSIL. The 
index lesion(s) are first identified. If a quadrant does not have a lesion and has not been 
biopsied at the BQE, a biopsy will be collected in this quadrant immediately prior to RFA. 
The anal canal is surveyed for lesions. 
 
• Any new lesions suspicious for HSIL are biopsied using standard forceps (one 
lesion per jar). 
 
• If any quadrant does not have any visible abnormalities and has not already 
been biopsied, one biopsy is taken randomly from that quadrant to define 
baseline histologic status prior to treatment. 
 
RFA 
 
• Circumferential RFA the entire anal canal as described RFA Procedure section. 
 
Condyloma in the ETZ < 1/2 cm in diameter judged not to be sufficiently treated by RFA are 
excised or cauterized immediately after application of RFA.  The anal canal is surveyed for 
lesions. 
 
 
Visit 2 Month 3 
 
The subject returns 3 months ± 2 weeks after Visit 1 for HRA (referred to as Visit 2) 
 
and the anal canal is surveyed to assess healing (epithelialization, granulation tissue, 
fissure, scar formation) and for possible recurrent dysplasia.  Patients are asked about 
potential AE’s and SAE’s. 
 
HRA and Biopsy – Quadrant 
 
• Biopsy only lesion suspicious for persistent/recurrent HSIL. 
 
• Biopsy any new lesion suspicious for HSIL. 
 
 
 
 
 
 

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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Visit 2.1 (within 4 weeks of Visit 2) 
 
If any ETZ biopsy is positive for HSIL, the patient returns 4 ± 2 weeks later for RFA.  
• Any new lesions suspicious for HSIL are biopsied using standard forceps (one 
lesion per jar). 
RFA 
If the HSIL-positive biopsy was obtained from a visible lesion, that lesion (and any 
additional ETZ HSIL) is focally ablated as described in the RFA Procedure section. If 
there are new lesion(s) suspicious for HSIL in the ETZ that were not seen at Visit 2.0, 
they should be biopsied and then treated with RFA immediately (same session). If a 
lesion had been present at V2 and is not visible at the time of RFA, then the quadrant 
where that lesion occurred is treated.  For any new lesion(s) suspicious for HSIL that 
are outside the ETZ, treatment is at the discretion and timing of the investigator.  
Visit 3 Month 6 
 
The subject returns 6 months ± 2 weeks after Visit 1 for HRA (referred to as Visit 3) 
 
and the anal canal is surveyed to assess healing (epithelialization, granulation tissue, 
fissure, scar formation) and for possible recurrent dysplasia. Patients are asked about 
potential AE’s and SAE’s. 
HRA and Biopsy – Quadrant 
 
• Biopsy only lesion suspicious for persistent/recurrent HSIL. 
 
• Biopsy any new lesion suspicious for HSIL. 
 
Visit 3.1 (within 4 weeks of Visit 3) 
 
If any ETZ biopsy is positive for HSIL, the patient returns 4 ± 2 weeks later for RFA.  
RFA 
If the HSIL-positive biopsy was obtained, that lesion (and any additional ETZ HSIL) 
is focally ablated as described in the RFA Procedure section. If there are new lesion(s) 
suspicious for HSIL in the ETZ that were not seen at Visit 3.0, they should be biopsied 
and then treated with RFA immediately (same session). If a lesion had been present at 
V3 and is not visible at the time of RFA, then the quadrant where that lesion occurred 
is treated.  For any new lesion(s) suspicious for HSIL that are outside the ETZ, 
treatment is at the discretion and timing of the investigator.  

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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Visit 4 Month 9 
 
The subject returns 9 months ± 2 weeks after Visit 1 for HRA (referred to as Visit 4) 
 
and the anal canal is surveyed to assess healing (epithelialization, granulation tissue, 
fissure, scar formation) and for possible recurrent dysplasia.  Patients are asked about 
potential AE’s and SAE’s. 
 
HRA and Biopsy – Quadrant 
 
• Biopsy only lesions suspicious for persistent/recurrent HSIL. 
 
• Biopsy any new lesion suspicious for HSIL. 
 
 
Visit 4.1 (within 4 weeks of Visit 4) 
 
If any ETZ biopsy is positive for HSIL, the patient returns 4 ± 2 weeks later for RFA.  
• Biopsy any new lesions suspicious for HSIL are biopsied using standard 
forceps (one lesion per jar). 
RFA 
If the HSIL-positive biopsy was obtained, that lesion (and any additional ETZ HSIL) 
is focally ablated as described in the RFA Procedure section. If there are new lesion(s) 
suspicious for HSIL in the ETZ that were not seen at Visit 4.0, they should be biopsied 
and then treated with RFA immediately (same session). If a lesion had been present at 
V4 and is not visible at the time of RFA, then the quadrant where that lesion occurred 
is treated.  For any new lesion(s) suspicious for HSIL that are outside the ETZ, 
treatment is at the discretion and timing of the investigator. 
 
Visit 5 Month 12 
 
The subject returns 12 months ± 2 weeks after Visit 1 for HRA (referred to as Visit 5), 
and the anal canal is surveyed to assess healing (epithelialization, granulation tissue, 
fissure, scar formation) and for possible recurrent dysplasia.  Patients are asked about 
potential AE’s and SAE’s. 
 
 
HRA and Biopsy – Quadrant 
 
• Biopsy all locations of HSIL, lesions indexed at BQE and at Visit 1, 2, 3 and 4. 


Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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• Biopsy any new lesion suspicious for HSIL. 
 
• Any quadrant without biopsy will undergo random biopsy. 
 
 
Study Endpoints 
 
Primary: 
 
1. 1.  Histologic clearance of all HSIL within the ETZ on a patient and lesion basis 
at 12 months from first RFA 
 Related adverse event (AE) incidence by subject (study device relationship 
considered related if categorized by Investigator as: definite, probably, 
possibly, or unknown) 
 
Secondary: 
 
2. Related adverse event (AE) incidence by subject (study device relationship 
considered related if categorized by Investigator as: definite, probably, possibly 
or unknown)  
3. Persistence of index HSIL at 3 months post RFA. 
.  
Hypothesis 
 
The study device RFA procedure will have an acceptable safety profile and demonstrate 
superior disease-free survival at 12 months over standard ablative techniques. 
 
 
3.   ADVERSE EVENTS 
 
 
General 
 
Transient side effects that can be expected after treatment include pain, fever, bleeding, and 
discharge. Adverse events may include anal stenosis, incontinence (including leakage), 
scarring, disfiguration, infection, abscess, fissure, or fistula formation. Potential serious 
adverse events (SAE’s) (although they have not been observed to date in Barrx™ Ablation 
System clinical trials) may include ablation-related visceral perforation, major bleeding or 
death. There is potential for the occurrence of adverse events or serious adverse events 
related to the monitored sedation. 
 
Reporting 
 
The Investigator will assess each adverse event with respect to severity and relationship to the  

Protocol 01-2017: RFA for Anal Intraepithelial Neoplasia 
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study device and make the determination regarding reportability of any adverse event to the 
Study Sponsor and GI Solutions, Covidien formerly BÂRRX Medical, Inc. (the manufacturer 
of the device) and the IRB according to the guidelines of the IRB. All reported events will be 
evaluated by GI Solutions, Covidien for determination of reporting requirements with respect 
to the FDA under the MDR reporting regulation in 21 C.F.R. Part 803.  All SAE’s deemed to 
be possibly, probably or definitely related to the RFA will be reported to the sponsor and IRB 
within 72 hours of discovery of the event. 
 
Reports relating to the subject’s subsequent medical course must be submitted to the GI 
Solutions, Covidien until the event has subsided or, in case of permanent impairment, until 
the event stabilizes and the overall clinical outcome has been ascertained. 
 
4.   STATISTICAL METHODS 
 
As this is a single arm, open label trial, no power calculations are applicable.  Baseline and 
study outcomes will be summarized using descriptive statistics. Lesion-free survival will be 
estimated using the Kaplan-Meier method, and multivariate analysis on HSIL persistent 
recurrence will be performed using Cox’s proportional hazards regression model. A p-value 
<0.05 is considered statistically significant. 
Assuming the usual binomial distribution assumptions, a 95% Wald/Laplace confidence 
interval is formed assuming an 80% expected proportion of healing. To form a confidence 
interval of width 20% (that is, 10% below and 10% above), and a dropout rate of 10%, we 
arrive at a sample size needed of 70. 
 
5.   STUDY MANAGEMENT 
 
Case Report Forms 
 
All study data will be recorded onto paper case report forms (CRF) and forwarded to the 
Principal Investigator as a scan or fax, where it will be entered into an internet-based 
secured data entry and processing system. Data entered onto CRF pages will be typed or 
legibly printed using a black ballpoint pen. Although CRF completion may be delegated to 
other study personnel, the Investigator remains responsible for the accuracy and integrity of 
all data entered on CRFs. 
 
Protocol Modification 
 
Significant changes to the protocol will require that the protocol be formally amended. 
Protocol amendments will be agreed upon by the Investigators and approved of by the IRB. 

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Administrative changes to the protocol such as a change that has no effect on the conduct of 
the study or risk to the subject will be documented in a memorandum. The IRB must be 
notified of administrative changes, but formal approval is not required unless it is deemed 
necessary under the standard IRB operating procedures. 
 
Study Data Collection 
 
Primary data collection based on source-documented chart reviews will be performed by 
 
study coordinators at the clinical site. All study data will be recorded on CRFs and sent to the 
Principal Investigator. As per the ICF, all subject confidentiality will be maintained. Data 
entered through the internet-based data entry system will reside in a secure password protected 
database. The database will be backed up periodically, and the backup files will be stored in a 
secure location. Any issues identified will be communicated back to the site for clarification. 
 
Data Handling and Record Keeping 
 
In order to assure adequate control and provide study data that are consistent and of the 
highest quality, the methods described in this section will be employed. Data will be handled 
by qualified personnel at the study site and the Principal Investigator. 
 
Data Receipt 
 
All CRFs and online data will be subjected to initial inspection for omitted data, gross data 
inconsistencies, and timeliness of reporting. Any deficiencies will be addressed with the 
clinical site. 
 
Data Entry 
 
Data will be entered into a secured database with limited access. 
 
 
Data Queries and Resolution 
 
If an issue is discovered at Principal Investigator, a data query will be made to the site. The 
issue will be stated and response will be requested from the site. Pending the nature of the 
query, a signature from site personnel or the Investigator may be required. 
 
Data Back-up 
 
Database back-ups will be performed regularly at the Principal Investigator. 
 
 
Data Analysis 
 

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All exported datasets for analyses will be inspected using edit checks so as to minimize 
errors. 
 
Data Ownership 
 
The Principal Investigator retains ownership of all clinical data generated in this study 
and controls the use of the data for purposes of regulatory submissions. 
 
Record Retention 
 
All documents related to this study must be retained by the Investigator for a minimum of 
two (2) years after the investigation has been completed or terminated. Study records that 
must be retained include, but are not limited to: 
 
• signed subject informed consent forms, 
 
• subject medical records including laboratory and diagnostic reports, 
 
• supporting documentation of all serious adverse events 
 
• study-related correspondence and study reports. 
 
6.   ETHICAL AND REGULATORY CONSIDERATIONS 
 
 
Institutional Review Board (IRB) Approval 
 
The Principal Investigator is responsible for submitting this protocol, the informed consent 
document, and all relevant supporting data to the IRB. IRB approval of the protocol, 
informed consent document, and any advertisement used to recruit study subjects must be 
obtained before the study may be initiated. 
 
The Principal Investigator is responsible for keeping the IRB advised of the progress of the 
study and of any changes made to the protocol as deemed appropriate, but in any case, at 
least once a year. The Principal Investigator is also responsible for notifying the IRB of any 
serious adverse events that occur during the study. 
 
Informed Consent 
 
Each subject will have the nature and the purpose of this investigation explained to him or her 
by the Investigator or another member of the investigative team at the place of care. The 
study will be explained to the subjects in lay language. Prior to entry into this investigation 
the subject must give voluntary, written informed consent to participate by signing the ICF. 
On the same occasion, the Investigator or another staff member responsible for the conduct of 

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the study (i.e. Sub-investigator) will also sign the ICF. The person responsible for obtaining 
the informed consent must also sign it if he/she is not the Investigator or Sub- investigator. 
The subject will receive a copy of the signed informed consent. 
 
Subject Identification 
 
All CRFs are coded with a site code and subject code, no subject identifying information will 
be contained on study documents. Further, no source documents will be provided to the 
Principal Investigator so subject confidentiality is completely protected in this study. All 
subjects are identified with a site code and a subject code. Sites will be assigned a unique 3 
digit number upon IRB approval. Each subject is uniquely identified by the site number 
followed by a 3 digit subject number. For example, subject 001 from site 002 is identified as 
002-001. 
 
7.   SUBJECT WITHDRAWAL AND STUDY TERMINATION 
 
 
Subject Withdrawal 
 
Any subject who wishes to withdraw from this investigation on his/her own accord and for 
whatever reason is entitled to do so without obligation and prejudice to further treatment. In 
addition, the Investigator may decide for reasons of medical prudence, to withdraw a subject. 
In either event, the Investigator will clearly document the date and reason(s) for the subject’s 
withdrawal from this investigation in the CRF and should indicate whether or not he considers 
it related to the device. 
 
Termination of Investigation 
 
The Principal Investigator reserves the right to terminate the study at any time. Should this 
be necessary, the Investigator and subject will arrange the procedures on an individual basis 
after review and consultation. In terminating the study, the Investigator will assure that 
adequate consideration is given to the protection of subject interests. 
 
8.   REGULATORY STANDARDS 
 
This protocol was designed using Regulatory Reference: ISO 14155:2011, and all applicable 
parts of 21CFR. 

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9. APPENDIX A. SCHEDULE OF VISITS AND TASKS 
 
Task↓    Visit → 
BQE 
V1 RFA 
V2-
3Mth 
V.2.1 
V3-6Mth 
V3.1 
V4-9Mth 
V4.1 
V5 Final-12 
Mth 
Informed Consent Signed 
 x 
Inclusion/Exclusion Criteria 
 x 
 x 
Confirm Baseline HSIL Dx 
 x 
Med. Hx Obtained from Subject 
 x 
Pregnancy Test on Female Subjects 
of Child Bearing Potential 
 x 
Digital Anorectal Exam 
 x 
 x 
 x 
 x 
 x  
 x 
 x  
 x 
 x 
HRA  
 x 
 x 
 x 
 x 
 x 
 x 
 x 
 x 
 x 
Map anal canal; identify lesions 
 x 
 x 
 x 
 x 
Designate quadrants (A,B,C,D) to 
be used throughout study 
 x 
Biopsy lesions suspicious of HSIL 
 x 
 x 
 x 
 x 
 x 
Biopsy new HSIL‐appearing lesions 
and send to local pathology 
 x 
 x 
 x  
 x 
x 
 x 
x 
 x 
Random biopsy of any quadrant 
without a biopsy and send to local 
pathology 
 x 
X(If not done 
@ BQE) 
 x 
Radiofrequency Ablation (RFA) 
Treatment with Barrx™ RFA 
System 
 X 
Circumferential  
X 
Focal 
X 
Focal 
X 
Focal 
Post Treatment Discharge 
Instructions and “As Needed” Pain 
Medications Addressed 
 x 
 x 
 x 
 x 
 x 
Adverse Event/SAE Inquiry 
 x 
 x 
 x 
 x 
 x 
 x 
 x 
 x 
 x