Significance of High-Risk Human Papillomavirus DNA Detection in Women 50 Years and Older With Squamous Cell Papanicolaou Test Abnormalities

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1 Significance of High-Risk Human Papillomavirus DNA Detection in Women 50 Years and Older With Squamous Cell Papanicolaou Test Abnormalities Chengquan Zhao, MD; Shuping Zhao, MD, PhD; Amer Heider, MD; R. Marshall Austin, MD, PhD N Context. Data on cytologic screening and follow-up disproportionately reflect findings from frequently screened younger women, and data from screened women 50 years and older using newer screening technologies remain limited. Objective. To better understand the utility of adjunctive high-risk human papillomavirus (hrhpv) testing for disease risk stratification in women 50 years and older with a range of liquid-based cytology, abnormal, squamous cell Papanicolaou test results. Design. Liquid-based cytology cases interpreted as high-grade squamous intraepithelial lesion (HSIL); lowgrade squamous intraepithelial lesion (LSIL); atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H); and atypical squamous cells of undetermined significance (ASC-US) at Magee-Womens Hospital (Pittsburgh, Pennsylvania) were retrospectively identified for a 36-month period between July 1, 2005, and June 30, 2008, from women 50 years and older who also had hrhpv DNA test results. Histopathologic follow-up diagnoses were analyzed. Accepted for publication December 31, From the Departments of Pathology (Drs C. Zhao, Heider, and Austin) and Obstetrics and Gynecology (Dr S. Zhao), Magee-Womens Hospital, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; and the Department of Gynecology and Obstetrics, Qingdao University Medical School, Qingdao, China (Dr S. Zhao). The authors have no relevant financial interest in the products or companies described in this article. Presented in part at the annual meeting of the American Society of Cytopathology, Orlando, Florida, November 7 11, 2008; and at the annual meeting of the College of American Pathologists, Washington, DC, October 11 14, Reprints: Chengquan Zhao, MD, Department of Pathology, Magee- Womens Hospital of the University of Pittsburgh Medical Center, 300 Halket St, Pittsburgh, PA ( zhaoc@upmc.edu). Results. During the study period, 4855 women 50 years and older had HSIL, LSIL, ASC-H, or ASC-US Papanicolaou test results and hrhpv testing. In 89.3% of HSIL cases, 71.0% of LSIL cases, 38.9% of ASC-H cases, and 14.2% of ASC-US cases, hrhpv test results were positive. The positive predictive value of a positive hrhpv test for histopathologic cervical intraepithelial neoplasia 2/3 + detection was 90.5% with HSIL, 15% with ASC-H, 9.8% with LSIL, and 3.2% with ASC-US. A negative hrhpv test result had a 100% negative predictive value for histopathologic cervical intraepithelial neoplasia 2/3 + in both LSIL and ASC-H cases. Conclusions. In women 50 years and older, a positive hrhpv test result significantly increased the likelihood of follow-up histopathologic diagnoses of cervical intraepithelial neoplasia 2 /3 + in patients with HSIL, LSIL, and ASC- H Papanicolaou test results compared with women with negative hrhpv test results. No cervical intraepithelial neoplasia 2/3 + diagnoses were documented in women 50 years and older with LSIL or ASC-H Papanicolaou test results and negative hrhpv test results. (Arch Pathol Lab Med. 2010;134: ) Cervical screening in older women presents many special challenges. 1 Many older women present themselves less consistently for screening than do younger women, who may have the added incentives for periodic office visits to obtain contraceptives or obstetric or other reproductive services. 2 Furthermore, interpretation of cervical cytology specimens in older women can be especially challenging, given the changes associated with progressive degrees of atrophy, 3 5 other aging-associated squamous atypias, 6,7 and changes associated with hormone replacement therapy. 8 Because of these challenges and the lower rates of human papillomavirus (HPV) infection documented in older populations, 9 the positive predictive value of an abnormal cytologic test result in the older population tends to be lower than for the same result in younger women Furthermore, colposcopic evaluations can be more difficult in older women as well. 15,16 Despite these problems, screening of older women remains important because cervical cancer rates overall remain relatively high in this population and death rates are disproportionately higher, 1 reflecting cervical cancers in women who have never been screened, who are infrequently screened, or who have a history of highgrade cervical dysplasia Since 2003, routine cotesting for high-risk (hr) HPV DNA along with cytology has been approved by the US Federal Drug Administration for women 30 years and older. 21 As a result, follow-up is now becoming available on a large group of older women with a wide range of cytologic findings not previously commonly associated with adjunctive reflex hrhpv testing. Because of this experience, data are accruing on the significance of hrhpv test results and associated histopathologic outcomes in large numbers of older women with a wide range of 1130 Arch Pathol Lab Med Vol 134, August 2010 HPV in Women 50 Years and Older Zhao et al

2 abnormal cytology results. Therefore, we analyzed followup histopathologic data from the cohort of patients 50 years and older with abnormal squamous cell cytologic findings and adjunctive HPV testing in our significantly older-than-average, metropolitan US population. We also assessed the effect on follow-up biopsy diagnoses of the presence or absence of an adequate endocervical cell/ transformation zone sample (EC/TZS) in Papanicolaou (Pap) test specimens from this selected older population. MATERIALS AND METHODS After obtaining institutional review board approval at the University of Pittsburgh Medical Center, a retrospective study was initiated. A computer-based search of the CoPath (Cerner Corporation, Kansas City, Missouri) laboratory information system database at Magee-Womens Hospital of the University of Pittsburgh Medical Center was carried out during a 36-month period between July 1, 2005, and June 30, 2008, to retrieve ThinPrep Pap test (TPPT; Hologic, Inc, Bedford, Massachusetts) results reported with the Bethesda System 2001 (TBS2001) 22 interpretations of high-grade squamous intraepithelial lesion (HSIL); low-grade squamous intraepithelial lesion (LSIL); atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H); and atypical squamous cells of undetermined significance (ASC-US), as well as negative results, in women 50 years and older who were also tested for hrhpv DNA. Vaginal Pap tests were excluded from this study. The Magee-Womens Hospital/University of Pittsburgh Medical Center cytopathology laboratory is a large, subspecialized, academic hospital laboratory that consistently reports more than Pap tests per year from a large, integrated 20-hospital health system that serves a metropolitan area with a significantly older age population profile than the national average. 23 The University of Pittsburgh Medical Center is a large, integrated private health system in which Pap tests are collected by a highly diverse group of clinical providers that includes gynecologists, family physicians, internists, nurse practitioners, physician assistants, and house-staff trainees. The reporting profile of the laboratory is now documented in numerous recent publications ThinPrep Processing ThinPrep Pap tests were prepared according to manufacturer s specifications from PreservCyt (Hologic) samples using an automated processor (ThinPrep 3000, Hologic). Location-guided computer-assisted screening of TPPT slides was accomplished using the ThinPrep Imaging System (Hologic). 31 The ThinPrep Imaging System (Hologic) performed analysis of batches of up to 250 TPPT slides with specialized imaging software. For each slide, the locations of 22 microscopic fields that contained cells or cell clusters of interest were recorded. The imaged TPPT slides were placed on cytotechnologist s ThinPrep Imaging System Review Scopes (Hologic), and the cytotechnologists reviewed the 22 fields in geographic order. If the cytotechnologists found no abnormalities on those 22 fields, the cytotechnologist could sign out the case results as negative. In all cases in which any of the 22 fields contained any abnormality, reactive or reparative cellular changes, or microorganisms, the cytotechnologists manually rescreened the entire TPPT slide. All cases interpreted by cytotechnologists as having abnormalities or as showing reactive or reparative changes were referred to a pathologist for review. High-Risk HPV DNA Testing High-risk HPV DNA testing was ordered by clinicians according to several ordering options, as follows: reflex triggered by indeterminate, abnormal, atypical squamous cell (ASC) Pap test results; cotesting with Pap tests in women 30 years and older; and cotesting regardless of age or Pap test results. If hrhpv DNA was detected in patients with negative Pap test results, the Pap test slides were routinely manually rescreened by the screening cytotechnologist, referred for further manual rescreening by a quality-assurance cytotechnologist, and then also reviewed by a pathologist. High-risk HPV DNA detection was performed by the commercially available, US Federal Drug Administration approved hybrid capture 2 (HC2) assay method (Digene Corporation, Gaithersburg, Maryland), 32 which tests for highrisk and intermediate-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. High-risk HPV detection rates were compared between women with either the presence or absence of an EC/TZS. Histopathologic Follow-up Histopathologic follow-up included endocervical curettage, cervical biopsy, cervical conization by loop electrosurgical excision procedure or cold knife. Time elapsed from the abnormal Pap test result until the colposcopic examination and cervical biopsy, the follow-up procedures, and the histologic findings were abstracted from record reviews. For women undergoing 2 or more procedures during the follow-up period, only the most abnormal histologic diagnosis was recorded. Throughout this article, cervical intraepithelial neoplasia (CIN) terminology is used exclusively for histopathology diagnoses, and TBS2001 terminology is used exclusively for cytology results. Statistical Analysis The 95% confidence interval for different frequencies of hrhpv DNA detection were obtained through a Wald test. The positive rate of hrhpv DNA for each age group was compared with that of the reference group. High-risk HPV detection rates between women with and without an EC/TZS were compared. Statistical analyses were performed by x 2 test or Fisher exact test for small sample size using the SAS 9.1 system (SAS Institute Inc, Cary, North Carolina). P values,.05 were considered statistically significant. RESULTS Prevalence of hrhpv During the study period, a total of cervical TPPT results were interpreted as HSIL, LSIL, ASC-H, and ASC- US or as negative were identified from women 50 years and older who were also tested for hrhpv DNA. The detection rate for hrhpv DNA among these women was 6.3%. The HC2 hrhpv DNA test results among women with different Pap test result categories are shown in Table 1. High-risk HPV prevalence was highest (89.3%) in women with HSIL Pap results and continuously declined for patients with LSIL, ASC-H, ASC-US, and negative Pap results. Only 1.7% of women with negative cytology results had a positive hrhpv test result (Table 1). High-risk HPV detection rates for women with ASC-US Pap results were 8 times higher than for women with negative Pap results. The differences in hrhpv DNA prevalence between all studied TBS2001 categories of abnormal squamous cell Pap results were statistically significant (P,.001). When hrhpv DNA detection rates in women 50 years and older with HSIL, LSIL, ASC-H, ASC-US, and negative TPPT results were compared between women with and without an EC/TZS, no statistically significant difference was found with one exception. High-risk HPV detection rates were significantly higher in ASC-US cases in which an EC/TZS was absent compared with ASC-US cases in which a EC/TZS was present (Table 2). Histopathologic Follow-up The 24 women with HSIL cytology results and the 106 women with LSIL cytology results all had at least one Arch Pathol Lab Med Vol 134, August 2010 HPV in Women 50 Years and Older Zhao et al 1131

3 Table 1. High-Risk Human Papillomavirus (hrhpv) DNA Detection Rates in Women 50 Years and Older With Negative and Abnormal Squamous Cell Papanicolaou (Pap) Test Results Pap Test Result hrhpv Test Results, No. hrhpv + Test Results, No. (%) 95% CI HSIL (89.3) LSIL (71.0) ASC-H (38.9) ASC-US (14.2) Negative (1.7) Abbreviations: ASC-H, atypical squamous cells, cannot exclude highgrade squamous intraepithelial lesion; ASC-US, atypical squamous cells of undetermined significance; CI, confidence interval; HSIL, highgrade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion. cervical biopsy during the study period and were included in this follow-up study. The average age of patients in the LSIL group with histopathologic follow-up was 56.2 years (range, years), and the average age of patients in the HSIL group with histopathologic follow-up was 59.3 years (range, years). The average interval between squamous intraepithelial lesion cytology results and an initial histopathologic diagnosis of CIN 2/3 was 2.6 months (range months) in women with HSIL and 4 months (range, months) in women with LSIL. The histopathologic findings are shown in Table 3. Histopathologic CIN 2/3 was detected in 84% of women 50 years or older with HSIL and in 7.6% of women 50 years and older with LSIL. Both CIN 2/3 and CIN 1 were identified in a significantly greater proportion of women with LSIL who had positive hrhpv DNA results than in women who had negative hrhpv DNA results. The CIN detection rate was 68.3% (56 of 82 cases) in patients with HPV + LSIL, significantly higher than the rate of 45.8% (11 of 24 cases) seen in patients with HPV 2 LSIL (P 5.04). In patients with HPV + LSIL, 8 of 82 patients (9.8%) had a subsequent histopathologic diagnosis of CIN 2/3. In comparison, no women in the HPV 2 LSIL group had a follow-up tissue diagnosis of CIN 2/3. Histopathologic follow-up results were assessed for women with ASC-H TPPT and concurrent hrhpv test results, 27 between July 2005 and June 2007, and for women with ASC-US TPPT and positive hrhpv test results, 28 between July 2005 and March In the patients 50 years and older with HPV + results, 15% (3 of 20) of women with ASC-H and 3.2% (3 of 94) of women with ASC-US had a subsequent histopathologic diagnosis of CIN 2/3. No cases of CIN 2/3 were detected in the 39 women 50 years and older with hrhpv 2 and ASC-H Pap results (Table 4). Positive Predictive Value, Negative Predictive Value, Sensitivity, and Specificity When squamous intraepithelial lesion and abnormal squamous cell cytology results were considered in conjunction with positive hrhpv test results, the positive predict value for CIN 2/3 detection was 90.5% with HSIL, 15% with ASC-H, 9.8% with LSIL, and 3.2% with ASC-US. A negative hrhpv test had a negative predictive value of 33.3% with HSIL and 100% with both LSIL and ASC-H. The sensitivity and specificity of hrhpv DNA testing for histopathologic detection of CIN 2/3 were 90.5% and 33.3% with HSIL, 100% and 24.5% with LSIL, and 100% and 15% with ASC-H, respectively. COMMENT Cervical cytologic screening in older women has long been recognized as particularly challenging because of the difficulties associated with interpretation of a wide variety of perimenopausal and postmenopausal squamous atypia. It is well known by cytopathologists that squamous atypia in older women can, on occasion, closely mimic high-grade squamous dysplasia or even invasive squamous carcinoma, 36,37 contributing to the observed low positive predictive value of squamous cell abnormalities Local estrogen application has often been relied on to see whether some worrisome squamous atypia might prove to be reversible. 37,38 More recently, there has been great interest in the utility of adjunctive HPV testing to help clarify abnormal squamous cell cytology findings in older women With the increasing use of routine Pap and HPV cotesting in the United States for women 30 years and older, both Pap and HPV test results are becoming more commonly available to guide follow-up considerations. 42 Our findings of 100% negative predictive value for histopathologic CIN 2/3 detection with negative HPV test results in women with ASC-H or LSIL Pap results are consistent with other reports 27,43 that have supported the option of reflex HPV testing as an age-selective alternative to routine colposcopic referral in older women. The 2006 American Society for Colposcopy and Cervical Pathology guidelines, 44 for example, recommended, for the first time, reflex HPV testing as an alternative to colposcopy for postmenopausal women with LSIL Pap results. Our data support this option and raise for consideration the Table 2. High-Risk Human Papillomavirus (hrhpv) DNA Detection Rates in Women 50 Years and Older With Negative and Abnormal Squamous Cell Papanicolaou (Pap) Test Results With and Without an Endocervical Cell/Transformation Zone Sample (EC/TZS) EC/TZS Present EC/TZS Absent Pap Test Result hrhpv Tested, No. Positive, No. (%) 95% CI hrhpv Tested, No. Positive, No (%) 95% CI P Value HSIL (90.0) (87.5) a LSIL (70.1) (72.1) ASC-H (37.0) (50) ASC-US (13.1) (18.9) ,.001 Negative (1.7) (2.0) Abbreviations: ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; ASC-US, atypical squamous cells of undetermined significance; CI, confidence interval; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion. a Fisher exact test Arch Pathol Lab Med Vol 134, August 2010 HPV in Women 50 Years and Older Zhao et al

4 Table 3. Histopathologic Cervical Intraepithelial Neoplasia (CIN) Diagnosed in Women 50 Years and Older With Abnormal Squamous Cell Papanicolaou (Pap) Test Results Source, y Pap Test Result Histopathologic Follow-up, No. CIN 1, No. (%) CIN 2/3 +, No. (%) This study HSIL 24 2 (8.3) 21 a (87.5) This study LSIL (55.7) 8 (7.5) Bandyopadhyay et al, ASC-H 59 9 (15.3) 3 (5.1) Armah et al, ASC-US b (38.3) 3 (3.2) Abbreviations: ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; ASC-US, atypical squamous cells of undetermined significance; hrhpv, high-risk papillomavirus; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion. a Includes one case of invasive squamous cell carcinoma. b All cases of ASC-US were positive for hrhpv. suggestion that a cutoff at age 50 years could represent a more practical and more specific age point at which clinicians could begin considering the option of HPV triage with LSIL Pap results. Our data are also consistent with other reports 27,45 supporting the utility of HPV triage for older women with ASC-H Pap test results. In this study, 38.9% of older women with ASC-H Pap results tested positive for hrhpv. Higher rates of HPV detection reported in a few studies with ASC-H Pap interpretations have reflected both significantly younger study populations and consensus panel cytology interpretations, rather than usual and routine practice where final interpretations of ASC-H are generally decided on by a single reviewing pathologist. 27 The increased positive predictive value for histopathologic CIN 2/3 associated with HC2 HPV + HSIL Pap tests in older women has not, to the best of our knowledge, been previously reported. This observation is consistent with numerous reports that have described postmenopausal atypia as particularly challenging mimics of high-grade squamous cell abnormalities. Although a small proportion of CIN 2/3 lesions may be associated with negative HC2 hrhpv test results, 46,47 our data indicate that an HPV + HSIL result gives added urgency to the need to locate and histopathologically document the origin of HSIL cells, even when initial colposcopic and histopathologic examinations may be negative. It is now well recognized that many such cytologic-histopathologic discrepancies reflect the limited sensitivity of colposcopy. 48 Our data also demonstrate that hrhpv DNA prevalence varies progressively with different degrees of cytologic squamous cell abnormalities identified in older women. The lowest prevalence of HPV was seen in women with negative-imaged liquid-based cytology results, 1.7% in the study group of women 50 years and older. Available older follow-up studies from North America suggest that cytology-negative, HC2 HPV + patients in North America have a very low (2%) long-term risk for CIN3 +, even using a study design where pre-tbs2001 conventional Pap test results reported as benign atypia were reclassified as negative We also document here an 8-fold higher HPV prevalence of 14.2% in study patients 50 years and older with ASC-US Pap test results, abnormal findings that, on histopathologic follow-up, largely reflected minor nonprecancerous epithelial cell abnormalities, changes presumably associated with mostly transient HPV infections. One other review 53 of US laboratory data reported that HPV detection rates with ASCUS Pap results declined significantly in women 30 years and older but did not look separately at women 50 years and older. The significantly higher rates of HPV detection associated with ASC-H Pap results and LSIL Pap results compared with findings of ASC-US have been reported in other studies. 27,28,54 56 Lower rates of CIN 2/3 identified here in older women after HPV + LSIL, ASC-H, and ASC-US results differ from results reported from the widely cited ASCUS LSIL Triage Study (ALTS) and likely reflect several important factors. After the ALTS, based on entry level, manually screened, community, conventional Pap test results from much younger women (mean, 29 years), 57 the definition of ASC-US shifted in TBS2001, and liquid-based cytology also largely replaced conventional Pap tests. The Bethesda System 2001 specifically eliminated the category atypical squamous cells of undetermined significance, favor reactive (ASCUS-R), encouraged pathologists to judiciously downgrade a portion of ASCUS-R cases, and separated from the ASC-US category the subset of atypical squamous cells cases now referred to as ASC-H. 22 In previously presented work using Bayesian network modeling of cervical cancer screening, 61 we reported that Table 4. Histopathologic Cervical Intraepithelial Neoplasia (CIN) Diagnosed in Women 50 Years and Older With Positive and Negative Results For High-Risk Human Papillomavirus (hrhpv) and Abnormal Squamous Cell Papanicolaou (Pap) Test Results Source, y Pap Test Results Histopathologic Follow-up, No. P Value of CIN 1, CIN 2/3, Histopathologic CIN 1, CIN 2/3, hrhpv + hrhpv No. (%) No. (%) Follow-up, No. No. (%) No. (%) CIN 1 3 This study HSIL 21 2 (9.5) 19* (90.5) (66.7).13 a This study LSIL (58.5) 8 (9.8) (45.8) 0.04 Bandyopadhyay et al, ASC-H 20 5 (25) 3 (15) 39 4 (10.3) 0.01 a Armah et al, ASC-US (38.3) 3 (3.2) N/A Abbreviations: ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; ASC-US, atypical squamous cells of undetermined significance; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion, N/A, not available. a Fisher exact test. Arch Pathol Lab Med Vol 134, August 2010 HPV in Women 50 Years and Older Zhao et al 1133

5 CIN 2/3 + risk, after specified current Pap and HPV results, varies considerably based on prior screening history. We believe that lower rates of CIN 2/3 + reported here after HPV + LSIL, ASC-H, and ASC-US in older women reflect not only post-alts era changes mentioned above but also the significantly lower risk for disease in older populations with low HPV infection rates and screening history (usually repeated) with liquid-based cytology and computer-assisted screening. 26 This is also consistent with available data comparing HPV prevalence in negative Pap test results, an objective measure of residual risk after cytologic screening. 29,30 The 1.7% HPV prevalence among cytology-negative older women in this study contrasts dramatically with the 32.7% HPV detection rate in cytology-negative cases reported in the ALTS. 57 Cervical intraepithelial neoplasia 2/3 + risk reflects infection rates, screening history, and treatment history in addition to current cytology and HPV results. Therefore, we concur with previous recommendations that autonomous investigations in diverse communities using a variety of contemporary screening test methodologies are needed to optimize local and national screening strategies. 53 The prevalent current use of liquid-based cytology, computerassisted screening, widespread, adjunctive hrhpv testing and the use of TBS2001 with an ASC-H category, all make the current US screening environment a very different world from the one studied and reported in the ALTS. It is not clear why hrhpv prevalence was higher in older patients with ASC-US but without an EC/TZS than in similarly aged patients with ASC-US and an EC/TZS. Several previous reports have documented that HPV prevalence for most categories of Pap test results and age groups are independent of sampling of the transformation zone We have no completely satisfactory explanation for this isolated finding, which may reflect some selection bias that we are currently unable to identify. 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