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Does high-risk human papilloma virus genotyping of abnormal anal cytology improve detection of high-grade anal intraepithelial neoplasia?
Journal of the American Society of Cytopathology (2014) xx, 1e8
Available online at www.sciencedirect.com
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Does high-risk human papilloma virus genotyping of abnormal anal cytology improve detection of high-grade anal intraepithelial neoplasia? Ann E. Walts, MDa,*, Pradip Manna, PhDb, Raymond C.-K. Chan, PhDa, Spencer Kerley, MDb, Shikha Bose, MDa a
Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California b Molecular Pathology, Physicians Reference Laboratory, Overland Park, Kansas Received 31 December 2013; received in revised form 31 March 2014; accepted 31 March 2014
KEYWORDS Human papilloma virus; Anal cytology; Genotypes; Anal intraepithelial neoplasia; Multiplex polymerase chain reaction
Introduction High-risk (HR) human papillomavirus (HPV) testing is accepted as the standard of care for surveillance of cervical cancer. Its role in anal cancer is not clear. This study was therefore designed to determine if HR HPV genotyping is a useful adjunct in management of abnormal anal Papanicolaou (Pap) tests. Materials and methods HR HPV genotyping and virus quantification was performed on 101 residual anal Pap test samples (28 negative, 25 atypical squamous cells of undetermined significance [ASC], 34 low-grade squamous intraepithelial lesion [LSIL], 6 atypical squamous cells of undetermined significance, cannot exclude high-grade squamous intraepithelial lesion, and 8 high-grade squamous intraepithelial lesion) using multiplex real-time polymerase chain reaction. Results were correlated with cytodiagnosis and follow-up. Results HR HPV was detected in 82% (50% negative, 84% ASC, and 100% LSIL and above) cases. Multiple genotypes were present in 71% of cases. Genotype number and viral load correlated with the degree of anal cytologic abnormality. HPV 16, 18, and 45 were the most frequent genotypes detected. The high frequency of HR HPV in abnormal anal cytologies limits its use as an adjunct test. Anal Pap test samples with anal intraepithelial neoplasia 2/3 (AIN 2/3) on follow-up were positive for HPV 16 and/or 18 (HPV 16/18þ) in 80% of cases. We hypothesize that testing for HPV 16/18 on the ASC and LSIL cases would have detected AIN 2/3 with a sensitivity of 81%, specificity of 43%, positive predictive value of 39%, and negative predictive value of 83%.
Portions of this work were presented at the United States and Canadian Academy of Pathology annual meeting, Washington DC, March 2010. *Corresponding author: Ann E. Walts, MD, Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048; Tel.: (310) 423-6626; Fax: (310) 248-6233. E-mail address: [email protected] (A.E. Walts). 2213-2945/$36 Ó 2014 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jasc.2014.03.012
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A.E. Walts et al. Conclusions Our results with a small cohort suggest that genotyping for HPV 16/18 may be effective in identifying patients at high risk for anal cancer and in reducing the number of anoscopy referrals. Prospective studies with follow-up are warranted. Ó 2014 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.
Introduction Anal intraepithelial neoplasia (AIN) and anal carcinoma occur with greater frequency in human immunodeficiency virusepositive (HIVþ) men and women, men who have sex with men (MSM), and organ transplant recipients. HIVþ MSM are also at increased risk for accelerated progression from low-grade to high-grade AIN1,2 and have a risk of anal cancer from 37 to 84 the risk in the general population.3,4 Highly active antiretroviral therapy has extended the lifespan of HIVþ individuals but appears ineffective against human papilloma virus (HPV)-induced AIN.5-7 Whereas other viral diseases are decreasing in HIVþ MSM patients, the incidence of AIN and anal cancer is increasing.5,6,8 Hence there is a need for guidelines aimed at effective surveillance and early detection of anal cancer in populations at increased risk. HPV has been established as the major cause of cervical and anal cancer and their precursor lesions. Based on epidemiologic studies and DNA analysis, 15 of the more than 100 known HPV genotypes are associated with carcinoma and therefore have been designated as high risk (HR).9 Two of these, HPV 16 and 18, cause 70% of cervical10,11 and 70% to 80% of anal carcinomas.12-14 HPV testing has become an integral part of cervical cancer screening. It is approved for triage of women with atypical squamous cells of undetermined significance (ASC) in their Papanicolaou (Pap) tests and, as a cotest for primary screening of women over 30 years of age. Studies have also shown that testing for HPV 16 and/or 18 (HPV 16/18) is useful in identifying women at greatest risk for cervical intraepithelial neoplasia 2/3.9 Accordingly, the American Society for Colposcopy and Cervical Pathology has incorporated HPV 16/18 testing into their guidelines for triaging women with a positive HPV test and a negative Pap tests to colposcopy.15 To date, there are no standardized recommendations for screening for anal carcinoma. Park and Palefsky16 proposed anoscopy with biopsy as follow-up for ASC and low-grade squamous intraepithelial lesion (LSIL), but the role for HPV genotyping in diagnosis and management of persons at increased risk for AIN is still controversial. We and others have shown that the anal Pap test is a relatively sensitive (69%-98%) method for detecting anal precancerous lesions, but its specificity for accurately predicting the severity of AIN detected on biopsy is low (32%59%), with cytology tending to underestimate the grade of AIN observed in tissue and to diagnose ASC in a substantial number of cases that are negative in subsequent biopsy.17,18 HPV testing offers the advantages of objectivity and high
specificity for the presence of HPV DNA. In this retrospective study, we used a multiplex real-time polymerase chain reaction (PCR) that simultaneously detects, types, and quantifies 15 HR HPV genotypes to determine the prevalence and viral load of HR HPV genotypes in a cohort of anal cytology samples and to assess the potential role of HR HPV genotyping as an adjunct in diagnosis and management of persons with abnormal anal cytology.
Materials and methods With institutional review board approval, 101 liquid-based anal cytology samples submitted to the cytology laboratory from 92 men and 9 women were analyzed. The patients ranged in age from 21 to 74 years (median 46.0 years) and were clinically considered to be at increased risk for AIN. Cases were selected to include a range of cytologic diagnoses. No additional selection criteria were applied. The samples were collected into BD SurePath liquid medium (Becton, Dickinson, and Company, Franklin Lakes, NJ) by several clinicians. Each specimen was processed according to our standard laboratory protocol and in accordance with the manufacturer’s recommendations to prepare 1 Pap-stained slide. The residual sample was used for HPV genotyping. Each slide was screened by an experienced cytotechnologist and reported by a cytopathologist using the Bethesda Reporting System. Cytologic diagnoses were 28 negative, 25 ASC, 34 LSIL, 6 atypical squamous cells of undetermined significance, cannot exclude high-grade squamous intraepithelial lesion (ASCH), and 8 high-grade squamous intraepithelial lesion (HSIL). One to 30 months (mean 3.8 months; median 4.0 months) after accession, our departmental files were searched for cytological and/or histological follow-up on each patient.
Detection, identification, and quantification of 15 HR HPV genotypes Automated DNA extraction DNA was extracted from 1 ml of each residual cytology sample using the NucliSens Easy MAG platform (BioMe’rieux, Durham, NC), a second-generation system for automated isolation of nucleic acid from clinical samples based on silica extraction technology, as previously described.19 Multiplex real-time PCR Multiplex real-time PCR for the simultaneous detection, typing, and quantification of the 15 HR HPV genotypes
HR HPV Genotyping in Abnormal Anal Cytology (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82) was performed on the DNA extracted from each sample. The assay used type-specific primers and probes that target a highly conserved region of the E7 oncogene, the presence of which is required for the development of cervical cancer. Four multiplex reactions (3 quadraplex and 1 pentaplex) were used to detect the 15 HR HPV and internal control beta globin. HPV 16 testing was performed in duplicate. The laboratory performing the assay was blinded to the cytological diagnoses. Briefly, 8 mL of extracted DNA was mixed with 2 mL of AssayMix and 10 mL of ProbesMaster (Roche Diagnostics, Indianapolis, IN) for each multiplex reaction, and subjected to real-time PCR on a LightCycler 480 instrument (Roche Diagnostics) with the following thermal profile: 95 C for 8 minutes, 40 cycles of 95 C for 45 seconds, 60 C for 1 minute 10 seconds, and 1 cycle of 40 C for 20 seconds. As a positive control and for quantitation of viral loads, a 100 copies of HPV genome equivalent standard for each of the 4 multiplex AssayMixes was included in the same run. Linear regression curves were established for each HR HPV genotype and for beta globulin and then used to determine viral/beta globulin levels in the samples. Normalized beta globulin levels enabled us to compare viral loads across diagnostic groups. The test detects 10 HPV genome equivalents for each genotype. The prevalence and distribution of HR HPV genotypes in each SurePath sample were recorded and correlated with cytodiagnosis and, where available, with follow-up. Estimated viral loads for all HR HPV, HPV 16, and combined HPV 16/18 were also correlated with cytodiagnosis.
Results Prevalence of HR HPV in anal Pap tests HR HPV DNA was detected in 83 of the 101 cases (82%) encompassing 14 of 28 cases (50%) with negative cytologic diagnosis, 21 of ASC cases (84%), and all of 34 LSIL, 6 ASCH, and 8 HSIL cases (100%). In the majority of cases (72 cases; 71%) multiple (from 2 to 9) HR HPV genotypes were detected. The average number of HR subtypes detected per case increased with severity of cytological diagnosis, ranging from a mean of 2 in negative cases up to 5 in ASCH and HSIL cases. Four or more HR HPV genotypes were detected in the majority of cases diagnosed as LSIL and above (62% LSIL, 67% ASCH, and 63% HSIL cases) and in 19% of the ASC cases. Only 1 HR genotype was detected in 17 cases (7 negative, 6 ASC, 3 LSIL, 1 ASCH), composing 17% of all cases in the study. The mean number of HR HPV types detected per case did not correlate with patient age but reached a maximum of 4 in patients between 41 and 50 years of age. Except for HR HPV 35 and HR HPV 82, all other of the 13 HR HPV genotypes were detected in the negative anal Pap tests as follows: HPV 16
3 in 6, HPV 18 in 4, HPV 31 in 4, HPV 33 in 2, HPV 39 in 1, HPV 45 in 2, HPV 51 in 1, HPV 52 in 1, HPV 56 in 3, HPV 58 in 2, HPV 59 in 3, HPV 68 in 3, and HPV 73 in 1 (Table 1).
Prevalence of HR HPV genotypes in anal Pap tests HPV 16, present in 44 cases (44% of all cases and 53% of the HR HPVþ cases) was the most frequent HR genotype detected, followed by HPV 18 and HPV 45, which were detected in 28% and 27% of the cases in the study. HPV 16/18 were detected in a total of 56 cases (55%) and coexisted in 16 cases (16%). Detection of HPV 16 and of HPV 16/18 increased with increasing severity of cytologic diagnosis (from 21% in negative cases up to 75% in HSIL cases and from 29% in negative cases up to 75% in HSIL, respectively). HPV 16 was also the most frequent genotype detected in each diagnostic category. The second most frequent HR genotype detected varied among the diagnostic groups, being HPV 45 in 28% of ASC, HPV 18 in 47% of LSIL, HPV 73 in 67% of ASCH, and HPV 58 in 63% of HSIL cases. Twenty-seven cases (6 negative, 9 ASC, 9 LSIL, 1 ASCH, 2 HSIL) contained only HR genotypes other than HPV 16/18, composing 27% of all and 33% of the HRþ cases. HPV 45 was the most frequent noneHPV 16/ 18 genotype detected (in 11 of these 27 cases) (Table 2).
HPV levels in anal Pap tests As shown in Table 3, the mean viral levels for all HR HPV genotypes, for HPV 16, and for HPV 16/18 increase with severity of cytodiagnosis, whereas the normalized beta globulin values remain constant across the diagnostic groups. Interestingly, a substantial (and the largest) increment in mean viral load for all HR genotypes, for HPV 16, and for HPV 16/18 is noted to occur between ASC/ASCH and LSIL. In cases with LSIL and HSIL, only the mean viral loads for HPV 16 and HPV 16/18 continue to show striking increments, suggesting that although noneHPV 16/18 HR genotypes are present, HPV 16 and HPV 16/18 genotypes are present in greatest numbers (Table 3).
HR HPV genotyping for triage of abnormal anal Pap tests Follow-up (cytology and/or tissue) became available within 30 months (range 1-30 months; mean 8.7 months) for 66 of the cases (65%), including 2 negative, 19 ASC, 32 LSIL, 6 ASCH, and 7 HSIL. For patients with >1 follow-up, the worst/highest grade AIN diagnosed within 30 months following the initial Pap test is presented as “the follow-up diagnosis.” A total of 43 AIN (18 AIN 1 and 25 AIN 2/3) were diagnosed at follow-up. AIN 2/3 was detected in 16% of ASC, 40% of LSIL, 67% of ASCH, and 71% of HSIL cases (Tables 4-6).
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A.E. Walts et al. Table 1
HR HPV detected in anal Pap tests (N Z 101).
Cytologic diagnosis (n)
Pap tests HR HPVþ, n (%)
HR HPV genotypes/cases, range
HR HPV genotypes/cases, mean
Negative (28) ASC (25) LSIL (34) ASCH (6) HSIL (8) All cases (101)
14 21 34 6 8 83
0e7 0e6 1e8 1e9 3e9 0e9
2 3 4 5 5 3.6
(50) (84) (100) (100) (100) (82)
Abbreviations: ASC, atypical squamous cells of undetermined significance; ASCH, atypical squamous cells of undetermined significance, cannot exclude high-grade squamous intraepithelial lesion; HPV, human papilloma virus; HR, high risk; HR HPVþ, positive for 1 HR HPV genotypes; HSIL, highgrade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; Pap, Papanicolaou.
HR HPV was detected in 82% of all anal samples including in 50% of negative cases and in 84% of abnormal cases (Table 1) in this study. Therefore, it is not useful for triage of abnormal Pap tests. Additionally, 67% of ASCH and 71% of HSIL cases were AIN 2/3 on follow-up and therefore are best subjected to anoscopy and biopsy as the next step in management. This prompted us to assess HPV 16/18 genotyping as an adjunct to cytologic diagnosis of ASC and LSIL for triaging patients to anoscopy. Among the 25 AIN 2/3 cases, 20 (80%) had HPV 16/18 in the preceding anal Pap test and 4 (16%) had only noneHPV 16/18 HR genotypes (including 31, 33, 35, 45, 51, 52, 58, 59, 68, 73). One AIN 2/3 was preceded by a HR HPV-Pap test that was diagnosed as ASC. Of the 51 ASC/LSIL anal Pap tests with follow-up, 33 (65%) were HPV 16/18þ and 18 (35%) were HPV 16/18e (of which 4 were HR HPVe). On follow-up, 13 (39%) of these HPV 16/18þ cases and 3 (17%) of these HPV 16/18e cases showed AIN 2/3, yielding a sensitivity of 81%, a specificity of 43%, a positive predictive value of 39%, and a negative predictive value of 83%. HSIL and ASCH cases usually have follow-up anoscopy with biopsy. If, as recommended by Park and Palefsky,16 all ASC and LSIL cases also were to have follow-up anoscopy with biopsy, then performing HPV 16/18 genotyping rather than HR HPV screen as the primary test would reduce the number of ASC and LSIL cases triaged for anoscopy from 84% to 48% and from 100% to 76%, respectively.
Table 2
Discussion In summary, 82% of the cases in our study were HR HPVþ with multiple genotypes detected in 71% of the cases including cases from each cytodiagnostic group (negative, ASC, LSIL, ASCH, HSIL). The prevalence of HR HPV, multiplicity of HR HPV genotypes, prevalence of HPV 16 and HPV 16/18, and HPV 16 and HPV 16/18 viral load each increased with increasing severity of cytodiagnosis. HPV 16 was the most frequent genotype detected in each diagnostic group. In addition to all HSIL and ASCH, Park and Palefsky16 recommend that all ASC and LSIL undergo anoscopy with biopsy. Based on our findings, we suggest that HPV 16/18 genotyping be used in lieu of HR HPV as the initial triage of ASC and LSIL cases for anoscopy/ biopsy. Using HPV 16/18 genotyping in place of HR HPV to triage ASC and LSIL cases would have reduced the number of cases triaged for anoscopy from 84% to 48% and from 100% to 76%, respectively. Interobserver variability in cytodiagnosis in the negative/ASC categories20 and limitations associated with anoscopy (difficulties visualizing lesions and morbidity associated with liberal sampling) further suggest that HPV 16/18 genotyping as a primary triage tool could improve management of these persons at increased risk for anal cancer. Our findings confirm the high prevalence of HR HPV (from 44% to 95%), the predominance of multiple HR genotypes (up to 88%), and HPV 16 as the most frequent HR
Distribution of HR HPV genotypes in anal Pap tests (N Z 101).
Cytologic diagnosis (n)
HR HPVþ
HPV 16þ
HPV 16/18þ
none HPV 16/18þ
HR HPVe
Most frequent HR genotypes
Negative (28) ASC (25) LSIL (34) ASCH (6) HSIL (8) All cases (101)
14 21 34 6 8 83
6 9 18 5 6 44
8 12 25 5 6 56
6 9 9 1 2 27
14 (50) 4 (16) 0 0 0 18 (18)
16 16 16 16 16 16
(50) (84) (100) (100) (100) (82)
(21) (36) (53) (83) (75) (44)
(29) (48) (74) (83) (75) (55)
(21) (36) (26) (17) (25) (27)
> > > > > >
18 45 18 73 58 18
Z 31 > 39 > 45 > 33 Z 35 > 68 > 45
Abbreviations: HPV 16þ, positive for HPV 16; HPV 16/18þ, positive for HPV 16 and/or 18; HR HPVe, negative for all 15 HR HPV genotypes; noneHPV 16/ 18þ, positive for HR HPV genotypes other than HPV 16 and 18; other abbreviations as in Table 1. Unless otherwise indicated, values are n (%).
HR HPV Genotyping in Abnormal Anal Cytology Table 3
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HPV levels in negative and abnormal anal Pap tests (N Z 101). Cytological Diagnosis
Viral Load Viral load all genotypes Mean (Cp) value Mean copies/1000 cells Viral load HPV 16/18 only Mean (Cp) value Mean copies/1000 cells Viral load HPV 16 only Mean (Cp) value Mean copies/1000 cells Quantity of cells Mean (Cp) value Mean cells Beta globin level Mean (Cp) value Mean copies/mL
Negative
ASC/ASCH
LSIL
HSIL
31.83 119
31.11 253
27.65 1142
27.67 780
33.26 51
30.35 398
26.2 2710
22.68 15,264
33.09 56
29.32 735
25.62 3830
21.98 23,166
27.09 18,241
27.64 13,143
26.71 22,877
26.09 33,104
27.09 40,129
27.64 28,913
26.71 50,329
26.09 72,828
Abbreviations: Cp, crossing point, the point at which fluoresence level crosses the baseline; other abbreviations as in Table 1.
genotype in anal samples from individuals at increased risk for AIN previously reported.5,18 We, like others,21,22 detected HR HPV (including HPV 16/18) in some cases with negative cytology. A literature review indicates that these observations are largely independent of collection method (swab versus brush), presence or absence of symptoms, and with a few exceptions, of geography.21-23 In the current study using multiplex PCR, we detected HR HPV in 84% ASC and 100% LSIL cases; these are higher percentages than we detected in similar case cohorts using the Digene Hybrid Capture II assay (55% ASC and 87% LSIL)24 or using the Invader test for HR HPV (81% ASC and 94% LSIL) (unpublished review of our laboratory’s experience). The higher prevalence of HR HPV detected by PCR could reflect increased sensitivity associated with this methodology. In a study of anal swab samples with ASC, Gohy et al22 found similar proportions of HR HPVþ cases
Table 4
in patients with AIN 1 and AIN 2/3 on biopsy and concluded that HR HPV testing was not useful to select ASC cases at highest risk for AIN 2/3. The high rate of HR HPV positivity observed by us and others in ASC and LSIL (which together compose the majority of abnormal anal cytodiagnoses) indicates that HR HPV testing is not an efficacious screen for AIN 2/3 in a population at increased risk for AIN.16,25 Multiplicity of HR HPV genotypes, detected in 71% of the cases in our study, has been reported in 12% to 88% of anal HPV infections in HIVþ individuals.5,26 Salit et al27 also reported a positive correlation between the number of HPV genotypes detected per cytology sample and the grade of AIN at biopsy. The number of HR HPV genotypes detected did not correlate with patient age in our study or in the study by Chin-Hong et al.28 Noting that the number of HPV genotypes detected in anal cytology samples (swabs)
Correlation of Pap test diagnosis with HPV genotypes detected in the Pap test and follow-up (n Z 66).
Cytologic diagnoses (n)
Cases with follow-up, n
HPV genotypes in Pap samples with follow-up
Follow-up diagnosis AIN 2/3
ASCH
AIN 1
ASC
Negative
Negative (28) ASC (25)
2 19
LSIL (34)
32
ASCH (6)
6
HSIL (8)
7
2 HPV 16/18þ 9 HPV 16/18þ 6 HR noneHPV 16/18þ 4 HR HPVe 24 HPV 16/18þ 8 HR noneHPV 16/18þ 5 HPV 16/18þ 1 HR noneHPV 16/18þ 5 HPV 16/18þ 2 HR noneHPV 16/18þ
0 2 0 1 11 2 3 1 4 1 25
0 1 0 0 2 1 0 0 0 0 4
1 2 4 1 4 4 1 0 1 0 18
1 1 1 1 4 1 1 0 0 1 11
0 3 1 1 3 0 0 0 0 0 8
Total cases (101)
66
Abbreviations: AIN, anal intraepithelial neoplasia; other abbreviations as in Tables 1 and 2.
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A.E. Walts et al. Table 5
Distribution of HR HPV genotypes in anal Pap tests with AIN 2/3 on follow-up (n Z 25).
Cytologic diagnosis
AIN 2/3 on follow-up
HPV 16/18þ Pap tests
HR noneHPV 16/18þ Pap tests
HR HPVePap tests
HR noneHPV 16/18 genotypes in Pap tests
Negative ASC LSIL ASCH HSIL Total
0 3 13 4 5 25
0 2 11 3 4 20 (80)
0 0 2 1 1 4 (16)
0 1 0 0 0 1 (4)
NA NA 35, 45, 58 33, 35, 45, 58, 68, 73 31, 33, 35, 45, 51, 52, 59, 68, 73
Abbreviations: NA, not applicable; other abbreviations as in Tables 1, 2, and 4. Values are n and n (%).ß
from HIVþ men exceeded the number detected in concurrent biopsies, Gohy et al22 suggested that cytology, by sampling a much larger area than a tiny biopsy, more accurately reflects in vivo conditions. Nevertheless, they concluded that the high prevalence of HR HPV and the multiplicity of HPV genotypes detected in cytology samples made it unlikely that HPV testing would become part of routine screening for AIN. The risk of progression to carcinoma varies among the HR HPV genotypes, but HPV 16 is known to be the most “virulent.” HPV 16 has been reported in up to 65% of anal HPV infections21 and in at least 66% of anal cancers.12-14,29 Using type-specific real-time PCR to evaluate HPV 16 viral load at different anogenital sites in asymptomatic men who tested positive for HPV 16, Flores et al30 found that normalized viral loads varied significantly by anatomic site and that the anal canal and the penile shaft had the highest viral loads. More than a decade ago, studies using quantitative typespecific PCR in cervical material showed that the viral load of HPV 16 reaches much higher levels than that of other HR genotypes and that the viral loads only for HPV 16 correlate with increased severity of dysplasia.31 The correlation of anal viral load, particularly HPV 16 load, with severity of AIN has been investigated with conflicting findings. We observed a positive correlation between HPV 16 and HPV 16/18 loads and severity of cytological abnormality in samples from the anal canal. Salit et al27 also reported positive correlations among high HPV 16 viral loads, high-
Table 6 Correlation of HR HPV genotypes in ASC and LSIL Pap tests (n Z 51) with AIN 2/3 on follow-up. HPV genotype
Pap tests positive
AIN 2/3 on follow-up
HPV 16/18þ HR noneHPV 16/18þ HR HPVe
33 (65) 14 (27) 4 (8)
13 (39) 2 (14) 1 (25)
Abbreviations as in Tables 1, 2, and 4. Values are n (%).
grade cytology, and high-grade histology in anal samples. However, in a recent report by Zhao et al,32 viral load did not vary significantly across different diagnostic groups, although the likelihood of a high viral load was significantly greater among patients with abnormal anal cytology than among patients with negative cytology. Devarajan et al33 also reported no correlation between HPV viral load and severity of cytodiagnosis in anal samples from HIVþ persons. Confounding host factors including HIV status/virus burden, CD4 count at study time or nadir count, persistent anal HPV infection, number of partners, prior therapy, and other factors might explain the discordant results. Using our case cohort, we queried the potential role of testing for HPV 16/18 in 4 scenarios: as a primary screen for AIN 2/3 and as a means to triage patients with ASC, with LSIL, or with ASC/LSIL on anal Pap test for anoscopy. We found that 39% of ASC/LSIL cases with HPV 16/18þ anal Pap test had AIN 2/3 on follow-up compared with 17% of those with noneHPV 16/18 and HR HPV cytology. Using HPV 16/18 testing as a primary screen for AIN 2/3 yielded a sensitivity of 80%. Using HPV 16/18 testing for triage of ASC or LSIL yielded sensitivities of 67% and 85%, respectively. Using HPV 16/18 testing to triage ASC/LSIL yielded a sensitivity of 81%, a specificity of 43%, a positive predictive value of 39%, and a negative predictive value of 83%. Limitations in our study include its retrospective nature, variability in number of data points and in intervals between data points, and incomplete follow-up especially among cases with negative cytology. Prospective studies with follow-up are warranted. While we recognize the potential role of confounding factors such as reinfection and spontaneous clearance of HPV, evaluation of these complex host-pathogen relationships is beyond the scope of our study. We hypothesize that testing for HPV 16/18 would capture 80% (20 of 25) whereas testing for 4 genotypes (HPV 16,18,35,45) would capture 96% (24 of 25) of our AIN 2/3 cases diagnosed at follow-up. However, this increase in sensitivity would result in a marked reduction in specificity (testing for HPV 16, 18, 35, 45 rather than only for HPV 16,
HR HPV Genotyping in Abnormal Anal Cytology 18 would also capture 78% [compared with 50%] of the AIN 1 diagnosed at follow-up and 88% [compared with 75%] of cases with negative follow-up). Although the addition of testing for HPV 35 and 45 captured the largest number of AIN 2/3 in our cohort, different HR genotypes were detected more frequently than were HPV 35 and 45 in AIN 2/3 and invasive anal cancer in other studies.3,23,24,34 In our study, no HPV genotype other than HPV 16/18 was significantly correlated with AIN 2/3. The potential value of HPV 16/18 genotyping in combination with HPV E6/E7 messenger RNA or p16/Ki-67 immunostains for anal cancer screening awaits further study.
Conclusions Although much of our knowledge about anal HPV is based on similarities with cervical HPV, differences between these 2 infections also need to be considered when developing guidelines for diagnosis and management of individuals with abnormal findings on anal cytology. Compared with cervical Pap tests, anal Pap tests are characterized by higher prevalence of HR genotypes including HPV 16/18 and greater multiplicity of HR genotypes. Similarly, a substantially higher rate of HR HPV was detected in negative anal Pap tests (50%) as compared to cervical Pap tests (20%). A possible explanation for this observation is that the negative anal cytology cases were not from a random patient population being screened for HR HPV but were biased toward a population who were clinically at increased risk for anal cytological abnormalities. The relationship between HPV genotypes, levels, and persistence to disease progression is less clear in anal cancer. Progression from low-grade to high-grade AIN has been reported to occur within 2 years in as many as 62% of HIVþ and 36% of HIVe at-risk men.1,2 This rapid progression may be due to an underlying poor immune response. Currently, little is known about viral interactions (HPV/HPV, HIV/HPV) and the role they might play in etiology or progression of squamous intraepithelial lesions. Additionally, whereas the cervical Pap test is a routine test recommended for all women, the anal Pap test is recommended predominantly for HIVþ individuals and/or MSM. Paucity of screening programs, limited resources, a persistent element of social stigma, and/or coexistent lifethreatening illnesses that take precedence contribute to suboptimal screening in populations at increased risk for AIN and invasive anal carcinoma.
Funding sources The human papilloma virus polymerase chain reaction test was provided at no cost by Physicians Reference Laboratory.
Conflict of interest disclosures The authors made no disclosures.
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References 1. Palefsky JM, Holly EA, Hogeboom CJ, et al. Virologic, immunologic, and clinical parameters in the incidence and progression of anal squamous intraepithelial lesions in HIV-positive and HIV-negative homosexual men. J Acquir Immune Defic Syndr Hum Retrovirol. 1998;17: 314e319. 2. Lacey HB, Wilson GE, Tilston P, et al. A study of anal intraepithelial neoplasia in HIV positive homosexual men. Sex Transm Infect. 1999; 75:172e177. 3. Frisch M, Bigger RJ, Goedert JJ. Human papillomavirus-associated cancers in patients with human immunodeficiency virus infection and acquired immunodeficiency syndrome. J Natl Cancer Inst. 2000;92: 1500e1510. 4. Melbye M, Cote TR, Kessler L, Gail M, Biggar RJ. High incidence of anal cancer among AIDS patients. The AIDS/Cancer Working Group. Lancet. 1994;343:636e639. 5. Palefsky JM, Holly EA, Efird JT, et al. Anal intraepithelial neoplasia in the highly active antiretroviral therapy era among HIV-positive men who have sex with men. AIDS. 2005;19:1407e1414. 6. Piketty C, Darragh TM, Heard I, et al. High prevalence of anal squamous intraepithelial lesions in HIV-positive men despite the use of highly active antiretroviral therapy. Sex Transm Dis. 2004; 31:96e99. 7. Fox P, Stebbing J, Portsmouth S, et al. Lack of response of anal intraepithelial neoplasia to highly active antiretroviral therapy. AIDS. 2003; 17:279e280. 8. Chaturvedi AK, Madeleine MM, Biggar RJ, Engels EA. Risk of human papillomavirus-associated cancers among persons with AIDS. J Natl Cancer Inst. 2009;101:1120e1130. 9. Muñoz N, Bosch FX, de Sanjosé S, et al, International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003;348:518e527. 10. Muñoz N, Bosch FX, Castellsagué X, et al. Against which human papillomavirus types shall we vaccinate and screen? The international perspective. Int J Cancer. 2004;111:278e285. 11. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet. 2007;370:890e907. 12. The National Cervical Cancer Coalition. HPV/Cervical Cancer Overview. 2013. Available at: www.nccc-online.org/index.php/overview. Accessed May 1, 2014. 13. Hoots BE, Palefsky JM, Pimenta JM, Smith JS. Human papillomavirus type distribution in anal cancer and anal intraepithelial lesions. Int J Cancer. 2009;124:2375e2383. 14. Abramowitz L, Jacquard AC, Jaroud F, et al. Human papillomavirus genotype distribution in anal cancer in France: the EDiTH V study. Int J Cancer. 2011;129:433e439. 15. American Society for Colposcopy and Cervical Pathology. Management Guidelines. 2013. Available at: www.asccp.org. Accessed May 1, 2014. 16. Park IU, Palefsky JM. Evaluation and management of anal intraepithelial neoplasia in HIV-negative and HIV-positive men who have sex with men. Curr Infect Dis Rep. 2010;12:126e133. 17. Arain S, Walts AE, Thomas P, Bose S. The anal Pap smear: cytomorphology of squamous intraepithelial lesions. Cytojournal. 2005;2:4. 18. Chiao EY, Giordano TP, Palefsky JM, Tyring S, El Serag H. Screening HIV-infected individuals for anal cancer precursor lesions: a systematic review. Clin Infect Dis. 2006;43:223e233. 19. Wong AK, Chan RC, Nichols WS, Bose S. Human papillomavirus (HPV) in atypical squamous cervical cytology: the Invader HPV test as a new screening assay. J Clin Microbiol. 2008;46:869e875. 20. Darragh TM, Tokugawa D, Castle PE, et al. Interrater agreement of anal cytology. Cancer Cytopathol. 2013;121:72e78. 21. Fox PA, Seet JE, Stebbing J, et al. The value of anal cytology and human papillomavirus typing in the detection of anal intraepithelial
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22.
23.
24. 25.
26.
27.
A.E. Walts et al. neoplasia: a review of cases from an anoscopy clinic. Sex Transm Infect. 2005;81:142e146. Gohy L, Gorska I, Rouleau D, et al, HIPVIRG Study Group. Genotyping of human papillomavirus DNA in anal biopsies and anal swabs collected from HIV-seropositive men with anal dysplasia. J Acquir Immune Defic Syndr. 2008;49:32e39. Tsai TF, Kuo GT, Kuo LT, Hsiao CH. Prevalence status and association with human papilloma virus of anal squamous proliferative lesions in a patient sample in Taiwan. Sex Transm Dis. 2008;35:721e724. Walts AE, Thomas P, Bose S. Anal cytology: is there a role for reflex HPV DNA testing? Diagn Cytopathol. 2005;33:152e156. Salit IE, Lytwyn A, Raboud J, et al. The role of cytology (Pap tests) and human papillomavirus testing in anal cancer screening. AIDS. 2010;24:1307e1313. Pierangeli A, Scagnolari C, Degener AM, et al. Type-specific human papillomavirus-DNA load in anal infection in HIV-positive men. AIDS. 2008;22:1929e1935. Salit IE, Tinmouth J, Chong S, et al. Screening for HIV-associated anal cancer: correlation of HPV genotypes, p16, and E6 transcripts with anal pathology. Cancer Epidemiol Biomarkers Prev. 2009;18: 1986e1992.
28. Chin-Hong PV, Vittinghoff E, Cranston RD, et al. Age-related prevalence of anal cancer precursors in homosexual men: the EXPLORE study. J Natl Cancer Inst. 2005;97:896e905. 29. Darragh TM, Winkler B. Anal cancer and cervical cancer screening: key differences. Cancer Cytopathol. 2011;119:5e19. 30. Flores R, Lu B, Nielson C, et al. Correlates of human papillomavirus viral load with infection site in asymptomatic men. Cancer Epidemiol Biomarkers Prev. 2008;17:3573e3576. 31. Burd EM. Human papillomavirus and cervical cancer. Clin Microbiol Rev. 2003;16:1e17. 32. Zhao C, Domfeh AB, Austin RM. Histopathologic outcomes and clinical correlations for high-risk patients screened with anal cytology. Acta Cytol. 2012;56:62e67. 33. Devarajan V, Robertson J, Smulian GA. Molecular makers for human papillomavirus (HPV) in HIV infected patients with and without anal dysplasia. Abstract #686 presented at: Infectious Diseases Society of America annual meeting, October 21-24, 2010; Infectious Diseases Society of America, Vancouver BC. 34. Valmary-Degano S, Jacquin E, Prétet JL, et al. Signature patterns of human papillomavirus type 16 in invasive anal carcinoma. Hum Pathol. 2013;44:992e1002.
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Does high-risk human papilloma virus genotyping of abnormal anal cytology improve detection of high-grade anal intraepithelial neoplasia? Ann E. Walts, MDa,*, Pradip Manna, PhDb, Raymond C.-K. Chan, PhDa, Spencer Kerley, MDb, Shikha Bose, MDa a
Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California b Molecular Pathology, Physicians Reference Laboratory, Overland Park, Kansas Received 31 December 2013; received in revised form 31 March 2014; accepted 31 March 2014
KEYWORDS Human papilloma virus; Anal cytology; Genotypes; Anal intraepithelial neoplasia; Multiplex polymerase chain reaction
Introduction High-risk (HR) human papillomavirus (HPV) testing is accepted as the standard of care for surveillance of cervical cancer. Its role in anal cancer is not clear. This study was therefore designed to determine if HR HPV genotyping is a useful adjunct in management of abnormal anal Papanicolaou (Pap) tests. Materials and methods HR HPV genotyping and virus quantification was performed on 101 residual anal Pap test samples (28 negative, 25 atypical squamous cells of undetermined significance [ASC], 34 low-grade squamous intraepithelial lesion [LSIL], 6 atypical squamous cells of undetermined significance, cannot exclude high-grade squamous intraepithelial lesion, and 8 high-grade squamous intraepithelial lesion) using multiplex real-time polymerase chain reaction. Results were correlated with cytodiagnosis and follow-up. Results HR HPV was detected in 82% (50% negative, 84% ASC, and 100% LSIL and above) cases. Multiple genotypes were present in 71% of cases. Genotype number and viral load correlated with the degree of anal cytologic abnormality. HPV 16, 18, and 45 were the most frequent genotypes detected. The high frequency of HR HPV in abnormal anal cytologies limits its use as an adjunct test. Anal Pap test samples with anal intraepithelial neoplasia 2/3 (AIN 2/3) on follow-up were positive for HPV 16 and/or 18 (HPV 16/18þ) in 80% of cases. We hypothesize that testing for HPV 16/18 on the ASC and LSIL cases would have detected AIN 2/3 with a sensitivity of 81%, specificity of 43%, positive predictive value of 39%, and negative predictive value of 83%.
Portions of this work were presented at the United States and Canadian Academy of Pathology annual meeting, Washington DC, March 2010. *Corresponding author: Ann E. Walts, MD, Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048; Tel.: (310) 423-6626; Fax: (310) 248-6233. E-mail address: [email protected] (A.E. Walts). 2213-2945/$36 Ó 2014 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jasc.2014.03.012
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A.E. Walts et al. Conclusions Our results with a small cohort suggest that genotyping for HPV 16/18 may be effective in identifying patients at high risk for anal cancer and in reducing the number of anoscopy referrals. Prospective studies with follow-up are warranted. Ó 2014 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.
Introduction Anal intraepithelial neoplasia (AIN) and anal carcinoma occur with greater frequency in human immunodeficiency virusepositive (HIVþ) men and women, men who have sex with men (MSM), and organ transplant recipients. HIVþ MSM are also at increased risk for accelerated progression from low-grade to high-grade AIN1,2 and have a risk of anal cancer from 37 to 84 the risk in the general population.3,4 Highly active antiretroviral therapy has extended the lifespan of HIVþ individuals but appears ineffective against human papilloma virus (HPV)-induced AIN.5-7 Whereas other viral diseases are decreasing in HIVþ MSM patients, the incidence of AIN and anal cancer is increasing.5,6,8 Hence there is a need for guidelines aimed at effective surveillance and early detection of anal cancer in populations at increased risk. HPV has been established as the major cause of cervical and anal cancer and their precursor lesions. Based on epidemiologic studies and DNA analysis, 15 of the more than 100 known HPV genotypes are associated with carcinoma and therefore have been designated as high risk (HR).9 Two of these, HPV 16 and 18, cause 70% of cervical10,11 and 70% to 80% of anal carcinomas.12-14 HPV testing has become an integral part of cervical cancer screening. It is approved for triage of women with atypical squamous cells of undetermined significance (ASC) in their Papanicolaou (Pap) tests and, as a cotest for primary screening of women over 30 years of age. Studies have also shown that testing for HPV 16 and/or 18 (HPV 16/18) is useful in identifying women at greatest risk for cervical intraepithelial neoplasia 2/3.9 Accordingly, the American Society for Colposcopy and Cervical Pathology has incorporated HPV 16/18 testing into their guidelines for triaging women with a positive HPV test and a negative Pap tests to colposcopy.15 To date, there are no standardized recommendations for screening for anal carcinoma. Park and Palefsky16 proposed anoscopy with biopsy as follow-up for ASC and low-grade squamous intraepithelial lesion (LSIL), but the role for HPV genotyping in diagnosis and management of persons at increased risk for AIN is still controversial. We and others have shown that the anal Pap test is a relatively sensitive (69%-98%) method for detecting anal precancerous lesions, but its specificity for accurately predicting the severity of AIN detected on biopsy is low (32%59%), with cytology tending to underestimate the grade of AIN observed in tissue and to diagnose ASC in a substantial number of cases that are negative in subsequent biopsy.17,18 HPV testing offers the advantages of objectivity and high
specificity for the presence of HPV DNA. In this retrospective study, we used a multiplex real-time polymerase chain reaction (PCR) that simultaneously detects, types, and quantifies 15 HR HPV genotypes to determine the prevalence and viral load of HR HPV genotypes in a cohort of anal cytology samples and to assess the potential role of HR HPV genotyping as an adjunct in diagnosis and management of persons with abnormal anal cytology.
Materials and methods With institutional review board approval, 101 liquid-based anal cytology samples submitted to the cytology laboratory from 92 men and 9 women were analyzed. The patients ranged in age from 21 to 74 years (median 46.0 years) and were clinically considered to be at increased risk for AIN. Cases were selected to include a range of cytologic diagnoses. No additional selection criteria were applied. The samples were collected into BD SurePath liquid medium (Becton, Dickinson, and Company, Franklin Lakes, NJ) by several clinicians. Each specimen was processed according to our standard laboratory protocol and in accordance with the manufacturer’s recommendations to prepare 1 Pap-stained slide. The residual sample was used for HPV genotyping. Each slide was screened by an experienced cytotechnologist and reported by a cytopathologist using the Bethesda Reporting System. Cytologic diagnoses were 28 negative, 25 ASC, 34 LSIL, 6 atypical squamous cells of undetermined significance, cannot exclude high-grade squamous intraepithelial lesion (ASCH), and 8 high-grade squamous intraepithelial lesion (HSIL). One to 30 months (mean 3.8 months; median 4.0 months) after accession, our departmental files were searched for cytological and/or histological follow-up on each patient.
Detection, identification, and quantification of 15 HR HPV genotypes Automated DNA extraction DNA was extracted from 1 ml of each residual cytology sample using the NucliSens Easy MAG platform (BioMe’rieux, Durham, NC), a second-generation system for automated isolation of nucleic acid from clinical samples based on silica extraction technology, as previously described.19 Multiplex real-time PCR Multiplex real-time PCR for the simultaneous detection, typing, and quantification of the 15 HR HPV genotypes
HR HPV Genotyping in Abnormal Anal Cytology (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82) was performed on the DNA extracted from each sample. The assay used type-specific primers and probes that target a highly conserved region of the E7 oncogene, the presence of which is required for the development of cervical cancer. Four multiplex reactions (3 quadraplex and 1 pentaplex) were used to detect the 15 HR HPV and internal control beta globin. HPV 16 testing was performed in duplicate. The laboratory performing the assay was blinded to the cytological diagnoses. Briefly, 8 mL of extracted DNA was mixed with 2 mL of AssayMix and 10 mL of ProbesMaster (Roche Diagnostics, Indianapolis, IN) for each multiplex reaction, and subjected to real-time PCR on a LightCycler 480 instrument (Roche Diagnostics) with the following thermal profile: 95 C for 8 minutes, 40 cycles of 95 C for 45 seconds, 60 C for 1 minute 10 seconds, and 1 cycle of 40 C for 20 seconds. As a positive control and for quantitation of viral loads, a 100 copies of HPV genome equivalent standard for each of the 4 multiplex AssayMixes was included in the same run. Linear regression curves were established for each HR HPV genotype and for beta globulin and then used to determine viral/beta globulin levels in the samples. Normalized beta globulin levels enabled us to compare viral loads across diagnostic groups. The test detects 10 HPV genome equivalents for each genotype. The prevalence and distribution of HR HPV genotypes in each SurePath sample were recorded and correlated with cytodiagnosis and, where available, with follow-up. Estimated viral loads for all HR HPV, HPV 16, and combined HPV 16/18 were also correlated with cytodiagnosis.
Results Prevalence of HR HPV in anal Pap tests HR HPV DNA was detected in 83 of the 101 cases (82%) encompassing 14 of 28 cases (50%) with negative cytologic diagnosis, 21 of ASC cases (84%), and all of 34 LSIL, 6 ASCH, and 8 HSIL cases (100%). In the majority of cases (72 cases; 71%) multiple (from 2 to 9) HR HPV genotypes were detected. The average number of HR subtypes detected per case increased with severity of cytological diagnosis, ranging from a mean of 2 in negative cases up to 5 in ASCH and HSIL cases. Four or more HR HPV genotypes were detected in the majority of cases diagnosed as LSIL and above (62% LSIL, 67% ASCH, and 63% HSIL cases) and in 19% of the ASC cases. Only 1 HR genotype was detected in 17 cases (7 negative, 6 ASC, 3 LSIL, 1 ASCH), composing 17% of all cases in the study. The mean number of HR HPV types detected per case did not correlate with patient age but reached a maximum of 4 in patients between 41 and 50 years of age. Except for HR HPV 35 and HR HPV 82, all other of the 13 HR HPV genotypes were detected in the negative anal Pap tests as follows: HPV 16
3 in 6, HPV 18 in 4, HPV 31 in 4, HPV 33 in 2, HPV 39 in 1, HPV 45 in 2, HPV 51 in 1, HPV 52 in 1, HPV 56 in 3, HPV 58 in 2, HPV 59 in 3, HPV 68 in 3, and HPV 73 in 1 (Table 1).
Prevalence of HR HPV genotypes in anal Pap tests HPV 16, present in 44 cases (44% of all cases and 53% of the HR HPVþ cases) was the most frequent HR genotype detected, followed by HPV 18 and HPV 45, which were detected in 28% and 27% of the cases in the study. HPV 16/18 were detected in a total of 56 cases (55%) and coexisted in 16 cases (16%). Detection of HPV 16 and of HPV 16/18 increased with increasing severity of cytologic diagnosis (from 21% in negative cases up to 75% in HSIL cases and from 29% in negative cases up to 75% in HSIL, respectively). HPV 16 was also the most frequent genotype detected in each diagnostic category. The second most frequent HR genotype detected varied among the diagnostic groups, being HPV 45 in 28% of ASC, HPV 18 in 47% of LSIL, HPV 73 in 67% of ASCH, and HPV 58 in 63% of HSIL cases. Twenty-seven cases (6 negative, 9 ASC, 9 LSIL, 1 ASCH, 2 HSIL) contained only HR genotypes other than HPV 16/18, composing 27% of all and 33% of the HRþ cases. HPV 45 was the most frequent noneHPV 16/ 18 genotype detected (in 11 of these 27 cases) (Table 2).
HPV levels in anal Pap tests As shown in Table 3, the mean viral levels for all HR HPV genotypes, for HPV 16, and for HPV 16/18 increase with severity of cytodiagnosis, whereas the normalized beta globulin values remain constant across the diagnostic groups. Interestingly, a substantial (and the largest) increment in mean viral load for all HR genotypes, for HPV 16, and for HPV 16/18 is noted to occur between ASC/ASCH and LSIL. In cases with LSIL and HSIL, only the mean viral loads for HPV 16 and HPV 16/18 continue to show striking increments, suggesting that although noneHPV 16/18 HR genotypes are present, HPV 16 and HPV 16/18 genotypes are present in greatest numbers (Table 3).
HR HPV genotyping for triage of abnormal anal Pap tests Follow-up (cytology and/or tissue) became available within 30 months (range 1-30 months; mean 8.7 months) for 66 of the cases (65%), including 2 negative, 19 ASC, 32 LSIL, 6 ASCH, and 7 HSIL. For patients with >1 follow-up, the worst/highest grade AIN diagnosed within 30 months following the initial Pap test is presented as “the follow-up diagnosis.” A total of 43 AIN (18 AIN 1 and 25 AIN 2/3) were diagnosed at follow-up. AIN 2/3 was detected in 16% of ASC, 40% of LSIL, 67% of ASCH, and 71% of HSIL cases (Tables 4-6).
4
A.E. Walts et al. Table 1
HR HPV detected in anal Pap tests (N Z 101).
Cytologic diagnosis (n)
Pap tests HR HPVþ, n (%)
HR HPV genotypes/cases, range
HR HPV genotypes/cases, mean
Negative (28) ASC (25) LSIL (34) ASCH (6) HSIL (8) All cases (101)
14 21 34 6 8 83
0e7 0e6 1e8 1e9 3e9 0e9
2 3 4 5 5 3.6
(50) (84) (100) (100) (100) (82)
Abbreviations: ASC, atypical squamous cells of undetermined significance; ASCH, atypical squamous cells of undetermined significance, cannot exclude high-grade squamous intraepithelial lesion; HPV, human papilloma virus; HR, high risk; HR HPVþ, positive for 1 HR HPV genotypes; HSIL, highgrade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; Pap, Papanicolaou.
HR HPV was detected in 82% of all anal samples including in 50% of negative cases and in 84% of abnormal cases (Table 1) in this study. Therefore, it is not useful for triage of abnormal Pap tests. Additionally, 67% of ASCH and 71% of HSIL cases were AIN 2/3 on follow-up and therefore are best subjected to anoscopy and biopsy as the next step in management. This prompted us to assess HPV 16/18 genotyping as an adjunct to cytologic diagnosis of ASC and LSIL for triaging patients to anoscopy. Among the 25 AIN 2/3 cases, 20 (80%) had HPV 16/18 in the preceding anal Pap test and 4 (16%) had only noneHPV 16/18 HR genotypes (including 31, 33, 35, 45, 51, 52, 58, 59, 68, 73). One AIN 2/3 was preceded by a HR HPV-Pap test that was diagnosed as ASC. Of the 51 ASC/LSIL anal Pap tests with follow-up, 33 (65%) were HPV 16/18þ and 18 (35%) were HPV 16/18e (of which 4 were HR HPVe). On follow-up, 13 (39%) of these HPV 16/18þ cases and 3 (17%) of these HPV 16/18e cases showed AIN 2/3, yielding a sensitivity of 81%, a specificity of 43%, a positive predictive value of 39%, and a negative predictive value of 83%. HSIL and ASCH cases usually have follow-up anoscopy with biopsy. If, as recommended by Park and Palefsky,16 all ASC and LSIL cases also were to have follow-up anoscopy with biopsy, then performing HPV 16/18 genotyping rather than HR HPV screen as the primary test would reduce the number of ASC and LSIL cases triaged for anoscopy from 84% to 48% and from 100% to 76%, respectively.
Table 2
Discussion In summary, 82% of the cases in our study were HR HPVþ with multiple genotypes detected in 71% of the cases including cases from each cytodiagnostic group (negative, ASC, LSIL, ASCH, HSIL). The prevalence of HR HPV, multiplicity of HR HPV genotypes, prevalence of HPV 16 and HPV 16/18, and HPV 16 and HPV 16/18 viral load each increased with increasing severity of cytodiagnosis. HPV 16 was the most frequent genotype detected in each diagnostic group. In addition to all HSIL and ASCH, Park and Palefsky16 recommend that all ASC and LSIL undergo anoscopy with biopsy. Based on our findings, we suggest that HPV 16/18 genotyping be used in lieu of HR HPV as the initial triage of ASC and LSIL cases for anoscopy/ biopsy. Using HPV 16/18 genotyping in place of HR HPV to triage ASC and LSIL cases would have reduced the number of cases triaged for anoscopy from 84% to 48% and from 100% to 76%, respectively. Interobserver variability in cytodiagnosis in the negative/ASC categories20 and limitations associated with anoscopy (difficulties visualizing lesions and morbidity associated with liberal sampling) further suggest that HPV 16/18 genotyping as a primary triage tool could improve management of these persons at increased risk for anal cancer. Our findings confirm the high prevalence of HR HPV (from 44% to 95%), the predominance of multiple HR genotypes (up to 88%), and HPV 16 as the most frequent HR
Distribution of HR HPV genotypes in anal Pap tests (N Z 101).
Cytologic diagnosis (n)
HR HPVþ
HPV 16þ
HPV 16/18þ
none HPV 16/18þ
HR HPVe
Most frequent HR genotypes
Negative (28) ASC (25) LSIL (34) ASCH (6) HSIL (8) All cases (101)
14 21 34 6 8 83
6 9 18 5 6 44
8 12 25 5 6 56
6 9 9 1 2 27
14 (50) 4 (16) 0 0 0 18 (18)
16 16 16 16 16 16
(50) (84) (100) (100) (100) (82)
(21) (36) (53) (83) (75) (44)
(29) (48) (74) (83) (75) (55)
(21) (36) (26) (17) (25) (27)
> > > > > >
18 45 18 73 58 18
Z 31 > 39 > 45 > 33 Z 35 > 68 > 45
Abbreviations: HPV 16þ, positive for HPV 16; HPV 16/18þ, positive for HPV 16 and/or 18; HR HPVe, negative for all 15 HR HPV genotypes; noneHPV 16/ 18þ, positive for HR HPV genotypes other than HPV 16 and 18; other abbreviations as in Table 1. Unless otherwise indicated, values are n (%).
HR HPV Genotyping in Abnormal Anal Cytology Table 3
5
HPV levels in negative and abnormal anal Pap tests (N Z 101). Cytological Diagnosis
Viral Load Viral load all genotypes Mean (Cp) value Mean copies/1000 cells Viral load HPV 16/18 only Mean (Cp) value Mean copies/1000 cells Viral load HPV 16 only Mean (Cp) value Mean copies/1000 cells Quantity of cells Mean (Cp) value Mean cells Beta globin level Mean (Cp) value Mean copies/mL
Negative
ASC/ASCH
LSIL
HSIL
31.83 119
31.11 253
27.65 1142
27.67 780
33.26 51
30.35 398
26.2 2710
22.68 15,264
33.09 56
29.32 735
25.62 3830
21.98 23,166
27.09 18,241
27.64 13,143
26.71 22,877
26.09 33,104
27.09 40,129
27.64 28,913
26.71 50,329
26.09 72,828
Abbreviations: Cp, crossing point, the point at which fluoresence level crosses the baseline; other abbreviations as in Table 1.
genotype in anal samples from individuals at increased risk for AIN previously reported.5,18 We, like others,21,22 detected HR HPV (including HPV 16/18) in some cases with negative cytology. A literature review indicates that these observations are largely independent of collection method (swab versus brush), presence or absence of symptoms, and with a few exceptions, of geography.21-23 In the current study using multiplex PCR, we detected HR HPV in 84% ASC and 100% LSIL cases; these are higher percentages than we detected in similar case cohorts using the Digene Hybrid Capture II assay (55% ASC and 87% LSIL)24 or using the Invader test for HR HPV (81% ASC and 94% LSIL) (unpublished review of our laboratory’s experience). The higher prevalence of HR HPV detected by PCR could reflect increased sensitivity associated with this methodology. In a study of anal swab samples with ASC, Gohy et al22 found similar proportions of HR HPVþ cases
Table 4
in patients with AIN 1 and AIN 2/3 on biopsy and concluded that HR HPV testing was not useful to select ASC cases at highest risk for AIN 2/3. The high rate of HR HPV positivity observed by us and others in ASC and LSIL (which together compose the majority of abnormal anal cytodiagnoses) indicates that HR HPV testing is not an efficacious screen for AIN 2/3 in a population at increased risk for AIN.16,25 Multiplicity of HR HPV genotypes, detected in 71% of the cases in our study, has been reported in 12% to 88% of anal HPV infections in HIVþ individuals.5,26 Salit et al27 also reported a positive correlation between the number of HPV genotypes detected per cytology sample and the grade of AIN at biopsy. The number of HR HPV genotypes detected did not correlate with patient age in our study or in the study by Chin-Hong et al.28 Noting that the number of HPV genotypes detected in anal cytology samples (swabs)
Correlation of Pap test diagnosis with HPV genotypes detected in the Pap test and follow-up (n Z 66).
Cytologic diagnoses (n)
Cases with follow-up, n
HPV genotypes in Pap samples with follow-up
Follow-up diagnosis AIN 2/3
ASCH
AIN 1
ASC
Negative
Negative (28) ASC (25)
2 19
LSIL (34)
32
ASCH (6)
6
HSIL (8)
7
2 HPV 16/18þ 9 HPV 16/18þ 6 HR noneHPV 16/18þ 4 HR HPVe 24 HPV 16/18þ 8 HR noneHPV 16/18þ 5 HPV 16/18þ 1 HR noneHPV 16/18þ 5 HPV 16/18þ 2 HR noneHPV 16/18þ
0 2 0 1 11 2 3 1 4 1 25
0 1 0 0 2 1 0 0 0 0 4
1 2 4 1 4 4 1 0 1 0 18
1 1 1 1 4 1 1 0 0 1 11
0 3 1 1 3 0 0 0 0 0 8
Total cases (101)
66
Abbreviations: AIN, anal intraepithelial neoplasia; other abbreviations as in Tables 1 and 2.
6
A.E. Walts et al. Table 5
Distribution of HR HPV genotypes in anal Pap tests with AIN 2/3 on follow-up (n Z 25).
Cytologic diagnosis
AIN 2/3 on follow-up
HPV 16/18þ Pap tests
HR noneHPV 16/18þ Pap tests
HR HPVePap tests
HR noneHPV 16/18 genotypes in Pap tests
Negative ASC LSIL ASCH HSIL Total
0 3 13 4 5 25
0 2 11 3 4 20 (80)
0 0 2 1 1 4 (16)
0 1 0 0 0 1 (4)
NA NA 35, 45, 58 33, 35, 45, 58, 68, 73 31, 33, 35, 45, 51, 52, 59, 68, 73
Abbreviations: NA, not applicable; other abbreviations as in Tables 1, 2, and 4. Values are n and n (%).ß
from HIVþ men exceeded the number detected in concurrent biopsies, Gohy et al22 suggested that cytology, by sampling a much larger area than a tiny biopsy, more accurately reflects in vivo conditions. Nevertheless, they concluded that the high prevalence of HR HPV and the multiplicity of HPV genotypes detected in cytology samples made it unlikely that HPV testing would become part of routine screening for AIN. The risk of progression to carcinoma varies among the HR HPV genotypes, but HPV 16 is known to be the most “virulent.” HPV 16 has been reported in up to 65% of anal HPV infections21 and in at least 66% of anal cancers.12-14,29 Using type-specific real-time PCR to evaluate HPV 16 viral load at different anogenital sites in asymptomatic men who tested positive for HPV 16, Flores et al30 found that normalized viral loads varied significantly by anatomic site and that the anal canal and the penile shaft had the highest viral loads. More than a decade ago, studies using quantitative typespecific PCR in cervical material showed that the viral load of HPV 16 reaches much higher levels than that of other HR genotypes and that the viral loads only for HPV 16 correlate with increased severity of dysplasia.31 The correlation of anal viral load, particularly HPV 16 load, with severity of AIN has been investigated with conflicting findings. We observed a positive correlation between HPV 16 and HPV 16/18 loads and severity of cytological abnormality in samples from the anal canal. Salit et al27 also reported positive correlations among high HPV 16 viral loads, high-
Table 6 Correlation of HR HPV genotypes in ASC and LSIL Pap tests (n Z 51) with AIN 2/3 on follow-up. HPV genotype
Pap tests positive
AIN 2/3 on follow-up
HPV 16/18þ HR noneHPV 16/18þ HR HPVe
33 (65) 14 (27) 4 (8)
13 (39) 2 (14) 1 (25)
Abbreviations as in Tables 1, 2, and 4. Values are n (%).
grade cytology, and high-grade histology in anal samples. However, in a recent report by Zhao et al,32 viral load did not vary significantly across different diagnostic groups, although the likelihood of a high viral load was significantly greater among patients with abnormal anal cytology than among patients with negative cytology. Devarajan et al33 also reported no correlation between HPV viral load and severity of cytodiagnosis in anal samples from HIVþ persons. Confounding host factors including HIV status/virus burden, CD4 count at study time or nadir count, persistent anal HPV infection, number of partners, prior therapy, and other factors might explain the discordant results. Using our case cohort, we queried the potential role of testing for HPV 16/18 in 4 scenarios: as a primary screen for AIN 2/3 and as a means to triage patients with ASC, with LSIL, or with ASC/LSIL on anal Pap test for anoscopy. We found that 39% of ASC/LSIL cases with HPV 16/18þ anal Pap test had AIN 2/3 on follow-up compared with 17% of those with noneHPV 16/18 and HR HPV cytology. Using HPV 16/18 testing as a primary screen for AIN 2/3 yielded a sensitivity of 80%. Using HPV 16/18 testing for triage of ASC or LSIL yielded sensitivities of 67% and 85%, respectively. Using HPV 16/18 testing to triage ASC/LSIL yielded a sensitivity of 81%, a specificity of 43%, a positive predictive value of 39%, and a negative predictive value of 83%. Limitations in our study include its retrospective nature, variability in number of data points and in intervals between data points, and incomplete follow-up especially among cases with negative cytology. Prospective studies with follow-up are warranted. While we recognize the potential role of confounding factors such as reinfection and spontaneous clearance of HPV, evaluation of these complex host-pathogen relationships is beyond the scope of our study. We hypothesize that testing for HPV 16/18 would capture 80% (20 of 25) whereas testing for 4 genotypes (HPV 16,18,35,45) would capture 96% (24 of 25) of our AIN 2/3 cases diagnosed at follow-up. However, this increase in sensitivity would result in a marked reduction in specificity (testing for HPV 16, 18, 35, 45 rather than only for HPV 16,
HR HPV Genotyping in Abnormal Anal Cytology 18 would also capture 78% [compared with 50%] of the AIN 1 diagnosed at follow-up and 88% [compared with 75%] of cases with negative follow-up). Although the addition of testing for HPV 35 and 45 captured the largest number of AIN 2/3 in our cohort, different HR genotypes were detected more frequently than were HPV 35 and 45 in AIN 2/3 and invasive anal cancer in other studies.3,23,24,34 In our study, no HPV genotype other than HPV 16/18 was significantly correlated with AIN 2/3. The potential value of HPV 16/18 genotyping in combination with HPV E6/E7 messenger RNA or p16/Ki-67 immunostains for anal cancer screening awaits further study.
Conclusions Although much of our knowledge about anal HPV is based on similarities with cervical HPV, differences between these 2 infections also need to be considered when developing guidelines for diagnosis and management of individuals with abnormal findings on anal cytology. Compared with cervical Pap tests, anal Pap tests are characterized by higher prevalence of HR genotypes including HPV 16/18 and greater multiplicity of HR genotypes. Similarly, a substantially higher rate of HR HPV was detected in negative anal Pap tests (50%) as compared to cervical Pap tests (20%). A possible explanation for this observation is that the negative anal cytology cases were not from a random patient population being screened for HR HPV but were biased toward a population who were clinically at increased risk for anal cytological abnormalities. The relationship between HPV genotypes, levels, and persistence to disease progression is less clear in anal cancer. Progression from low-grade to high-grade AIN has been reported to occur within 2 years in as many as 62% of HIVþ and 36% of HIVe at-risk men.1,2 This rapid progression may be due to an underlying poor immune response. Currently, little is known about viral interactions (HPV/HPV, HIV/HPV) and the role they might play in etiology or progression of squamous intraepithelial lesions. Additionally, whereas the cervical Pap test is a routine test recommended for all women, the anal Pap test is recommended predominantly for HIVþ individuals and/or MSM. Paucity of screening programs, limited resources, a persistent element of social stigma, and/or coexistent lifethreatening illnesses that take precedence contribute to suboptimal screening in populations at increased risk for AIN and invasive anal carcinoma.
Funding sources The human papilloma virus polymerase chain reaction test was provided at no cost by Physicians Reference Laboratory.
Conflict of interest disclosures The authors made no disclosures.
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