PAX1 methylation as a potential biomarker for cervical cancer screening.

ORIGINAL STUDY

PAX1 Methylation as a Potential Biomarker for Cervical Cancer Screening Yuen-Yee Kan, MD,* Yu-Ligh Liou, MS,Þ Huei-Jen Wang, PhD,Þ Chiao-Ying Chen, BS,* Li-Chi Sung, BS,* Chi-Feng Chang, PhD,Þ and Cheng-I Liao, MDþ

Objectives: DNA methylation is a potential biomarker for early cancer detection. Previous studies suggested that the methylations of several genes are promising markers for the detection of cervical intraepithelial neoplasia at grade III or worse (CIN3+). The purpose of the present study was to explore the feasibility of these DNA methylation testing in cervical cancer screening. Methods: A total of 443 women were recruited from the Yuan’s General Hospital. Cervical scrapings were collected for Papanicolaou (Pap) test by using cervical brushes, and the cytological data were used for analysis. The residual cells on the brush were preserved in phosphate-buffered saline solution at 4-C until DNA extraction. Then, the extracted DNA were used for molecular tests, which included human papillomavirus typing and quantification of the methylation levels for PAX1, SOX1, and NKX6-1 genes. Subjects who had abnormal Pap test results underwent colposcopy or biopsy with subsequent conization or major surgery when biopsy results revealed CIN2+. The final diagnosis for this group was confirmed by colposcopy or pathological examination. The study was approved by the institutional review board of Yuan’s General Hospital, and all the molecular tests were performed by ISO17025 certified laboratories. Results: The sensitivity of PAX1m and SOX1m was greater than 80%, and the specificity of PAX1m and NXK6-1m was greater than 80% for the detection of CIN3+ lesions. PAX1m detection alone had a sensitivity and specificity of 86% and 85%, respectively, whereas when used as a cotest with the Pap test, the sensitivity and specificity were 89% and 83%, respectively. Conclusions: PAX1m showed great potential as a biomarker for cervical cancer screening. When incorporating PAX1m detection into current screening protocol, the efficacy of screening could be greatly improved. Moreover, unnecessary referral for colposcopy and biopsy could be reduced up to 60%. However, prospective population-based studies are necessary for further implementation of this screening program. Key Words: Papanicolaou test (Pap), Human papillomavirus (HPV), Paired box gene 1 (PAX1), Sex determining region Y-box 1 (SOX1), NK6 transcription factor-related locus 1 (NKX6-1) Received October 08, 2013, and in revised form March 23, 2014. Accepted for publication March 23, 2014. (Int J Gynecol Cancer 2014;24: 928Y934) *Department of Obstetrics and Gynecology, Yuan’s General Hospital, Kaohsiung; †iStat Biomedical Co, Ltd, Taipei; and ‡Department of Obstetrics and Gynecology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan. Address correspondence and reprint requests to Yuen-Yee Kan, MD, Department of Obstetrics and Gynecology, Yuan’s General Copyright * 2014 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000155

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Hospital, Kaohsiung, Taiwan, No.162 Cheng Kung 1st Rd, Kaohsiung 80249, Taiwan. E-mail: [email protected]; Cheng-I Liao, MD, Department of Obstetrics and Gynecology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, 386 Ta-Chung 1st Rd, Kaohsiung, Taiwan 81346. E-mail: [email protected]. This research was funded by iStat Biomedical Co, Ltd, Taipei, Taiwan. The authors declare no conflicts of interest. Y.-Y.K. and C.-I.L. contributed equally to this work.

International Journal of Gynecological Cancer

& Volume 24, Number 5, June 2014

Copyright © 2014 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.



cancer is the second most common cancer among C ervical women worldwide and a major cause of death in devel-

oping countries.1 Approximately 500,000 new cases of cervical cancer and 20,000 related deaths were reported in 2010. Since the introduction of Papanicolaou (Pap) test decades ago, the mortality and morbidity for patients with invasive cervical cancer have reduced greatly, especially in developed countries. Thus, the contribution of the Pap test to public health is evident.2Y4 The Pap test can identify cervical cancer or its precursor lesions via the microscopic inspection of exfoliated cervical cells. However, the effectiveness of cytological screening varies according to the performance of health care infrastructures. Consequently, the sensitivity of the Pap test ranged from approximately 50% to 80% but could be as low as 20%,5Y8 which limits the efficacy of cancer detection. Moreover, many developing countries do not have the infrastructure for Pap screening. Because persistent infection of oncogenic human papillomavirus (HPV) causes cervical cancer,9Y11 HPV DNA testing has become appealing. Despite the higher sensitivity of HPV DNA testing compared with the Pap test, less than 1% of women infected with HPV progress to cervical cancer. Human papillomavirus testing may lead to unnecessary referrals for colposcopy and unwarranted concern, which decrease its value in cervical cancer screening.12 Therefore, the identification of novel biomarkers for cervical cancer screening that can be used alone or in combination with current cytology or virus-based methods is important. Previous epigenetic studies demonstrated that DNA methylation could be a signature event of carcinogenesis.13,14 For example, septin 9 methylation is used as a biomarker for the detection of colorectal cancer, and SHOX2 methylation levels in bronchial fluid indicate the presence of malignant

Methylation Markers for Cervical Cancer

lung disease, thus facilitating a fast and reliable diagnosis.15,16 Several DNA methylation biomarkers have been identified, which possess the clinical potential to facilitate the early diagnosis of cervical cancer.17Y20 For cervical cancer, the methylation of paired box gene 1 (PAX1),21 sex determining region Y-box 1 (SOX1), and NK6 transcription factor-related locus 1(NKX6-1) were found to be promising markers for the detection of cervical intraepithelial neoplasia (CIN) lesions at grade III or worse (CIN3+).22,23 To validate the clinical feasibility of these methylated genes, a cross-sectional prevalidation study was conducted. The results of standardized methylation quantitative assays and HPV typing were compared.24

MATERIALS AND METHODS Study Participants and Specimen Collection The cross-sectional validation studies were conducted following Good Clinical Practice guidelines. The institutional review board of Yuan’s General Hospital approved the clinical protocols. The inclusion criteria were female patients older than 20 years with a history of sexual activity. Patients with a history of reproductive tractYrelated cancer, those who had received treatment for cervical lesions or HPV vaccination, and those with confirmed pregnancy were excluded from the study. All participating investigators are board-certified gynecologic oncologists. As indicated in Figure 1, the study included patients with a normal uterine cervix (n = 247) and patients with abnormal Pap test results (n = 172) of different stages. After signing the informed consent, all the participants were examined and treated according to the approved protocols.

FIGURE 1. Procedures used in the study. * 2014 IGCS and ESGO


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Patient enrollment, diagnosis, treatment, and tissue banking were all performed at Yuan’s General Hospital. Patients who had low- and high-grade lesions, as determined by cytology, underwent colposcopic cervical biopsy. When the biopsy results revealed grade 2 or worse CIN, patients underwent conization or major surgery. In the abnormal Pap test result group, the final diagnosis was made by tissue-proven pathology, and the patient distribution was as follows: CIN1, n = 76; CIN2, n = 7; CIN3/carcinoma in situ (CIS), n = 32; and squamous cell carcinoma (SCC), n = 4. The clinical, histological, and pathological characteristics of the patients are summarized in Table 1.

DNA Preparation, HPV Tests, and DNA Methylation All molecular tests were performed in laboratories certified by ISO 17025 and the Taiwan Accreditation Foundation. Cervical scrapings for Pap test were collected by using a cervical brush during routine examination or before the biopsy. The residual cells on the brush were preserved in a TABLE 1. Characteristic of subjects Characteristics

n*

Age (n = 419) Mean (SD) [range] 48.4 (12.0) [21.8Y90.9] G30 21 30Y50 215 Q50 183 Cytology results (n = 419) HSIL/SCC/CIS 31 LSIL 38 AGC 19 ASCUS 84 Normal 247 Pathology results (n = 419) SCC/AC 4 CIN3/CIS 32 CIN2 7 CIN1 76 Normal 300 HPV (n = 419)† Positive 87 Negative 332

Percentage

5.0 51.3 43.7

7.4 9.1 4.5 20.1 59.0

1.0 7.6 1.7 18.1 71.6 20.8 79.2

*A total of 443 subjects were initially recruited; 24 were excluded, 6 had poor specimen quality, and 18 did not fit the exclusion criteria. †Subjects with high-risk oncogenic types were designated as HPVpositive (types 16, 18, 31, 33, 35, 38, 45, 51, 52, 56, 58, 59, and 68). AC, adenocarcinoma; HSIL, high-grade squamous intraepithelial lesion.

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FIGURE 2. Validation of the cut-off value for the methylated genes.

phosphate-buffered saline solution at 4-C for subsequent use in DNA methylation and HPV tests. Genomic DNA was extracted from the collected cells with a QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. A NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Newark, Del) was used to quantify the amount of extracted DNA. Seminested polymerase chain reaction (PCR) and reverse hybridization were used for HPV typing.24 Quantitative methylation-specific PCR was used for DNA methylation tests. Briefly, 500-ng genomic DNA was subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kits (Zymo Research, Irvine, Calif ), according to the manufacturer’s recommendations. The methylation levels of SOX1, PAX1, and NKX6-1 were then determined using the Q-PCR kits developed by iStat Biomedical Co, Ltd, and the Type II Collagen gene (COL2A) was used as the internal reference by amplifying non-CpG sequences (iStat Biomedical Co, Ltd, New Taipei City, Taiwan). The registered Caski and C33A cancer cell lines were used as the methylation controls, and the methylation levels of the 3 genes in the cancer cell lines, expressed as percentages, were reconfirmed by pyrosequencing analysis. PCR reactions consisted of an initial incubation at 95-C for 10 minutes, followed by 50 cycles of denaturation at 95-C for 10 seconds, annealing and extension at 60-C for 40 seconds. Two crossing point (Cp) values were obtained, one from the target gene and one from COL2A. The DNA methylation level was subsequently determined from the difference of the 2 Cp values ($Cp = Cptarget gene j CpCOL2A).

Statistical Analysis

Categorical variables were compared using the W2 test, and 2-sample t tests were used for comparison of continuous variables between the case and control groups. The cutoff values for each gene were the data generated from the first 100 subjects, including 50 subjects with normal Pap test results and 50 subjects with abnormal Pap test results. Receiver operating characteristic (ROC) curves were generated, and the area under the ROC curve (AUC) of each genes was calculated for the detection of the CIN3+ lesions (CIN3+). The * 2014 IGCS and ESGO




TABLE 2. AUC for PAX1m, SOX1m, and NKX6-1m for the diagnosis of CIN3+ Genes

Sensitivity

Specificity

AUC

92% 92% 54%

83% 30% 82%

0.97 0.76 0.73

PAX1m SOX1m NKX6-1m

accuracy are comparable with those generated by the Taiwanese Gynecologic Oncology Group (TGOG) studies.25 For the l analysis, sensitivity and specificity with a 95% confidence interval (CI) for grade CIN3+ lesions were obtained using different combinations of testing methods. SAS software (version 9.2; SAS Institute, Ltd, Cary, NC) was used for all statistical analyses. All differences were considered 2 sided and statistically significant at P G 0.05.

RESULTS Enrollment and Characteristics of Subjects Between June 2011 and November 2012, 443 women were enrolled from the Yuan’s General Hospital. Eighteen patients were excluded based on the exclusion criteria, and 6 were excluded because of poor-quality DNA specimens. Thus, data from 419 patients were included in the final statistical analysis. The demographic characteristics and basic clinical information of these patients are shown in Table 1.

Methylation of PAX1, SOX1, and NKX6-1 The cutoff values for the methylation of PAX1, SOX1, and NKX6-1 were based on the data generated from the first 100 subjects, as shown in Figure 2. The current study targeted the detection of CIN3+ lesions. The AUCs of the ROC plots


for the methylated genes PAX1m, SOX1m, and NKX6-1m were 0.97, 0.76, and 0.73, respectively. The sensitivity and specificity at the cutoff values are indicated in Table 2. The distribution of the methylated genes according to the different levels of disease severity is shown in Figure 3 and Table 3. The sensitivities for PAX1m, SOX1m, and NKX6-1m were 86%, 89%, and 58% and the specificities were 85%, 39 % and 79%, respectively. The overall accuracy values for PAX1m, SOX1m, and NKX6-1m were 84%, 41%, and 78%, respectively. However, when the methylation test targeted the detection of CIN2 or more severe neoplasms, the sensitivity values for PAX1m, SOX1m, and NKX6-1m decreased to 77.4%, 86%, and 48.8% and the specificity to 84.6%, 39.6%, and 79.0%, respectively (Table 4).

Triage of Women With Minor Cervical Lesions In 141 women, cervical cytology results corresponded to minor cervical lesions including atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), and atypical glandular cells (AGCs). After confirming by colposcopy, only 15 women had CIN3 or worse lesions. Triage with molecular tests revealed that there were 18% PAX1mYpositive and 38% high-risk (HR) HPVYpositive patients. The sensitivities of PAX1m and HRHPV testing were 73% and 87% and the specificities were 88% and 67%, respectively (Table 5).

DISCUSSION In 2012, the American Cancer Society proposed new screening guidelines that included age-adjusted screening methodology as well as the use of cytological and HPV cotesting. HPV 16 and 18 typing is recommended for HRHPVYpositive women. However, geographic variation in the distribution of HPV genotypes has been reported. In Asia, in addition to HPV types 16 and 18, types 52 and 58 are frequently

TABLE 3. Positive rate of PAX1, SOX1, and NKX6-1 methylation at various disease stages Pathology for the Subjects with Abnormal Pap Test Results (n = 172)

Normal Pap Test Result (n = 247)

Biopsy Target Gene m

PAX1 (+)

42 (17%) 164 (66%) 47 (19%) 247

SOX1m(+) NKX6-1m(+) Total no. subjects

Normal

CIN1

CIN2

CIN3/CIS

SCC/AC

6 (11%) 24 (45%) 14 (26%) 53

10 (13%) 39 (51%) 18 (24%) 76

1 (14%) 5 (71%) 0 (0%) 7

27 (84%) 28 (88%) 20 (63%) 32

4 (100%) 4 (100%) 1 (25%) 4

AC, adenocarcinoma.

* 2014 IGCS and ESGO


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TABLE 4. Sensitivity and specificity for the methylated genes analyzed

HPV-HR PAX1m SOX1m NKX6-1m PAX1m or PAP§ PAX1m or (HPV 16 or 18) PAX1m or (HPV16, 18, 52, or 58) SOX1m or PAP SOX1m or (HPV 16 or 18) SOX1m or (HPV 16, 18, 52, or 58) NKX6-1m or PAP NKX6-1m or (HPV 16 or 18) NKX6-1m or (HPV 16, 18, 52, or 58)

Sensitivity, %

Specificity, %

P*

78 86 89 58 89 89 94 92 92 97 78 72 86

85 85 39 79 83 83 78 38 38 36 77 78 73

G0.0001 G0.0001 0.0008 G0.0001 G0.0001 G0.0001 G0.0001 0.0003 0.0004 0.0002 G0.0001 G0.0001 G0.0001

OR (95% CI)† 38.5 32.6 4.8 5.5 40.0 40.0 65.3 6.6 6.4 9.4 13.2 9.8 19.3

OR (95% CI)‡

(15.9Y93.4) (12.0Y87.9) (1.6Y13.8) (2.7Y11.3) (13.5Y118.1) (13.5Y118.1) (15.3Y279.1) (2.0Y21.9) (1.9Y21.3) (2.2Y40.0) (5.7Y30.3) (4.5Y21.3) (7.2Y51.4)

97.2 46.9 5.4 1.3 44.6 44.6 34.3 5.4 5.2 5.0 3.8 1.3 3.1

(5.1Y1857.4) (2.5Y884.4) (0.3Y100.7) (0.1Y12.8) (2.4Y842.2) (2.4Y842.2) (1.8Y646.4) (0.3Y100.7) (0.3Y97.9) (0.3Y93.8) (0.5Y27.2) (0.1Y12.3) (0.4Y22.5)

*Based on W2 test. †Odds ratio of CIN3+. ‡Odds ratio of SCC/AC. §The results of PAP smear are HSIL/or SCC/or CIS. AC, adenocarcinoma; HSIL, high-grade squamous intraepithelial lesion.

observed in patients with invasive cervical cancers. A similar trend was observed in the present study. In CIN3 or more severe lesions (n = 36), HPV 16 and HPV 58 (25%) were the most common types, followed by HPV 52 (19%), HPV 33 (16%), and HPV 18 (2%). Therefore, the combination of Pap test and HPV 16/18 typing may not be sufficient for cervical cancer screening in Asia. DNA methylation has been identified as a potential biomarker for cancer detection. However, accurate cancer screening is challenging because of the complex etiology of the disease, which could lead to heterogeneous cancer types or uneven methylation at different CpG islands even in the same gene. In clinical cervical cancer screening, the assessment of DNA methylation in cervical scrapings rather than in

tissues may be more effective. In the present study, PAX1m showed the greatest clinical potential as a screening biomarker among the 3 methylated genes. PAX1m alone showed sensitivity and specificity values greater than 80% for the detection of CIN3 or worse lesions. We also observed low PAX1m in the CIN2 group (approximately 16%). It is known that CIN2 lesion is an equivocal diagnosis of which most were likely to regress and only approximately 16% has a risk of progression to severe dysplasia or worse.26 The PAX1m might be associated with the carcinogenesis of the cervix dysplasia. Despite a reduction in the sensitivity of PAX1m to 77.4% for the detection of CIN2 or worse lesions, this methylated gene test may still be an effective tool for cervical cancer screening. In countries with good cytology infrastructures, the addition

TABLE 5. Performance of PAX1m and HPV tests in triage for the abnormal Pap test result group Pathology

Triage Test

Normal (n = 53)

CIN1 (n = 76)

CIN2 (n = 7)

CIN3/CIS (n = 32)

SCC/AC (n = 4)

Total (n = 172)

HSIL or above

PAX1m HPV-HR HPV 16/18 Total

0 (0%) 1 (50%) 0 (0%) 2

1 (33%) 2 (67%) 0 (0%) 3

1 (20%) 4 (80%) 1 (20%) 5

18 (95%) 13 (68%) 4 (21%) 19

2 (100%) 2 (100%) 1 (50%) 2

22 (71%) 22 (71%) 6 (19%) 31

Minor cervical lesions: LSIL, ASCUS, and AGC

PAX1m HPV-HR HPV 16/18 Total

6 (12%) 13 (25%) 4 (8%) 51

9 (12%) 27 (37%) 6 (8%) 73

0 (0%) 1 (50%) 0 (0%) 2

9 (69%) 11 (85%) 4 (31%) 13

2 (100%) 2 (100%) 0 (0%) 2

26 (18%) 54 (38%) 14 (10%) 141

Abnormal Pap Test Result Group

AC, adenocarcinoma; HSIL, high-grade squamous intraepithelial lesion.

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FIGURE 3. Methylation distribution at various disease stages. The y-axis is the $Cp, which represents the DNA methylation level of the target gene ($Cp = Cptarget gene j CpCOL2A). The dashed line represents the cutoff with a $Cp value of 11. of PAX1m detection may improve the sensitivity of cytology alone without compromising its specificity. Moreover, for minor cervical lesions or in cases of controversial Pap results such as ASCUS, LSIL, and AGC, the sensitivity and specificity of PAX1m were 73% and 87%, respectively. Compared with HR-HPV tests, the use of PAX1m testing as part of the triage protocol could reduce 60% of referrals for colposcopy/ biopsy (from 33% to 13%). This combined triage approach would be more suitable in developed countries, where the Pap screening infrastructure for cancer is well established. In countries lacking a satisfactory cytology-based screening infrastructure, visual inspection of cervix after applying acetic acid (VIA) was the most common screening strategy. Moreover, VIA combined with immediate treatment was thought to be the most cost-effective approach.27 However, the sensitivity and specificity of VIA are only approximately 65%, which might result in overtreatment. To pursue a better quality of screening method, the molecular test would be a more reasonable choice because of the possibility of selfsampling and a central laboratory operating system. HPV tests are appealing because of the clear etiological role of HPV in cervical cancer. However, the HPV test, when used alone, has low specificity and is not recommended for screening purposes. The high sensitivity and specificity of PAX1m testing could make it the preferred choice for molecular screening. Moreover, when combined with HPV 16/18 typing, PAX1m testing showed a sensitivity of almost 90% and a specificity of 83%. Analytically, traditional procedures for methylation analysis are tedious and labor intensive. High-level laboratory skills are crucial to obtain reproducible results. The quantitative measurement of gene methylation by Taqman QPCR allows a highly specific recognition of methylated sequences. In the present study, methylated CpG sites were recognized using specific primers and Taqman probes. Furthermore, the detection of fluorescence-labeled amplicons allows the sensitive and real-time measurement of the amplicons, thus enabling accurate quantitation. In the present study, we used standardized and simplified quantitative methylation-specific PCR

kits to test for the presence of PAX1m, SOX1m, and NKX6-1m in an independent clinical cohort. In conclusion, this prospective case control study showed the potential of the methylation of a single gene, PAX1m, as a biomarker for clinical application in cervical cancer screening. The combination of PAX1m testing with cytology or HPV typing may significantly improve the efficacy of cervical cancer screening. However, prospective population-based studies are necessary for further implementation of this screening protocol.

ACKNOWLEDGMENTS Drs Pi-Sen Yu, Tsung-Fu Chen, Young-Chang Lee, and Chin-Ming Wu, and Ms Shu-Min Lin assisted in the patient recruitment for the study. Ms Yu-Hsin Chen assisted in the data analysis.

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18. Wang KL, Chang TC, Jung SM, et al. Primary treatment and prognostic factors of small cell neuroendocrine carcinoma of the uterine cervix: a Taiwanese Gynecologic Oncology Group study. Eur J Cancer. 2012;48:1484Y1494. 19. Chung MT, Lai HC, Sytwu HK, et al. SFRP1 and SFRP2 suppress the transformation and invasion abilities of cervical cancer cells through Wnt signal pathway. Gynecol Oncol. 2009;112:646Y653. 20. Lai HC, Lin YW, Chang CC, et al. Hypermethylation of two consecutive tumor suppressor genes, BLU and RASSF1A, located at 3p21.3 in cervical neoplasias. Gynecol Oncol. 2007;104:629Y635. 21. Chao TK, Ke FY, Liao YP, et al. Triage of cervical cytological diagnoses of atypical squamous cells by DNA methylation of paired boxed gene 1 (PAX1). Diagn Cytopathol. 2013;41:41Y46. 22. Lai HC, Lin YW, Huang RL, et al. Quantitative DNA methylation analysis detects cervical intraepithelial neoplasms type 3 and worse. Cancer. 2010;116:4266Y4274. 23. Lai HC, Lin YW, Huang TH, et al. Identification of novel DNA methylation markers in cervical cancer. Int J Cancer. 2008;123:161Y7. 24. Lin H, Moh JS, Ou YC, et al. A simple method for the detection and genotyping of high-risk human papillomavirus using seminested polymerase chain reaction and reverse hybridization. Gynecol Oncol. 2005;96:84Y91. 25. Lai HC, Ou YC, Chen TC, et al. PAX1/SOX1 DNA methylation and cervical neoplasia detection: a Taiwanese Gynecologic Oncology Group (TGOG) study. Cancer Medicine. 2014 (accepted). 26. Holowaty P, Miller AB, Rohan T, et al. Natural history of dysplasia of the uterine cervix. J Natl Cancer Inst. 1999;91:252Y258. 27. Mandelblatt JS, Lawrence WF, Gaffikin L, et al. Costs and benefits of different strategies to screen for cervical cancer in less-developed countries. J Natl Cancer Inst. 2002;94:1469Y1483.



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