Key Words: cervix; atypical squamous cells of undetermined significance (ASCUS); ASC-H; epigenetics; methylation

Transcription

1 Triage of Cervical Cytological Diagnoses of Atypical Squamous Cells by DNA Methylation of Paired Boxed Gene 1 (PAX1) Tai-Kuang Chao, M.D., Ph.D., 1 Feng-Yi Ke, M.S., 1 Yu-Ping Liao, M.S., 2 Hui-Chen Wang, B.S., 3 Cheng-Ping Yu, M.D., Ph.D., 1 and Hung-Cheng Lai, M.D., Ph.D. 2,3,4 * Detection of cervical high-grade squamous intraepithelial lesions (HSIL) in patients with equivocal cytological abnormalities, such as atypical squamous cells (ASC) of undetermined significance (ASCUS) or inability to exclude high-grade squamous intraepithelial lesions (ASC-H) is still a challenge. This study tested the efficacy of PAX1 methylation analysis in the triage of cervical ASCUS and ASC-H and compared its performance with Hybrid Capture 2 (HC2) HPV test. A hospital-based case control study was conducted. Cervical scrapings from patients with ASCUS or ASC-H were used for the quantitative methylation analysis of PAX1 methylation by MethyLight and HPV testing by HC2. Patients with ASC-H or ASCUS with repeated abnormal smears underwent colposcopic biopsy and subsequent therapies. Diagnoses were made by histopathology at a follow-up of 2 years. The efficacies of detecting high-grade lesions were compared. Fifty-eight cervical scrapings with cytological diagnosis of ASCUS (n ¼ 41) and ASC- H(n¼ 17) were analyzed. One of the 41 (2.4%) ASCUS patients and seven of 17 (41.2%) ASC-H patients were confirmed to have 1 Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China 2 Department of Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China 3 Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China 4 Lab of Epigenetics and Cancer Stem Cells, National Defense Medical Center, Taipei, Taiwan, Republic of China Contract grant sponsor: Tri-Service General Hospital, Taipei, Taiwan, Republic of China; Contract grant number: TSGH-C98-10-S05, TSGH- C S05, TSGH-C S01, and TSGH-C S02; Contract grant sponsor: National Science Council; Contract grant number: NSC B MY3. *Correspondence to: Hung-Cheng Lai, M.D., Ph.D., Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, 5F, 325, Sec 2, Cheng-Gong Rd., Neihu District, Taipei City 114, Taiwan, Republic of China. hclai@ndmctsgh.edu.tw Received 10 February 2011; Accepted 11 May 2011 DOI /dc Published online 27 June 2011 in Wiley Online Library (wileyonlinelibrary.com). HSIL. After dichotomy of the PMR, PAX1 methylation rates were significantly higher in ASC developing HSIL compared with those developing reactive atypia (87.5% vs. 12.5%, P < 0.001). Testing PAX1 methylation in cervical swabs of patients with ASC confers better sensitivity (87.5% vs. 62.5%) and specificity (98.0% vs. 86.0%) than HC2 HPV testing. We show for the first time that PAX1 hypermethylation analysis may be a better choice than HC2 in the triage of ASCUS and ASC-H. Diagn. Cytopathol. 2013;41: ' 2011 Wiley Periodicals, Inc. Key Words: cervix; atypical squamous cells of undetermined significance (ASCUS); ASC-H; epigenetics; methylation Screening by the cytological test developed by George Papanicolaou has led to a remarkable reduction in the incidence and mortality of cervical cancer over the past 40 years. 1 The 2001 Bethesda System (TBS) classifies as atypical squamous cells (ASC) smears those with cellular abnormalities, suggesting the possible presence of underlying cervical intraepithelial neoplasia (CIN) without clear-cut obvious cytological abnormalities. In the case of a ASC smear, the pathologist has to specify either ASCUS (of undetermined significance) or ASC-H (cannot exclude a high-grade lesion). 2 ASCUS and ASC-H smears need further investigation to identify patients likely to have or to develop a high-grade squamous intraepithelial lesion (HSIL). Because a small percentage of HSIL may be hidden within a large number of equivocal abnormal smears, substantial expense and effort are required to identify these HSIL. Infection with high-risk types of human papillomavirus (HR-HPV) is the most significant risk factor in the etiology of cervical cancer, and is present in nearly all cervical cancers. 3 In consequence, as recommended by TBS, Hybrid Capture 2 (HC2) HPV testing has been widely adopted for the triage of patients with ASCUS. 4 For ASC-H smears, the recommendation is colposcopy, with punch biopsy specimens taken from the areas colposcopically suspicious for CIN. The sensitivity of HPV testing is good but the ' 2011 WILEY PERIODICALS, INC. Diagnostic Cytopathology, Vol 41, No 1 41

2 CHAO ET AL. high prevalence of transient HPV infections limits the specificity of this approach. 5 Thus, there is a need for other markers to identify women with ASC cytology harboring an underlying HSIL. Recently, epigenetic alterations, such as DNA methylation, have been recognized as important driving forces for cancer. 6 Epigenetic silencing of tumor suppressor genes by promoter hypermethylation is commonly seen in many human cancers. 7 Our previous work using differential methylation hybridization identified paired boxed gene 1 (PAX1) as a novel methylation-silenced gene in cervical 8 and ovarian cancer. 9 The PAX1 gene belongs to a highly conserved family of developmentally controlled genes that encode transcription factors and play a role in pattern formation during embryogenesis in vertebrates. 10 Specifically, PAX1 is expressed during the development of the skeleton, thymus, and parathyroid glands. 11,12 Although the functional role of PAX1 in cancer biology remains unknown, our recent work demonstrated that quantitative methylation analysis of PAX1 is a potential biomarker for the detection of CIN grade 3 or higher (CIN3+). 8,13,14 In this study, we examined the performance of PAX1 gene methylation testing for the triage of ASCUS and ASC-H cervical scrapings, and compared the results with those of HPV DNA testing. Methods Patient Samples A series of 58 ASCUS (n ¼ 41) and ASC-H (n ¼ 17) cytology specimens were retrieved from the tissue bank of the National Defense Medical Center, Taipei, Taiwan, as described previously. 15 All patients were treated and followed by the standard clinical protocol. Patients with ASCUS underwent a repeat Papanicolaou (Pap) smear within 3 6 months. Patients with repeated abnormal Pap smears and all patients with ASC-H at the first visit underwent colposcopy-directed cervical biopsy and subsequent therapy when indicated. The final diagnoses used in the analysis were determined by the histopathology. Patients with ASCUS in resolution as documented by two consecutive Pap smears were classified as reactive atypia without further biopsy. Cervical scrapings collected at the first visit were used for the analysis. Cytology and Histology Cervical scrapings were collected for cytological diagnosis by gynecologists. Slides were prepared and stained by the Pap method according to the usual laboratory protocol. 16 The screening of cytology slides was first performed by the pool of cytotechnologists, and a pathologist always confirmed abnormal results. Cytology was performed using TBS For all cases in the study with initial ASC-H and ASCUS and a repeat abnormal Pap smear, biopsies were performed and paraffin-embedded. Biopsies were cut and stained with hematoxylin and eosin (H&E), read by a pathologist, and subsequently confirmed by a second independent reading. HPV Test Infection with HR-HPV was detected by the HC2 test (Digene, Silver Spring, MD) according to the manufacturer s protocol. In brief, the DNA in a specimen was denatured and hybridized with a cocktail of RNA probes directed against a panel of 13 HR-HPVs. The RNA-DNA hybrids were then captured by hybrid-specific antibodies and detected by alkaline phosphatase-linked second antibody and chemiluminescence. Samples with a RUL ratio higher than 1.0 were called positive. Bisulfite Conversion and Quantitative Methylation-Specific PCR (QMSP) Assay DNA was extracted from cervical scrapings and bisulfite converted using the modification kit (Chemicon International, Temeculla, CA) according to the manufacturer s recommendations. TaqMan-based QMSP (MethyLight) was performed on denatured genomic DNA after bisulfite treatment. 14,17 The primers and probe sequences will be provided on request. ThetypeIIcollagengene(COL2A)wasusedasaninternal reference gene by amplifying non-cpg sequences. Each sample was analyzed in duplicate. Genomic DNA methylated in vitro with M.Sss I methyltransferase (New England Biolabs, Beverly, MA) was used as a positive control that was considered to give 100% methylation of each gene. QMSP was done in a total volume of 20 ll containing 2 ll of modified template DNA, 1 ll of203 Custom TaqMan reagent, and 10 ll Universal PCR Master Mix (No AmpErase UNG, # , Applied Biosystems, Foster City, CA). The reactions were subjected to an initial incubation at 958C for 10 minutes, followed by 50 cycles of 958C for 15 s, annealing for 1 minute at 608C, and extension at the appropriate temperature, and then detected using the Applied Biosystems 7500 Real-time PCR System. DNA methylation level was assessed as the percentage of methylation references (PMR) using the formula: [sample_gene/sample_col2a] 3 100/ [SssI_gene/SssI_COL2A]. 17,18 Testing results with Ct-values for COL2A greater than 40 were defined as detection failures. Representative positive and negative QMSP results were shown in Figure 1. Statistical Analysis GraphPad Prism (version 5.0a) software was used for statistical analyses and data plotting. Where indicated, the PMR values obtained by QMSP were transformed as a function of ln(pmr + 1) to fulfill the assumption of normality and plotted according to clinical status to obtain the cut-off point that could best discriminate disease versus nondisease. Fisher s exact test and Chi-square test for 42 Diagnostic Cytopathology, Vol 41, No 1

3 PAX1 METHYLATION DETECTION IN ASCUS FOR HIGH GRADE LESIONS Fig. 1. Representative positive and negative PAX1 detection by real time PCR. The threshold cycle of PAX1 gene is shown in the upper panel. The control COL2A gene is shown at the lower panel. DCt means the threshold cycle difference between PAX1 and COL2A. The smaller DCt indicates the higher PMR in a testing sample. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Fig. 2. Pathological diagnosis in patient with ASCUS or ASC-H. Patients with ASCUS (A) or ASC-H (C) may be diagnosed as reactive atypia (squamous metaplasia) (B) or squamous cell carcinoma in situ (D). Magnification [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Diagnostic Cytopathology, Vol 41, No 1 43

4 CHAO ET AL. Table I. PAX1 Methylation and HPV Testing in ASC ASCUS ASC-H ASC Age (Mean 6 SD) a Patient No Reactive atypia HSIL PMR (Mean 6 SD) b Reactive atypia c d HSIL Methylation positivity 2.4% (1/41) 41.1% (7/17) 13.8% (8/58) Reactive atypia 0% (0/40) 10% (1/10) 2% (1/50) HSIL 100% (1/1) 85.7% (6/7) 87.5% (7/8) HR-HPV positivity 7.3% (3/41) e 52.9% (9/17) 20.7% (12/58) Reactive atypia 5% (2/40) f 50% (5/10) g 14% (7/50) h HSIL 100% (1/1) 57.1% (4/7) 62.5% (5/8) a P ¼ P ¼ P ¼ P < e P < 0.001, comparing ASC-US and ASC-H. f P ¼ g P ¼ 1. h P ¼ 0.007, comparing reactive atypia and HSIL. trend were used to analyze the status of PAX1 methylation or HPV infection in different groups. Results Methylation Status of PAX1 in ASC The spectrum of pathological diagnosis in patients with cytological ASCUS and ASC-H are exampled in Figure 2. Of the 41 patients with ASCUS smears, only one (2.4%) displayed HSIL (CIN2) in a two-year follow-up. Seven of the 17 patients with ASC-H (41.2%) demonstrated HSIL (CIN3) (Table I). TheageandPMRofthe58patientswithASCarelistedin Table I and Figure 3. The mean age is significantly higher in ASC-H patients (P ¼ 0.002). In contrast to only one in 41 (2.4%) ASCUS patients diagnosed as HSIL, seven of the 17 (41.2%) ASC-H patients were diagnosed as HSIL. The PMR of ASC-H ( ) was significantly higher than that of ASCUS ( ) (P ¼ 0.001). The PMRs of ASCUS or ASC-H developing HSIL were 74.9 and The PMRs of ASCUS or ASC-H resulting in reactive atypia were and , respectively. Collectively, the PMRs were significantly higher in ASC developing HSIL than in those with reactive atypia ( vs , P < 0.001) (Table I). At a PMR cutoff value of 73, the methylation rates in ASC-H and ASCUS are 41.1 and 2.4%, respectively (P < 0.001). The methylation rates are significantly higher in ASC developing HSIL compared with those with reactive atypia (87.5% vs. 12.5%, P < 0.001). PAX1 Methylation Test and HC2 HPV Test in the Detection of HSIL in Patients With ASC HR-HPV could be detected in 62.5% of patients with ASC developing HSIL, significantly higher than the detection Fig. 3. The PMRs in cervical swabs of ASCUS and ASC-H diagnosed subsequently with reactive atypia or HSIL. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Table II. Performance of PAX1 QMSP and HC2 Tests in Detection of HSIL Methods Sensitivity Specificity PPV NPV Accuracy PAX1 QMSP 87.5% 98.0% 87.5% 98.0% 96.6% HPV test 62.5% 86.0% 41.7% 93.5% 82.8% PPV, positive predictive value; NPV, negative predictive value. rate in those with reactive atypia (14%, P ¼ 0.007, Table I). We compared the performance of QMSP for PAX1 methylation testing to the HC2 HPV test in the detection of HSIL (Table II). The sensitivity and specificity of PAX1 methylation testing were 87.5 and 98%, compared with 62.5 and 86%, respectively, for detection by HPV testing. The positive predictive value (PPV), negative predictive value (NPV), and accuracy of PAX1 methylation testing are all superior to those of the HC2 HPV test. Discussion The Pap smear has become the most successful cancer screening method in the world and has reduced cervical cancer death by 74% since its widespread adoption in the 1950s in the U.S. 19 The TBS for reporting cervical cytological diagnoses was introduced in 1988 and revised in In TBS 2001, ASC was divided into two subcategories: ASCUS and ASC-H. ASC-H is the less common qualifier, accounting for 5 to 10% of all ASC cases, but the risk of an underlying high-grade lesion is higher in this category than in ASCUS. Diagnosis of ASC requires nuclear shape, size, and chromatin abnormalities, 2 but 44 Diagnostic Cytopathology, Vol 41, No 1

5 PAX1 METHYLATION DETECTION IN ASCUS FOR HIGH GRADE LESIONS ASC is still a problematic category because of poor interobserver reproducibility. 21,22 Indeed, the diagnosis of ASC has been a gray area and is the most difficult of all cervical smear diagnoses for pathologists to reproduce. Today, TBS classification guidelines recommend different follow-up for women with ASCUS and ASC-H smears. According to the annual report of the cervical cancer screening program in Taiwan in 2007 that included more than 2 million women, 1.6% of all smears were reported to be ASCUS and 0.1% were ASC-H, meaning that these classifications were more common than all types of abnormal smears (1.4%) including low-grade squamous epithelial lesions (LSIL), HSIL, and cancer. Ten percent of women with ASCUS were found to have HSILs, whilst 30% of women with ASC-H were reported to do so ( ISSN; ). HR-HPV testing is the preferred method for the triage of ASCUS/ASC-H. 4 Adding a high-risk HPV test in secondary screening increased the identification of women with CIN2-3 lesions by 33% in comparison with repeat cytology. 23 Patients with positive HR-HPV should undergo immediate colposcopic examination, whereas negative HR-HPV is a predictor of the absence of HSIL. 24 Despite the reported high sensitivity (86%) and NPV (82%) of HR-HPV testing, 25 some HSIL can still be missed. 4,26,27 The low specificity (31%) and PPV (37%) even make the situation worse because they lead to more patients undergoing unnecessary referrals. 25 P16 detection is an alternative. The qualitative assessment of P16 detected by immunohistochemistry (IHC) has been used to detect HSIL in ASC. 28 However, the sensitivities and specificities of p16 IHC and HC2 were not significantly different. 29 This study demonstrates that PAX1 hypermethylation is better than HC2 HR-HPV testing in the triage of ASC. The use of DNA methylation as a molecular marker for cervical cancer screening is being investigated. Recently, we reported PAX1 as a potential biomarker for the detection of CIN3+. 8,13,14 In a recent triage of ASCUS Pap smear results, WT1 and PCDH10 methylation detection is better than HPV test. 30 Here, we further expand the clinical application of PAX1 gene hypermethylation in the triage of ASCUS and ASC-H. To the best of our knowledge, this is the first report with promising results addressing the application of quantitative DNA methylation in the triage of ASC patients. The cost-effectiveness of using methylation biomarker as a screening or a triage of these mildly abnormal Pap smears remains to be investigated in a larger community-based study. As the PCRbased testing evolutes rapidly, the cost-down for a widely used could be achieved in the future. The role of PAX1 in cancer is still unclear. The PAX gene family has been classified into four groups according to the gene structure. 31 Group II and III with a homeodomain and an octapeptide are thought to be tumor promoting Other groups are much less often linked with cancer. PAX1 is categorized in group I without the homeodomain and is implicated as a tumor suppressor according to our studies. 8,13,14 The functional study of PAX1 in cervical cancer is ongoing and might in the future shed new light on our understanding of cervical carcinogenesis. In conclusion, the PAX1 methylation testing of cervical ASC smears may have a better performance than HPV testing in the detection of HSIL. It could help reduce the number of patients with ASC who require colposcopy and further clinical workup without compromising the detection rate of HSIL. Because it is limited by the sample size, this study may not be appropriate to be applied directly to the whole population. However, our results do provide a basis for an ongoing multicenter trial comparing the performance of a standardized PAX1 methylation analysis assay and HR-HPV testing in the triage of ASC in Taiwan, and may provide a new molecular method of triage for these ambiguous smears. References 1. Cannistra SA, Niloff JM. Cancer of the uterine cervix. N Engl J Med 1996;334: Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: Terminology for reporting results of cervical cytology. JAMA 2002;287: Parkin DM. The global health burden of infection-associated cancers in the year Int J Cancer 2006;118: Arbyn M, Buntinx F, Van Ranst M, Paraskevaidis E, Martin-Hirsch P, Dillner J. Virologic versus cytologic triage of women with equivocal Pap smears: A meta-analysis of the accuracy to detect highgrade intraepithelial neoplasia. J Natl Cancer Inst 2004;96: Dehn D, Torkko KC, Shroyer KR. Human papillomavirus testing and molecular markers of cervical dysplasia and carcinoma. 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