Can HPV-16 Genotyping Provide a Benchmark for Cervical Biopsy Specimen Interpretation?

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1 Anatomic Pathology / Monitoring HPV-16 Fractions in CIN Can HPV-16 Genotyping Provide a Benchmark for Cervical Biopsy Specimen Interpretation? Mary T. Galgano, MD, 1 Philip E. Castle, PhD, MPH, 2 Mark H. Stoler, MD, 1 Diane Solomon, MD, 3 and Mark Schiffman, MD, MPH 2 Key Words: Cervical intraepithelial neoplasia; Human papillomavirus; HPV; Colposcopy; Biopsy; Quality assurance; Benchmark DOI: /A8MFC18TWANC8QFH Abstract Human papillomavirus (HPV) detection is an objective quality assurance benchmark for cervical cytology. There is no comparable metric for cervical biopsies despite biopsies having similar interobserver variability. Because HPV-16 positivity increases with the severity of cervical abnormality, we explored whether HPV-16 detection might be a useful metric for distinguishing a diagnosis of less than cervical intraepithelial neoplasia (CIN) 2 from CIN 2 or worse (representing the clinical threshold for treatment). By using Atypical Squamous Cells of Undetermined Significance Low-Grade Squamous Intraepithelial Lesion Triage Study data, we compared biopsy diagnoses of the 10 clinical center (CC) pathologists with the quality control (QC) group. Although the percentage of HPV-16 correlated with severity of diagnoses, there was great variability between the rates for each individual CC pathologist. Agreement between individual CC pathologists and the QC group on the percentage of CIN 2 or worse containing HPV-16 correlated weakly with agreement on whether the diagnosis was less than CIN 2 or CIN 2 or worse. Thus the HPV-16 fraction was not found to be broadly useful as a quality assurance metric for biopsy diagnosis in this analysis. The 2001 Bethesda category of atypical squamous cells of undetermined significance (ASC-US) indicates the presence of cells suggestive but not diagnostic of squamous dysplasia. This equivocal interpretation provides higher sensitivity required for primary cervical cytologic screening than using more severe cytologic categories as the threshold for colposcopy. However, fewer than a third of all ASC-US cases have histologically confirmed cervical intraepithelial neoplasia (CIN) 2 or worse. 1-4 The poor predictive value of an ASC-US diagnosis has been somewhat modulated by the availability of assays for detection of carcinogenic human papillomavirus (HPV), based on the necessary role of these viruses in cervical carcinogenesis. Carcinogenic HPV testing can be used to triage an equivocal Papanicolaou (Pap) smear by distinguishing between carcinogenic HPV+ ASC-US that is equivalent to low-grade squamous intraepithelial lesion (LSIL) for risk for precancer and morphologic look-alikes that bear no risk. In fact, several studies have shown that HPV molecular testing in the triage of ASC-US is highly sensitive for identifying women at risk for cervical precancerous lesions, while only sending approximately half of women to colposcopy. 2-4 This strategy for clinical management has been adopted into national guidelines. 5-7 The detection of carcinogenic HPV DNA not only serves to direct the management of individual women with an ASC- US result, but it can also be used as a quality assurance metric for cytologic interpretations of ASC-US. The percentage of HPV+ ASC-US cases provides an objective calibrator for laboratories and individual cytopathologists or cytotechnologists to monitor use of ASC-US. 8,9 Typically, approximately half of ASC-US cases are positive for carcinogenic types of HPV. Much larger percentages of carcinogenic HPV+ cases Am J Clin Pathol 2008;130: DOI: /A8MFC18TWANC8QFH 65

2 Galgano et al / Monitoring HPV-16 Fractions in CIN for ASC-US cytology can indicate insensitive use of the ASC- US interpretation. On the other hand, much lower HPV prevalences with large percentages of ASC-US suggest overuse of the interpretation. Although carcinogenic HPV testing is not clinically indicated in the context of other abnormal cytologic interpretations such as LSIL and high-grade squamous intraepithelial lesion (HSIL), HPV positivity may also provide a quality assurance benchmark if results are available because two thirds or more of LSIL cases 10 and virtually all HSIL cases are typically positive for carcinogenic HPV types. 4,11 Histologic interpretation of cervical biopsy specimens and loop electrosurgical excision procedure (LEEP) specimens has high interobserver variability comparable to cytology. 12 CIN 1 is very poorly reproducible, and the phrase histologically confirmed CIN 1 might be questioned. Of more importance, CIN 2 is only poorly or moderately reproducible, 13 and, yet, it is an actionable threshold for treatment per current guidelines. Beyond masked review of tissue specimens, there is no well-accepted quality assurance metric for histology analogous to testing HPV+ cases for ASC-US. Most CIN of any grade is positive for carcinogenic HPV, 8,14,15 so the presence of one of the group of carcinogenic types is not a useful metric to evaluate or compare pathologists grading of CIN. However, the prevalence rate of the most carcinogenic genotype, HPV- 16, directly correlates with the histologic severity. 13,16 Given the significant variability and subjectivity of diagnostic interpretations of cervical biopsy specimens, HPV-16+ fraction might be considered a quality assurance metric to evaluate and provide educational feedback to pathologists about their histologic grading of cervical tissue, analogous to monitoring the percentage of ASC-US cases positive for high-risk HPV. We, therefore, sought to evaluate the possible usefulness of HPV-16 detection as a tool for quality assurance in the ASCUS and LSIL Triage Study (ALTS) Materials and Methods Study Design and Population ALTS was a randomized trial comparing 3 management strategies for 5,060 women with ASC-US (n = 3,488) or LSIL (n = 1,572): (1) immediate colposcopy (referral to colposcopy regardless of enrollment test results), (2) HPV triage (referral to colposcopy if enrollment HPV result by Hybrid Capture 2 [hc2; Digene, Gaithersburg, MD] was positive or missing or if the enrollment cytologic result was HSIL), or (3) conservative management (referral to colposcopy if the enrollment cytologic result was HSIL). At enrollment, all women underwent a pelvic examination with collection of 2 cervical specimens; the first specimen in PreservCyt for ThinPrep cytology (Cytyc, Marlborough, MA) and the second in specimen transport medium (STM; Digene). Women in all 3 arms of the study were reevaluated by cytology every 6 months for 2 years and sent to colposcopy if the cytologic result was HSIL. An exit examination with colposcopy was scheduled for all women, regardless of study arm or prior procedures, at the completion of follow-up. We refer readers to other references for details on randomization, examination procedures, patient management, and laboratory and pathology methods The National Cancer Institute and local institutional review boards approved the study, and all participants provided written informed consent. HPV DNA Testing Residual PreservCyt specimens, after being used for liquid-based cytology, were tested by hc2, 2 a pooled-probe, signal-amplification DNA test that targets a group of 13 HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). 22 The hc2 does not distinguish which individual HPV genotypes are present. HPV genotyping was performed on the STM specimen as previously described using the Line Blot Assay (LBA; Roche Molecular Systems, Alameda, CA), an L1 consensus primer-based PCR assay that uses a primer set designated PGMY09/11. 23,24 Amplimers were subjected to reverseline blot hybridization for detection of 27 individual HPV genotypes (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51-59, 66, 68, 73 [PAP238a], 82 [W13b], 83 [Pap291], and 84 [PAP155]). We also tested for an additional 11 noncarcinogenic HPV genotypes (61, 62, 64, 67, 69-72, 81, 82 variant [IS39], and 89 [CP6108]) in approximately half of the specimens (58%) at enrollment and in all specimens collected at the follow-up visits. Pathology and Treatment All pathology diagnoses were masked to HPV status. Clinical management was based on the clinical center (CC) pathologists cytologic and histologic diagnoses. In addition, referral smears, ThinPreps, and histologic slides were sent to the Pathology Quality Control (QC Pathology) based at Johns Hopkins Hospital, Baltimore, MD, for review and secondary diagnoses. A percentage of the cytologic slides from followup were screened by computer-assisted review (Neopath, TriPath Imaging, Burlington, NC). A CIN 2 or worse diagnosis based on the clinical center pathology or a CIN 3 or worse diagnosis based on the QC pathology review triggered treatment by LEEP. In addition, women with persistent LSIL or carcinogenic HPV+ ASC-US at the time of the exit from the study were offered LEEP. Based on the QC algorithm, we considered the QC Pathology consensus review to be the reference standard in this study because it was used as the trial end point and exhibited somewhat less interpretive variability and the highest correlation with HPV prevalence. 12,21 66 Am J Clin Pathol 2008;130: DOI: /A8MFC18TWANC8QFH

3 Anatomic Pathology / Original Article Statistical Analysis We first compared the diagnoses by the CC pathologists as a group with the QC Pathology diagnoses. For paired diagnostic levels (eg, CIN 2 by the CC pathologists but CIN 1 by QC Pathology), we examined the percentage of hc2+ and the percentage of HPV-16+. We used a test of trend to examine the relationships of percentage hc2+ and the percentage HPV- 16+ with the severity of diagnosis by one pathology group given a fixed diagnosis by the other pathology group. Ten CC pathologists had interpreted the colposcopic biopsy specimen(s) from more than 75 women at their CC, and the data were included in our next analysis. We examined the variability of the percentage of HPV-16+ cases by diagnostic category for each individual pathologist. For simplicity, we excluded biopsy diagnoses of atypical, which was not uniformly used by pathologists and had limited clinical usefulness. Each pathologist read a different group of biopsy specimens that varied in spectrum of severity. We wanted to understand their relative performance, with reference to QC Pathology. Therefore, we compared the diagnoses by each CC pathologist with those of QC Pathology, with regard to HPV- 16 positivity, for each diagnostic category separately. Finally, we reached our main analytic goal. We examined whether any aspect of HPV-16 positivity, absolute or relative to QC Pathology, for less than CIN 2 or CIN 2 or worse, predicted agreement on histologic grading (less than CIN 2 vs CIN 2 or worse) of the CC pathologists with QC Pathology. Results ztable 1z shows the comparison of the CC pathology review vs the QC Pathology review of all biopsy specimens obtained during ALTS in relation to the percentage of hc2+ (ie, any carcinogenic type) and percentage of HPV-16+ as detected by LBA. As previously reported for biopsy specimens obtained at enrollment, 13 there was a strong tendency for the percentage of HPV-16+ cases to increase with increasingly severe diagnosis by one pathology group for a given diagnosis on the same tissues by the other pathology group. When considering patients with CIN 1 or worse diagnosed by both pathology groups, the test of trend was much stronger for percentage of HPV-16+ cases compared with the percentage of hc2+ cases. The percentages of HPV-16+ cases by diagnostic category for each CC pathologist are given in zfigure 1z. HPVzTable 1z Comparison of Paired Biopsy Diagnoses by Two Pathology Groups in Relation to the Percentage of hc2+ and the Percentage of HPV-16+ as Detected by the Line Blot Assay * QC Pathology Clinical Center Pathology Negative Atypical CIN 1 CIN 2 CIN 3+ Total P Negative No. (%) of cases 2,002 (41) 9 (0) 24 (0) 15 (0) 4 (0) 2,054 (42) hc2+ (%) HPV-16+ (%) Atypical No. (%) of cases 402 (9) 35 (1) 45 (1) 11 (0) 7 (0) 500 (10) hc2+ (%) HPV-16+ (%) CIN 1 No. (%) of cases 568 (12) 22 (0) 798 (16) 115 (2) 23 (0) 1,526 (31) hc2+ (%) HPV-16+ (%) CIN 2 No. (%) of cases 35 (1) 13 (0) 110 (2) 241 (5) 147 (3) 546 (11) hc2+ (%) HPV-16+ (%) <.0001 CIN 3+ No. (%) of cases 7 (0) 4 (0) 6 (0) 62 (1) 210 (4) 289 (6) hc2+ (%) HPV-16+ (%) Total No. (%) of cases 3,014 (61) 83 (2) 983 (20) 444 (9) 391 (8) 4,915 hc2+ (%) HPV-16+ (%) P hc2+ (%) HPV-16+ (%) <.0001 < CIN, cervical intraepithelial neoplasia; hc2, Hybrid Capture 2; HPV, human papillomavirus; QC, quality control. * P values are for trend tests among women who had at least CIN 1 as diagnosed by both pathology groups. Am J Clin Pathol 2008;130: DOI: /A8MFC18TWANC8QFH 67

4 Galgano et al / Monitoring HPV-16 Fractions in CIN 16+ fractions, expressed as percentages, ranged from 4.7% to 11.6% for negative histologic results, 9.3% to 23.1% for CIN 1, 29.5% to 60.0% for CIN 2, and 47.7% to 79.2% for CIN 3. Different pathologists read different biopsies, but the QC pathologists read all slides. Therefore, we attempted to normalize the percentages of HPV-16 positivity for CC pathologists by dividing the percentage of HPV-16+ cases for a pathologist for a given diagnosis by the percentage of % HPV-16 Positive Ratio (CC/QC) of % HPV-16 Positive CC1 CC3 CC5 CC7 CC9 CC2 CC4 CC6 CC8 CC10 Negative CIN 1 CIN 2 Biopsy Diagnosis CC1 CC3 CC5 CC7 CC9 CC2 CC4 CC6 CC8 CC10 Negative CIN 1 CIN 2 Biopsy Diagnosis CIN 3 zfigure 1z Comparison of the percentage of human papillomavirus 16 (HPV-16)-positive cases with the biopsy diagnoses from 10 clinical center (CC) pathologists participating in the Atypical Squamous Cells of Undetermined Significance Low-Grade Squamous Intraepithelial Lesion Triage Study. CIN, cervical intraepithelial neoplasia. CIN 3 zfigure 2z The ratio of the percentage of human papillomavirus 16 (HPV-16) positivity (individual Clinical Center [CC] pathologists compared with the quality control [QC] pathology review standard) by diagnostic category. CIN, cervical intraepithelial neoplasia. κ HPV-16+ cases for the same category of diagnosis by QC Pathology review zfigure 2z. These ratios ranged from 0.61 to 1.13 for normal, 0.38 to 1.45 for CIN 1, 0.80 to 1.90 for CIN 2, and 0.75 to 1.65 for CIN 3. Finally, we compared the agreement of individual clinical pathologists with QC Pathology review for biopsy diagnoses of less than CIN 2 or CIN 2 or worse (translating clinically into no treatment vs treatment). We examined several possible uses of HPV-16 testing, looking for a simple metric that would predict agreement between pathologists, but found none. Among all parameters examined, we observed that the absolute difference in the percentage of HPV-16 diagnoses between CC pathologists and QC Pathology correlated with the difference in diagnostic readings as measured by κ values zfigure 3z. The κ values ranged from 0.65 to There was a significant trend (P =.01) for larger κ values, implying better agreement, with smaller differences (better agreement) in the percentage of HPV-16 diagnoses among diagnoses of CIN 2 or worse. A similar, albeit statistically nonsignificant trend (P =.1) was observed for the percentage of HPV-16 diagnoses among diagnoses of less than CIN 2. Discussion We examined whether using HPV-16 detection might be useful as a metric for quality assurance for cervical histopathologic diagnoses of biopsy specimens, which are critical for decisions about clinical management including treatment CIN 2+ <CIN 2 Linear (CIN 2+) Linear (<CIN 2) % 2% 4% 6% 8% 10% 12% 14% Absolute Difference (QC CC) in % HPV-16 Positive zfigure 3z Correlation of agreement, as measured by κ values, for biopsy diagnoses categorized as less than cervical intraepithelial neoplasia (CIN) 2 or CIN 2 or worse by the clinical center (CC) pathologists and the quality control (QC) pathology reading with the differences in the percentage of human papillomavirus 16 positive for pathology readings of less than CIN 2 and CIN 2 or worse. Less than CIN 2, P =.01; CIN 2 or worse, P = Am J Clin Pathol 2008;130: DOI: /A8MFC18TWANC8QFH

5 Anatomic Pathology / Original Article This is analogous to using carcinogenic HPV testing to monitor the use of ASC-US cytologic interpretations. In particular, we are concerned that variability in the diagnosis of CIN 2 might lead to unnecessary treatment. We made the following observations: First, as previously observed, 13 there was generally a strong trend of an increasing fraction of HPV-16+ diagnoses with increasing severity of the CIN grade, and this trend was stronger than it was for the detection of carcinogenic HPV. (Note: We used hc2 in this analysis because hc2 data might be routinely available to clinical pathologists.) The percentage of HPV-16+ diagnoses tended to have an intermediate value when the 2 pathology reviews did not agree. For example, diagnoses of CIN 1/ CIN 2 and CIN 2/CIN 1 (CC pathologists/qc pathologists) had percentages of HPV-16+ of 21% and 31%, respectively, which were intermediate to the 17% for consensus CIN 1 diagnosis and 44% for the consensus CIN 2 diagnosis. One obvious interpretation of these data is that diagnoses by both pathology reviews include error. Second, we found great variability between CC pathologists in the percentage of HPV-16+ diagnoses in biopsy diagnoses of CIN 1 and CIN 2, the 2 diagnoses with the poorest interpathologist agreement. 12 Interestingly, we noted a qualitative difference in the variability of the diagnosis of CIN 2 relative to percentage of HPV-16+ compared with other diagnoses. Four pathologists diagnosed CIN 2 with a very high percentage of HPV-16+ (Figure 2, relative to QC), and 6 pathologists diagnosed CIN 2 with a percentage of HPV-16+ that was similar or lower than the QC Pathology review. This may reflect differences in criteria for the diagnosis of CIN 2. Finally, we showed that agreement of CC pathologists with QC Pathology review was somewhat related to differences in the percentage of HPV-16+ diagnoses between the 2 reviews. As this difference became small, agreement with QC Pathology improved. However, our finding is too weak, we believe, to find broad clinical use. The clarification of CIN 2 using molecular tools will require very strong, simple triage algorithms. Otherwise, pathologists are correctly likely to trust instead in the visual reality of what they see. A potential limitation is that HPV-16 genotyping was conducted on cervical specimens that were temporally correlated with the corresponding biopsy specimen rather than HPV genotyping of the biopsy specimen itself. Given that many HPV genotypes can cause CIN 1 and that multitype infection is commonly found in patients with LSIL, with no way to attribute the CIN 1 on biopsy to any one HPV type, it seems likely that our analysis overestimates the fraction of HPV-16 in CIN 1 and normal biopsy specimens. This bias could have partially obscured the relationship of the percentage of HPV-16 and agreement on diagnoses. We had hoped that HPV-16 might prove a useful metric for measuring interpathologist tendencies in diagnosing CIN 2. No simple measure suitable for easy clinical adoption proved useful, eg, percentage of HPV-16 in CIN 2 or ratio of HPV-16 percentages in CIN 2 vs CIN 1. We continue to search for a potential quality assurance benchmark for the clinically significant problem of interpretive variability of cervical biopsy specimens. HPV RNA expression or other markers such as p might better allow the separation of cervical biopsy specimens into more biologically and clinically meaningful categories of normal, productive infection, and true precancer. From the 1 Robert E. Fechner Laboratory of Surgical Pathology, University of Virginia Health System, Charlottesville; and Divisions of 2 Cancer Epidemiology and Genetics and 3 Cancer Prevention, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD. Supported in part by the intramural research program of the National Cancer Institute. Some of the equipment and supplies used in these studies were donated or provided at reduced cost by Digene, Gaithersburg, MD; Cytyc, Boxborough, MA; National Testing Laboratories, Fenton, MO; DenVu, Tucson, AZ; TriPath Imaging, Burlington, NC; and Roche Molecular Systems, Alameda, CA. ALTS was supported by the National Cancer Institute, National Institutes of Health Department of Health and Human Services contracts CN-55153, CN-55154, CN-55155, CN-55156, CN-55157, CN-55158, CN-55159, and CN Address reprint requests to Dr Stoler: Dept of Pathology, University of Virginia Health System, 1215 Lee St, HEP Office 3032, Charlottesville, VA Acknowledgments: We thank the ALTS Group Investigators for their help in planning and conducting the trial and Franklin Demuth, Information Management Services, Rockville, MD, for programming support. References 1. Pitman MB, Cibas ES, Powers CN, et al. Reducing or eliminating use of the category of atypical squamous cells of undetermined significance decreases the diagnostic accuracy of the Papanicolaou smear. Cancer. 2002;96: Solomon D, Schiffman M, Tarone R, et al. Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: baseline results from a randomized trial. 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