ORIGINAL CONTRIBUTION

HIV-Infected Patients With Anal Cancer Precursors:

Clinicopathological Characteristics and Human
Papillomavirus Subtype Distribution
Yuxin Liu, M.D., Ph.D.1 • Keith M. Sigel, M.D., Ph.D.2 • William Westra, M.D.1
Melissa R. Gitman, M.D., M.P.H.1 • Wenxin Zheng, M.D.3 • Michael M. Gaisa, M.D., Ph.D.4
1 Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, New York
2 Division of General Internal Medicine, Icahn School of Medicine at Mount Sinai, New York, New York
3 Department of Pathology, Obstetrics and Gynecology, Simon Comprehensive Cancer Center, University of Texas
Downloaded from http://journals.lww.com/dcrjournal by BhDMf5ePHKbH4TTImqenVL56SvJs3yjmJdNKesj41jqQ7KxdFCQCuJxjQlNl7CjPa5qMS8dYRHE= on 06/06/2020

Southwestern Medical Center, Dallas, Texas

4 Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, New York

BACKGROUND:  People living with HIV have high rates of
anal human papillomavirus infection and anal precancer/
cancer.
OBJECTIVE:  This study aims to: 1) determine human
papillomavirus subtype distribution among people living
with HIV with anal high-grade squamous intraepithelial
lesions; 2) compare the clinicopathological characteristics
of patients with anal high-grade squamous intraepithelial
lesions by human papillomavirus 16 status; and 3)
investigate high-risk human papillomavirus negative anal
high-grade squamous intraepithelial lesion cases.
DESIGN:  In this retrospective study, 700 people living
with HIV who have biopsy-proven anal high-grade
squamous intraepithelial lesions were reviewed for
demographics, cytological diagnoses, and human
papillomavirus testing results for human papillomavirus
16, 18, and 12 other high-risk types. For human
papillomavirus-negative subjects, corresponding biopsies
were genotyped by using real-time polymerase chain
reaction.
Funding/Support: Research support was provided by the National In-
stitutes of Health (K07CA180782 to Dr Sigel).
Financial Disclosures: None reported.
Presented at the meeting of the American Society for Colposcopy and
Cervical Pathology, Atlanta, GA, April 4 to 7, 2019.
Correspondence: Yuxin Liu, M.D., Ph.D., Department of Pathology,
Icahn School of Medicine at Mount Sinai, One Gustave Levy Place, New
York, NY 10029. E-mail: Yuxin.liu@mountsinai.org
Dis Colon Rectum 2020; 63: 890–896
DOI: 10.1097/DCR.0000000000001671
© The ASCRS 2020
890
SETTINGS:  This study was conducted in a large urban
HIV clinic system and major referral center for anal
cancer screening.
PATIENTS:  Median age was 46 years (range, 20–76).
Ninety-one percent of the patients were men who have
sex with men.
MAIN OUTCOME MEASURES:  The primary outcome
measure was the association between demographic
variables and human papillomavirus 16 status.
RESULTS:  Anal cytology was unsatisfactory (5%),
benign (13%), atypical squamous cells of undetermined
significance (35%), low-grade squamous intraepithelial
lesion (36%), and high-grade squamous intraepithelial
lesions (11%). Human papillomavirus cotesting results
were negative (n = 38, 5%), human papillomavirus 16
(n = 303, 43%), human papillomavirus 18 (n = 78, 11%),
or exclusively non-16/18 types (n = 281, 40%). Human
papillomavirus 16 positivity was associated with ≥3 high-
grade lesions and ≥ low-grade squamous intraepithelial
lesion cytology (p < 0.001). Age, race/ethnicity, sex,
smoking, CD4+ T-cell count, and HIV viral load did
not differ by status of human papillomavirus 16 (p >
0.05). For human papillomavirus-negative cases, human
papillomavirus genotyping of biopsies was positive for
high-risk (n = 14, 36%) or possibly carcinogenic types (n
= 12, 32%), or negative
(n = 12, 32%).
LIMITATIONS:  This was a retrospective data analysis,
and it pooled the results for 12 high-risk human
papillomavirus types rather than individual types.
CONCLUSIONS:  Nearly all people living with HIV and
anal high-grade squamous intraepithelial lesions test
positive for high-risk human papillomavirus on anal
swabs; negative results may be due to sampling error,
DISEASES OF THE COLON & RECTUM VOLUME 63: 7 (2020)

Copyright © The American Society of Colon & Rectal Surgeons, Inc. Unauthorized reproduction of this article is prohibited.
 DISEASES OF THE COLON & RECTUM VOLUME 63: 7 (2020)
L1-based polymerase chain reaction assay, or human
papillomavirus types not captured by standard clinical
assays. Patients who have human papillomavirus
16-positive anal high-grade squamous intraepithelial
lesions are indistinguishable from others based on
demographic and clinical characteristics, underscoring
the potential role of human papillomavirus testing for
anal cancer screening. See Video Abstract at http://links.
lww.com/DCR/B208.
PACIENTES PORTADORES DE VIH CON PRECURSORES
DE CÁNCER DE ANO: CARACTERÍSTICAS
CLINICOPATOLÓGICAS Y DISTRIBUCIÓN DEL SUBTIPO
VPH
ANTECEDENTES:  Los pacientes portadores de VIH
tienen altas tasas de infección por VPH y alto riesgo de
desarrolar lesiones precáncerosas / cáncerosas del ano.
OBJETIVO:  (1) Determinar la distribución del subtipo
de VPH entre las personas portadoras de VIH con
lesiones intraepiteliales escamosas anales de alto grado.
(2) Comparar las características clinicopatológicas de
pacientes con lesiones intraepiteliales escamosas anales
de alto grado del subtipo VPH 16. (3) Investigar casos de
lesiones intraepiteliales escamosas anales de alto grado
negativas para el VPH de alto riesgo.
DISEÑO:  Estudio retrospectivo sobre 700 personas
portadoras de VIH con lesiones intraepiteliales escamosas
anales de alto grado confirmadas por biopsia. Los
datos fueron revisados para determinar información
demográfica, diagnósticos citológicos y resultados de
tipización en el VPH subtipos 16 y 18, y otros 12 tipos
de alto riesgo. Para los individuos negativos al VPH,
se analizó el genotipo en las biopsias correspondientes
mediante test de PCR en tiempo real.
AJUSTES:  Extenso sistema de clinicas urbanas tratando
VIH y un importante centro de referencia para la
detección del cáncer anal
PACIENTES:  la mediana de edad poblacional fue de 46
años (rango, 20–76). 91% eran hombres que tenían sexo
con hombres.
PRINCIPALES RESULTADOS:  Asociación entre las variables
demográficas y el estado del VPH subtipo16.
RESULTADOS:  la citología anal fue insatisfactoria (5%),
benigna (13%), células escamosas atípicas de importancia
indeterminada (35%), lesión intraepitelial escamosa de
bajo grado (36%) y lesiones intraepiteliales escamosas de
alto grado (11%). Los resultados de la prueba conjunta
del VPH fueron negativos (n = 38, 5%), el virus del
VPH subtipo 16 (n = 303, 43%), el VPH subtipo 18
(n = 78, 11%) o los subtipos exclusivamente no 16/18
(n = 281, 40%). La positividad del VPH subtipo 16 se
Copyright © The American Society of Colon & Rectal Surgeons, Inc. Unauthorized reproduction of this article is prohibited.
891
encotraba asociado con ≥3 lesiones de alto grado y ≥
células escamosas atípicas en la prueba de citología de
indeterminada importancia (p < 0.001). La edad, la raza /
etnia, el sexo, el tabaquismo, el recuento de células T CD4
+ y la carga viral del VIH no difirieron según el estado
del VPH subtipo 16 (p > 0.05). Para los casos negativos al
VPH, el genotipo del virus del papiloma humano de las
biopsias fue positivo para los tipos de alto riesgo
(n = 14, 36%) o posiblemente carcinogénicos (n = 12,
32%), o negativo (n = 12, 32%).
LIMITACIONES:  Análisis de datos retrospectivos, con
resultados agrupados para 12 tipos de VPH de alto riesgo
en lugar de tipos individuales.
CONCLUSIONES:  Casi todas las personas portadoras de
VIH con lesiones intraepiteliales escamosas anales de
alto grado dan positivo para el VPH de alto riesgo al
muestreo de hisopos anales; Los resultados negativos
pueden deberse a un error en el muestreo y al análisis de
PCR basado en L1 o subtipos de VPH no obtenidos en
los ensayos clínicos estándar. Los pacientes con lesiones
intraepiteliales escamosas anales de alto grado positivas
para el VPH subtipo 16 no son identificables de los
demás, en función de las características demográficas y
clínicas, lo que minimiza el rol potencial de la prueba
del VPH en la detección del cáncer anal. Consulte
Video Resumen en http://links.lww.com/DCR/B208.
(Traducción—Dr. Xavier Delgadillo)
KEY WORDS:  Anal cancer; Anal high-grade squamous
intraepithelial lesions; Human papillomavirus; People
living with human immunodeficiency virus.
P
eople living with the human immunodeficiency
virus (PLWH), especially men who have sex with
men, have a high prevalence of human papilloma-
virus (HPV) infection of the anal canal and subsequent
anal precancer/cancer.1,2 Anal cancer risk remains elevated
even for those who have achieved systemic HIV virologic
control with antiretroviral therapy.3 Although the inci-
dence of AIDS-defining cancers has dropped significantly,
anal cancer is now the second most common non-AIDS-
defining cancer and is linked to increased morbidity and
mortality.4,5
Anal cancer screening has become an important com-
ponent in the comprehensive care of PLWH, aiming to de-
tect and treat cancer precursor lesions (ie, anal high-grade
squamous intraepithelial lesions [HSIL]) before malig-
nant transformation occurs.6 Although anal cytology is
currently the mainstay of proposed screening protocols,
its performance is suboptimal as evidenced by its wide
range of sensitivity (69%–93%) and relatively low speci-
ficity (32%–59%).7,8 High-grade squamous intraepithelial
lesions and even invasive cancer are frequently missed by
 892
anal cytology, emphasizing the need for better risk assess-
ments and screening modalities.
Anal HSILs are caused by high-risk HPV (HR-
HPV) types belonging to alpha-papillomavirus species 9
(HPV16, 31, 33, 35, 52, 58) or species 7 (HPV18, 39, 45,
59, 68).9 Among these types, HPV16 is by far the most car-
cinogenic in the anus, accounting for 67% of anal cancers
in HIV-infected men, whereas HPV18 or other types ac-
count for only 14% and 26%.10 Evidently, HPV16-induced
HSILs carry a significantly higher risk of progression than
those induced by other oncogenic HPV types. Therefore,
HPV genotyping could provide a valuable, additional risk
assessment for patients who have developed HSIL.11
Human papillomavirus testing on anal swab or bi-
opsy samples has not gained wide acceptance in clinical
practice. A meta-analysis of 2024 anal HSIL cases ex-
tracted from 95 studies calculated that HPV prevalence is
96% in HIV-infected men with HSIL, specifically HPV16
(51%), HPV18 (20%), and HPV31/33/45/52/58 (57%).10
Although most studies in the meta-analysis used cytolog-
ical samples, a few tested biopsy samples.12–14 In a previ-
ous study, we reported a 90% HR-HPV detection rate in
histological HSIL samples, of which 39% were HPV16
and 3% were HPV18.15 Others reported lower rates (80%
and 67%).16,17 In other words, 10% to 33% of HSIL biopsy
samples did not have detectable HR-HPV for reasons that
are poorly understood.
Herein, we conducted a cross-sectional study of a
large cohort of PLWH with biopsy-proven anal HSIL
(AHSIL) aiming to 1) determine HPV subtypes detected
in anal swab samples by using the Cobas 4800 HPV test,
2) compare clinicopathological characteristics of patients
with HSIL according to HPV16 infection status, and 3) in-
vestigate Cobas hR-HPV-negative HSIL cases.
MATERIALS AND METHODS
Patient Selection and Demographics
This study was approved by the Institutional Review Board
of the Icahn School of Medicine. We queried the clinical
high-resolution anoscopy (HRA) database between Janu-
ary 2012 and January 2018 for all PLWH who had con-
comitant anal swabs, HPV genotyping, and HRA-guided
biopsy-proven AHSIL obtained during the same visit. We
reviewed electronic medical records and recorded patient
age, race/ethnicity, sex, HIV status, smoking history, his-
tory of anogenital condylomata, CD4+ T-cell count, and
HIV-1 RNA level most closely associated with the visit.
Anal Cytology
Anal swab samples were obtained by inserting a moist-
ened, nonlubricated cytobrush blindly into the anal ca-
nal in an effort to collect epithelial cells from above the
squamocolumnar junction to the anal verge. Samples were
Copyright © The American Society of Colon & Rectal Surgeons, Inc. Unauthorized reproduction of this article is prohibited.
LIU ET AL: HPV SUBTYPES IN HIV+ ANAL HSIL PATIENTS
then transferred to a liquid-based cytology preservation
medium for ThinPrep (Cytyc Corporation, Boxborough,
MA) and were subsequently stained by using the Papa-
nicolaou stain. Cytological diagnoses were rendered by
cytopathologists from the Mount Sinai Hospital follow-
ing criteria and categories of the 2001 Bethesda System:
unsatisfactory; benign; atypical squamous cells of unde-
termined significance (ASCUS); low-grade squamous in-
traepithelial lesion (LSIL); atypical squamous cells, cannot
exclude HSIL (ASC-H); and HSIL.18
HPV DNA Testing on Anal Swabs
Regardless of cytological diagnosis, aliquots of anal cytol-
ogy samples were subsequently tested for HPV DNA using
the Cobas 4800 HPV test (Roche Diagnostics, Indianapo-
lis, IN) following manufacturer instructions.19 The assay
reports HPV16 and 18 individually, and aggregate results
for 12 oncogenic HR-HPV types: 31, 33, 35, 39, 45, 51, 52,
56, 58, 59, 66, and 68.
HRA and Biopsy
High-resolution anoscopy procedures and biopsies were
performed in an office setting following the standard, pre-
viously described techniques.20 The perianal area, anal ca-
nal, and squamocolumnar junction were pretreated with
5% acetic acid and subsequently examined under a high-
resolution colposcope at 15-fold magnification and addi-
tional staining with Lugol iodine. Targeted biopsies were
obtained from mucosal areas suspicious for HSIL or can-
cer, whereas random biopsies were not performed.
Histological Diagnosis
Biopsy samples were processed following standard histo-
logical protocols. Each sample was sectioned in 3 to 5 lev-
els and stained with hematoxylin and eosin. All samples
were diagnosed by surgical pathologists from the Mount
Sinai Hospital using standard morphological criteria as
outlined in the Lower Anogenital Squamous Terminology
(LAST) project.21 The designation of HSIL required dys-
plastic squamous cells with nuclear enlargement, coarse
chromatin, and irregular nuclear membrane present in
the middle (anal intraepithelial neoplasia 2 [AIN 2]) or
top third of the epithelium (AIN 3). P16 immunohisto-
chemistry (IHC) was performed on ≈30% of the cohort
to differentiate between HSIL (strong and diffuse positive
staining) and LSIL or benign squamous epithelium (weak,
patchy, or negative staining).22 Following the LAST recom-
mendations, our pathologists used p16 IHC primarily to
triage intermediate lesions (formerly classified as AIN 2)
into LSIL versus HSIL categories. In addition, p16 IHC
was used to differentiate HSILs from their benign mimics
such as inflammatory atypia. At the time of initial diagno-
sis, consensus for Cobas-negative HSILs was reached be-
tween 2 or 3 pathologists at a multiheaded microscope. All
 DISEASES OF THE COLON & RECTUM VOLUME 63: 7 (2020) 893

are specialized gynecological pathologists with 10+ years TABLE 1.   Patient clinicopathological characteristics (n = 700)
experience in diagnosing HPV-related anogenital disease.
Characteristics n (%)
To prevent internal bias, an external expert pathologist
(W.Z.) independently reviewed and confirmed the HSIL Age, median (range), y 46 (20–76)
diagnosis for the cases included in the study. Sex
 MSM 637 (91)
 HM 4 (0.6)
HPV Genotyping on Biopsy Sample  Female 59 (8.4)
DNA was extracted from tissue sections using The Max- Race/ethnicity
well 16 FFPE Tissue LEV DNA Kit (Promega, Madison,  White 247 (35)
 Hispanic 189 (27)
WI) according to the manufacturer’s instructions. We   African American 159 (23)
measured the concentrations of extracted DNAs using a  Other 82 (12)
Nano-Drop ND-2000 spectrophotometer (Thermo Fisher  Unknown 23 (3)
Scientific Inc, Fair Lawn, NJ). Real-time polymerase chain Smoking history
reactions (PCRs), performed with Roche Light cycler 480  Current 181 (26)
 Former 173 (25)
and a High-Resolution Melting Master kit, used PCR  Never 342 (49)
primers (GP5+ and GP6+) specifically targeting the L1 re-  Unknown 4 (0.6)
gion.23 The β-actin gene served as the quality control for CD4+ T-cell, cells/mm3
DNA extraction and PCR. After initial confirmation of the  <500 238 (34)
presence of HPV DNA, type-specific primers and probes  ≥500 438 (63)
 Unknown 24 (3)
for HPV16 or 18 (targeting the E6 region) were used for HIV viral load, copies/mL
real-time PCR. The probe pairs were labeled with fluores-  <100 610 (87)
cein at the 3′ end and LightCycler-Red 640 for HPV16 or  ≥100 81 (12)
LightCycler-Red 670 for HPV18 at the 5′ end. If samples  Unknown 9 (1)
tested negative during HPV16/18-specific PCR, we per- Anal cytology
 Unsatisfactory 34 (5)
formed Sanger sequencing to detect other HPV types  Benign 89 (13)
after amplification with GP5+ and GP6+ primer pairs.  ASCUS 247 (35)
HPV types other than 16/18 were determined by using  LSIL 253 (36)
GenBank.  ASC-H, HSIL 77 (11)
Cobas4800 HPV testing
 Negative 38 (5)
Statistical Analysis  Positive 662 (95)
Using the Fisher exact test, the prevalence of HR-HPV,  HPV16/18/others 303 (43)
HPV16, and non-16 types was compared between HIV-  HPV18/others 78 (11)
 Others 281 (40)
infected men and women. Age distribution was compared
between HPV16, non-16 HR-HPV, and negative HPV ASC-H = atypical squamous cells, cannot exclude HSIL; ASCUS = atypical squamous
cells of undetermined significance; HM = heterosexual men; HPV = human papil-
groups using the Wilcoxon test. Other clinicopathologi- lomavirus; HSIL = high-grade squamous intraepithelial lesions; LSIL = low-grade
cal variables were compared using the χ2 test. All analy- squamous intraepithelial lesion; MSM = men who have sex with men.
ses were performed using STATA 15 (Stata Corp, College
Station TX). A total of 281 (40%) swabs were positive exclusively for
non-16/18 HR-HPV types. HIV-infected men and women
revealed similar overall HR-HPV prevalence and type dis-
RESULTS tribution (p > 0.05).
In the study period, 700 PLWH with biopsy-proven AH- Patients were categorized into 3 groups based on
SIL had concomitant HPV testing performed on anal swab HR-HPV types detected in anal swab samples: HPV16,
samples. All participants were prescribed antiretroviral non-16 HR-HPV, and HR-HPV negative. There was no
therapy at the time of HRA. Table 1 shows patient demo- significant difference in age, sex, race/ethnicity, smoking
graphics, HIV parameters, anal cytology, and Cobas HPV history, CD4+ T-cell count, HIV viral load, or history of
testing results. condylomata between the 3 groups (p > 0.2). When cyto-
HR-HPV was positive in 662 (95%) swabs but nega- logical diagnoses were considered (Table 2), the HPV16
tive in 38 (5%). Human papillomavirus 16 was detected in group had significantly more ≥ LSIL abnormalities than
303 (43%) swabs in the following combinations: HPV16 the non-16 and negative groups (57%, 41%, and 22%;
alone (n = 23, 3%), HPV16 and 18 (n = 1), HPV16 and p < 0.001). With the use of ASCUS or worse as a cutoff,
others (n = 213, 30%), or HPV16, 18, and others (n = 66, the sensitivity of anal cytology to detect HSIL was higher
10%). Isolated HPV18 was detected in 3 (0.4%); coin- in the HPV16 group (265/303, 87%) than in the non-16
fection of HPV18 and others was detected in 75 (11%). group (285/359, 79%).

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 894 LIU ET AL: HPV SUBTYPES IN HIV+ ANAL HSIL PATIENTS

TABLE 2.   Comparing pathological findings among patients with HPV16, non-16 HR-HPV, or negative for HR-HPV
HR-HPV types
HPV16 Non-16 Negative
Characteristics (n = 303) (n = 359) (n = 38) p
Anal cytology <0.001
 Unsatisfactory 11 (4) 19 (5) 4 (11)
 Benign 27 (9) 55 (15) 7 (17)
 ASCUS 90 (30) 138 (38) 19 (50)
 LSIL 131 (42) 118 (33) 4 (11)
 ASC-H, HSIL 44 (15) 29 (8) 4 (11)
No. of biopsy-proven HSIL
 1 or 2 216 (71) 296 (82) 37 (97) <0.001
 3–6 87 (29) 63 (18) 1 (3)
Severity of dysplasia
 AIN 2 210 (69) 278 (77) 29 (76) 0.06
 AIN 3 93 (31) 81 (23) 9 (24)
Data given are number of cases (percentage).
AIN = anal intraepithelial neoplasia; ASC-H = atypical squamous cells, cannot exclude HSIL; ASCUS = atypical squamous cells of undetermined significance; HPV = human
papillomavirus; HR-HPV = high-risk human papillomavirus; HSIL = high-grade squamous intraepithelial lesion; and LSIL = low-grade squamous intraepithelial lesion.

Among individuals with unsatisfactory or benign cy- 51, and 73) and 2 lesions harbored 2 types (HPV67/82;
tology, 112 of 123 (91%) were positive for HR-HPV, in- HPV73/82).
cluding HPV16 (31%) and non-16 HR-HPV types (60%).
HPV16 positivity was associated with a higher number of
concomitant HSILs (p < 0.001). The prevalence of AIN 3
DISCUSSION
was comparable among the 3 groups: 31%, 23%, and 24% This study comprised a large cohort of HIV-infected indi-
(p = 0.06). viduals with histologically proven AHSIL. The vast major-
For Cobas-negative swabs, corresponding HSIL bi- ity (95%) tested HR-HPV positive on anal swab samples
opsy specimens (n = 38) were genotyped using real-time collected at the time of HSIL diagnosis. HPV16 and 18
PCR (Table 3). Real-time PCR results were negative for were positive in 43% and 11% of swabs, primarily as coin-
HR-HPV (n = 12), positive for possibly carcinogenic HPV fection with other HR-HPV types. Forty percent tested
strains (n = 12, HPV53, 67, 69, 73, and 82), or positive positive for non-16/18 HR-HPV types alone, whereas 5%
for established HR-HPV subtypes that should have been were negative for all 14 types included in the Cobas panel.
detected by the Cobas assay (n = 14, HPV16, 18, 31, 33, HIV-infected women (8% of our cohort) revealed similar
35, 45, 51, 52, 58, and 59). Although most lesions harbored HPV-type distribution in anal swab samples compared to
a single viral type, one lesion harbored 3 types (HPV35, HIV-infected men. In comparison, HPV prevalence rates
from a large meta-analysis were slightly higher than ours:
96% in HIV-infected men with AHSIL, specifically HPV16
TABLE 3.   HPV genotyping of biopsy specimens from patients (51%), HPV18 (20%), and HPV31/33/45/52/58 (57%).10
with Cobas HPV-negative swabs (n = 38) Given the eminent role of HPV16 in anal carcinogen-
HPV types No. of lesions esis, we analyzed multiple clinicopathological character-
istics of patients with HSIL according to HPV16 status.
Negative 12
High-risk HPV 14
The only significant difference was that HPV16-positive
 16 3 patients tended to have multiple concurrent high-grade
 18 2 lesions (≥3) corroborating the particular pathogenicity
 45 2 of HPV16. Using ASCUS or worse as threshold, the sen-
 58 2
sitivity of anal cytology to detect HSIL was higher in the
 31; 33; 35/51/73a; 52; 59 5
Possibly carcinogenic HPV 12 HPV16 group than in the non-16 group (87% vs 79%),
 53  2 likely because of the presence of multiple lesions that are
 67 5 more likely to be sampled by a blindly performed anal
 69 1 swab. No other variable we studied proved to be specific
 73 1
for HPV16-positive patients with HSIL.
 82 1
 67/82a 1 The Cobas 4800 HPV assay was initially validated by
 73/82a 1 the multicenter, prospective ATHENA study involving
HPV = human papillomavirus. 47,208 women.24 Among 1083 women with ASCUS and
a
Lesion harboring 2 or 3 HPV subtypes. negative HPV results, 8 were found to have CIN 2/3 on

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 DISEASES OF THE COLON & RECTUM VOLUME 63: 7 (2020)
biopsy, resulting in a false-negative rate of 0.7%. Multiple
factors may cause false-negative or invalid results, such as
the number of viral copies present in the specimen, spec-
imen collection methods, stage of infection, and the pres-
ence of PCR inhibitors.25
Because HSIL generally implies persistent HR-HPV
infection, occasional instances of HPV-negative results are
to some degree unexpected. Such cases constituted 5% of
our cohort. All but one case involved patients with only
1 or 2 high-grade lesions, suggesting that anal swabbing
might have failed to collect sufficient lesional cells to yield
viral copies above the detection threshold.26 When HSIL
biopsy specimens were genotyped, 14 (37%) of the Cobas-
negative cases were indeed found to harbor HR-HPV.
Twelve additional specimens tested positive for HPV
types belonging to the International Agency for Research
on Cancer category 2B group (HPV53, 67, 69, 73, and
82).27 These HPV strains are currently considered “possi-
bly carcinogenic” because of their low incidence in cervi-
cal cancer (≤1% each) and therefore are not included in
primary screening tests.28
Last, 12 patients with HSIL tested negative for HR-
HPV on both anal swab and biopsy. Several potential fac-
tors may be considered: 1) Improper fixation, storage, or
laboratory processing yielded a low quantity or poor qual-
ity of DNA; 2) HR-HPV spontaneously cleared after ini-
tiation and promotion of the oncogenic process29; and 3)
L1-based HPV tests produced false-negative results. Both
Cobas and the tissue-genotyping method used in our
study are PCR-based assays designed to amplify the HPV
L1 gene. It has been shown that the L1 gene is occasion-
ally lost when the HPV genome integrates into the host
DNA.30 Theoretical calculations indicate that as many as
8.3% of HPV16 and 27.9% of HPV18 infections may be
missed by L1-based tests.31,32
In a large retrospective study, Sambursky et al33 re-
ported that, for each cytological category, the presence
of HPV16/18 was associated with a higher prevalence of
HSILs. The authors showed that HPV16/18 positivity en-
tails a 31-fold increased risk of HSIL in the benign cytol-
ogy category and an 18-fold increased risk in the ASCUS
category. More than half of our anal swabs were deemed
ASCUS or less on cytological review (35% ASCUS, 13%
benign, and 5% unsatisfactory). Of them, 34% tested posi-
tive for HPV16 and nearly all (92%) tested positive for HR-
HPV. By supplementing standard anal cytology screening
with HPV testing, at least one-third (using HPV16 as a
referral indicator) or nearly all (HR-HPV as an indicator)
of these individuals would undergo HRA. Recently, the Se-
VIHanal Study Group published similar observations and
recommended that HIV-infected men who have sex with
men with benign cytology but HR-HPV infection should
be considered for HRA.34
High-risk individuals with unsatisfactory cytology
would benefit from HPV cotesting. According to the 2001
Copyright © The American Society of Colon & Rectal Surgeons, Inc. Unauthorized reproduction of this article is prohibited.
895
Bethesda guidelines, cytology samples require a minimum
of 2000 to 3000 nucleated squamous cells to be considered
adequate, a figure subjectively estimated by cytotechnolo-
gists.18 The Cobas 4800 HPV Test is a fully automated mul-
tiplex real-time PCR assay with 150 viral copies/mL as its
lower limit of detection.19 Given the inherent differences
between these 2 technologies, theoretically, an inadequate
cytology sample could well be adequate for the purpose of
Cobas testing. In our experience, Cobas succeeds in ≈90%
of cytology samples deemed inadequate (unpublished
data). Studies from cervical screening trials have shown
that using HPV testing to triage inadequate cervical cytol-
ogy samples is feasible and cost-effective.35
Strengths of our study include the substantial num-
ber of cases, histological confirmation of HSIL, and HPV
cotesting of all samples at the time of cytology and biopsy.
Our study is limited by its retrospective nature as well as
the clinical nature of data collection whereby HPV geno-
typing is routinely reported as pooled results for 12 non-
16/18 HR-HPV types rather than individual types.
CONCLUSIONS
In summary, nearly all HIV-infected patients with AHSIL
tested HR-HPV positive in concurrently collected anal
swab samples. Because HPV16-associated AHSILs are
more prone to malignant transformation, the clinically
relevant question is whether this subgroup can be iden-
tified based on demographic or clinical characteristics.
We found no unique characteristics or predictors, with
the exception of larger numbers of HSILs associated with
HPV16 as opposed to other oncogenic subtypes. Our find-
ings therefore support the incorporation of HPV testing
into anal cancer screening protocols. Future studies are
needed to explore the optimal use of HPV testing whether
as cotesting with cytology or as a reflex test.
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