JCT  Vol.4 No.5 A , May 2013
Promoter Methylation, BRAF Mutation Analysis and Topoisomerase IIa Expression for the Detection of Endometrial Carcinoma in Liquid Based Cytology Samples
Abstract: Cancer of the corpus uteri remains the most common gynecological related cancer in developed countries. Cytology, after the induction of liquid based cytology, has reemerged as a possible first line non-interventional diagnostic procedure with promising results. Apart from slide preparation for cytology diagnosis, LBC allows the application of elaborate molecular tests on the residual material. Samples from 74 symptomatic women were collected in ThinPrep PreservCyt medium, from witch immunocytochemical and molecular tests were performed. Final diagnosis of 39 endometrioid carcinomas, 20 non-endometrioid carcinomas and 15 non-malignant was set after hysterectomy. Topoisomerase IIa expression was common (42%) in both types of cancer. Promoter methylation analysis revealed that hMLH1 is commonly methylated in cancers (52.7%), CDKN2A and MGMT less often (27.1%) and RARB rarely methylated (8.4%). BRAF activating mutation V600E was a rare event (8.4%) only found in low grade endometrioid carcinomas. Topoisomerase IIa expression correlated with BRAF mutations, hMLH1 and to lesser extent with CDKN2A methylation. Almost none of the biomarkers were positive in cytological negative or hyperplastic without atypia samples. Detection of methylation in any gene displayed sensitivity, specificity, PPV and NPV similar to cytology of cancer. However, inclusion of cytology diagnosis of hyperlasias with atypia increased sensitivity and NPV of cytology outperforming methylation of any gene. Further evaluation of the panel of promoter methylation, especially in cytology diagnoses of hyperplasia with or without atypia should be evaluated since initial results are promising. Even though methylation of MGMT and RARB are rare events, some patients could be benefit from specific chemotherapeutics that target either of them or the more frequently expressed topoisomerase IIa.
Cite this paper: A. Spathis, V. Sioulas, E. Aga, D. Aninos, C. Kottaridi, E. Terzakis, C. Chrelias and P. Karakitsos, "Promoter Methylation, BRAF Mutation Analysis and Topoisomerase IIa Expression for the Detection of Endometrial Carcinoma in Liquid Based Cytology Samples," Journal of Cancer Therapy, Vol. 4 No. 5, 2013, pp. 19-27. doi: 10.4236/jct.2013.45A004.

[1]   A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward and D. Forman, “Global Cancer Statistics,” CA: A Cancer Journal for Clinicians, Vol. 61, No. 2, 2011, pp. 69-90. doi:10.3322/caac.20107

[2]   F. Bray, I. Dos Santos Silva, H. Moller and E. Weiderpass, “Endometrial Cancer Incidence Trends in Europe: Underlying Determinants and Prospects for Prevention,” Cancer Epidemiology, Biomarkers & Prevention, Vol. 14, No. 5, 2005, pp. 1132-1142. doi:10.1158/1055-9965.EPI-04-0871

[3]   A. S. Felix, L. S. Cook, M. M. Gaudet, T. E. Rohan, L. J. Schouten, V. W. Setiawan, et al., “The Etiology of Uterine Sarcomas: A Pooled Analysis of the Epidemiology of Endometrial Cancer Consortium,” British Journal of Cancer, Vol. 108, No. 3, 2013, pp. 727-734. doi:10.1038/bjc.2013.2

[4]   J. V. Bokhman, “Two Pathogenetic Types of Endometrial Carcinoma,” Gynecologic Oncology, Vol. 15, No. 1, 1983, pp. 10-17. doi:10.1016/0090-8258(83)90111-7

[5]   J. Prat, “Prognostic Parameters of Endometrial Carcinoma,” Human Pathology, Vol. 35, No. 6, 2004, pp. 649-662. doi:10.1016/j.humpath.2004.02.007

[6]   M. K. McConechy, J. Ding, M. C. Cheang, K. C. Wiegand, J. Senz, A. A. Tone, et al., “Use of Mutation Profiles to Refine the Classification of Endometrial Carcinomas,” The Journal of Pathology, Vol. 228, No. 1, 2012, pp. 20-30.

[7]   T. Okuda, A. Sekizawa, Y. Purwosunu, M. Nagatsuka, M. Morioka, M. Hayashi, et al., “Genetics of Endometrial Cancers,” International Journal of Gynecology & Obstetrics, Vol. 2010, 2010, Article ID: 984013.

[8]   A. Yeramian, G. Moreno-Bueno, X. Dolcet, L. Catasus, M. Abal, E. Colas, et al., “Endometrial Carcinoma: Molecular Alterations Involved in Tumor Development and Progression,” Oncogene, Vol. 32, No. 4, 2013, pp. 403-413. doi:10.1038/onc.2012.76

[9]   G. L. Mutter, T. A. Ince, J. P. Baak, G. A. Kust, X. P. Zhou and C. Eng, “Molecular Identification of Latent Precancers in Histologically Normal Endometrium,” Cancer Research, Vol. 61, No. 11, 2001, pp. 4311-4314.

[10]   L. Jia, Y. Liu, X. Yi, A. Miron, C. P. Crum, B. Kong, et al., “Endometrial Glandular Dysplasia with Frequent p53 Gene Mutation: A Genetic Evidence Supporting Its Precancer Nature for Endometrial Serous Carcinoma,” Clinical Cancer Research, Vol. 14, No. 8, 2008, pp. 2263-2269. doi:10.1158/1078-0432.CCR-07-4837

[11]   F. Amant, P. Moerman, P. Neven, D. Timmerman, E. Van Limbergen and I. Vergote, “Endometrial Cancer,” Lancet, Vol. 366, No. 9484, 2005, pp. 491-505. doi:10.1016/S0140-6736(05)67063-8

[12]   J. D. Wright, N. I. Barrena Medel, J. Sehouli, K. Fujiwara and T. J. Herzog, “Contemporary Management of Endometrial Cancer,” Lancet, Vol. 379, No. 9823, 2012, pp. 1352-1360. doi:10.1016/S0140-6736(12)60442-5

[13]   F. Zagouri, G. Bozas, E. Kafantari, M. Tsiatas, N. Nikitas, M. A. Dimopoulos, et al., “Endometrial Cancer: What Is New in Adjuvant and Molecularly Targeted Therapy?” International Journal of Gynecology & Obstetrics, Vol. 2010, 2010, Article ID: 749579.

[14]   L. G. Koss, K. Schreiber, S. G. Oberlander, M. Moukhtar, H. S. Levine and H. F. Moussouris, “Screening of Asymptomatic Women for Endometrial Cancer,” CA: A Cancer Journal for Clinicians, Vol. 31, No. 5, 1981, pp. 300-317. doi:10.3322/canjclin.31.5.300

[15]   I. Symonds, “Ultrasound, Hysteroscopy and Endometrial Biopsy in the Investigation of Endometrial Cancer,” Best Practice & Research Clinical Obstetrics & Gynaecology, Vol. 15, No. 3, 2001, pp. 381-391. doi:10.1053/beog.2000.0183

[16]   M. M. Tahir, M. A. Bigrigg, J. J. Browning, S. T. Brookes and P. A. Smith, “A Randomised Controlled Trial Comparing Transvaginal Ultrasound, Outpatient Hysteroscopy and Endometrial Biopsy with Inpatient Hysteroscopy and Curettage,” BJOG: An International Journal of Obstetrics & Gynaecology, Vol. 106, No. 12, 1999, pp. 1259-1264. doi:10.1111/j.1471-0528.1999.tb08179.x

[17]   S. B. Sams, H. S. Currens and S. S. Raab, “Liquid-Based Papanicolaou Tests in Endometrial Carcinoma Diagnosis. Performance, Error Root Cause Analysis, and Quality Improvement,” American Journal of Clinical Pathology, Vol. 137, No. 2, 2012, pp. 248-254. doi:10.1309/AJCPLFBK1A2XJDQI

[18]   J. Watanabe, Y. Nishimura, S. Tsunoda, M. Kawaguchi, I. Okayasu and H. Kuramoto, “Liquid-Based Preparation for Endometrial Cytology—Usefulness for Predicting the Prognosis of Endometrial Carcinoma Preoperatively,” Cancer, Vol. 117, No. 4, 2009, pp. 254-263.

[19]   A. M. Buccoliero, C. F. Gheri, F. Castiglione, F. Garbini, A. Barbetti, M. Fambrini, et al., “Liquid-Based Endometrial Cytology: Cyto-Histological Correlation in a Population of 917 Women,” Cytopathology, Vol. 18, No. 4, 2007, pp. 241-249. doi:10.1111/j.1365-2303.2007.00463.x

[20]   Y. Norimatsu, H. Kouda, T. K. Kobayashi, K. Shimizu, K. Yanoh, C. Tsukayama, et al., “Utility of Liquid-Based Cytology in Endometrial Pathology: Diagnosis of Endometrial Carcinoma,” Cytopathology, Vol. 20, No. 6, 2009, pp. 395-402. doi:10.1111/j.1365-2303.2008.00589.x

[21]   M. Papaefthimiou, H. Symiakaki, P. Mentzelopoulou, A. E. Giahnaki, Z. Voulgaris, E. Diakomanolis, et al., “The Role of Liquid-Based Cytology Associated with Curettage in the Investigation of Endometrial Lesions from Postmenopausal Women,” Cytopathology, Vol. 16, No. 1, 2005, pp. 32-39. doi:10.1111/j.1365-2303.2004.00224.x

[22]   C. Remondi, F. Sesti, E. Bonanno, A. Pietropolli and E. Piccione, “Diagnostic Accuracy of Liquid-Based Endometrial Cytology in the Evaluation of Endometrial Pathology in Postmenopausal Women,” Cytopathology, 2012.

[23]   E. J. Fox, D. T. Leahy, R. Geraghty, H. E. Mulcahy, D. Fennelly, J. M. Hyland, et al., “Mutually Exclusive Promoter Hypermethylation Patterns of hMLH1 and O6-Methylguanine DNA Methyltransferase in Colorectal Cancer,” Journal of Molecular Diagnostics, Vol. 8, No. 1, 2006, pp. 68-75. doi:10.2353/jmoldx.2006.050084

[24]   A. Spathis, E. Aga, M. Alepaki, A. Chranioti, C. Meristoudis, I. Panayiotides, et al., “Promoter Methylation of p16(INK4A), hMLH1, and MGMT in Liquid-Based Cervical Cytology Samples Compared with Clinicopathological Findings and HPV Presence,” Infectious Diseases in Obstetrics and Gynecology, Vol. 2011, 2011, Article ID: 927861. doi/10.1155/2011/927861

[25]   A. K. Virmani, C. Muller, A. Rathi, S. Zoechbauer-Mueller, M. Mathis and A. F. Gazdar, “Aberrant Methylation during Cervical Carcinogenesis,” Clinical Cancer Research, Vol. 7, No. 3, 2001, pp. 584-589.

[26]   T. Ivanova, A. Petrenko, T. Gritsko, S. Vinokourova, E. Eshilev, V. Kobzeva, et al., “Methylation and Silencing of the Retinoic Acid Receptor-Beta 2 Gene in Cervical Cancer,” BMC Cancer, Vol. 2, 2002, p. 4.

[27]   A. Spathis, J. Georgoulakis, P. Foukas, M. Kefala, K. Leventakos, A. Machairas, et al., “KRAS and BRAF Mutation Analysis from Liquid-Based Cytology Brushings of Colorectal Carcinoma in Comparison with Formalin-Fixed, Paraffin-Embedded Tissue,” Anticancer Research, Vol. 30, No. 6, 2010, pp. 1969-1975.

[28]   M. Kapali, N. P. Agaram, D. Dabbs, A. Kanbour, S. White and R. M. Austin, “Routine Endometrial Sampling of Asymptomatic Premenopausal Women Shedding Normal Endometrial Cells in Papanicolaou Tests Is Not Cost Effective,” Cancer, Vol. 111, No. 1, 2007, pp. 26-33. doi:10.1002/cncr.22424

[29]   B. R. Kipp, F. Medeiros, M. B. Campion, T. J. Distad, L. M. Peterson, G. L. Keeney, et al., “Direct Uterine Sampling with the Tao Brush Sampler Using a Liquid-Based Preparation Method for the Detection of Endometrial Cancer and Atypical Hyperplasia: A Feasibility Study,” Cancer, Vol. 114, No. 4, 2008, pp. 228-235. doi:10.1002/cncr.23636

[30]   A. R. Williams, S. Brechin, A. J. Porter, P. Warner and H. O. Critchley, “Factors Affecting Adequacy of Pipelle and Tao Brush Endometrial Sampling,” BJOG, Vol. 115, No. 8, 2008, pp. 1028-1036. doi:10.1111/j.1471-0528.2008.01773.x

[31]   M. Papaefthimiou, H. Symiakaki, P. Mentzelopoulou, A. Tsiveleka, A. Kyroudes, Z. Voulgaris, et al., “Study on the Morphology and Reproducibility of the Diagnosis of Endometrial Lesions Utilizing Liquid-Based Cytology,” Cancer, Vol. 105, No. 2, 2005, pp. 56-64. doi:10.1002/cncr.21025

[32]   Y. Norimatsu, S. Sakamoto, H. Ohsaki, S. Ozaki, T. Yokoyama, K. Shimizu, et al., “Cytologic Features of the Endometrial Adenocarcinoma: Comparison of ThinPrep and BD Surepath Preparations,” Diagnostic Cytopathology, 2013.

[33]   A. Di Lorito, S. Rosini, E. Falo, S. Gustapane, M. Gomes, J.L. Costa, et al., “Molecular Alterations in Endometrial Archived Liquid-Based Cytology,” Diagnostic Cytopathology, 2012.

[34]   A. Mourtzikou, K. Kosmas, A. Marouga, M. Stamouli, A. Pouliakis and P. Karakitsos, “The Use of an Immunocytochemical Double-Labeling Staining Can Display the Distribution of Bcl-2/Ki-67 Cells in Endometrial Adenocarcinomas as well as in Normal Endometrium,” Clinical Laboratory, Vol. 58, No. 1-2, 2012, pp. 133-144.

[35]   G. E. Kim, S. S. Kweon, J. S. Lee, J. H. Lee, J. H. Nam and C. Choi, “Quantitative Assessment of DNA Methylation for the Detection of Cervical and Endometrial Adenocarcinomas in Liquid-Based Cytology Specimens,” Analytical and Quantitative Cytology and Histology, Vol. 34, No. 4, 2012, pp. 195-203.

[36]   D. Furlan, I. Carnevali, B. Marcomini, R. Cerutti, E. Dainese, C. Capella, et al., “The High Frequency of de Novo Promoter Methylation in Synchronous Primary Endometrial and Ovarian Carcinomas,” Clinical Cancer Research, Vol. 12, No. 11, 2006, pp. 3329-3336.

[37]   L. M. Peterson, B. R. Kipp, K. C. Halling, S. E. Kerr, D. I. Smith, T. J. Distad, et al., “Molecular Characterization of Endometrial Cancer: A Correlative Study Assessing Microsatellite Instability, MLH1 Hypermethylation, DNA Mismatch Repair Protein Expression, and PTEN, PIK3CA, KRAS, and BRAF Mutation Analysis,” International Journal of Gynecological Pathology, Vol. 31, No. 3, 2012, pp. 195-205. doi:10.1097/PGP.0b013e318231fc51

[38]   L. Catasus, A. Gallardo, M. Cuatrecasas and J. Prat, “PIK3CA Mutations in the Kinase Domain (Exon 20) of Uterine Endometrial Adenocarcinomas Are Associated with Adverse Prognostic Parameters,” Modern Pathology, Vol. 21, No. 2, 2008, pp. 131-139.

[39]   H. B. Salvesen, S. Das and L. A. Akslen, “Loss of Nuclear p16 Protein Expression Is Not Associated with Promoter Methylation But Defines a Subgroup of Aggressive Endometrial Carcinomas with Poor Prognosis,” Clinical Cancer Research, Vol. 6, No. 1, 2000, pp. 153-159.

[40]   B. J. Rimel, P. Huettner, M. A. Powell, D. G. Mutch and P. J. Goodfellow, “Absence of MGMT Promoter Methylation in Endometrial Cancer,” Gynecologic Oncology, Vol. 112, No. 1, 2009, pp. 224-228. doi:10.1016/j.ygyno.2008.08.038

[41]   F. Garcia, B. Barker, J. Davis J, et al., “Thin Layer Cytology and Histopathology in the Evaluation of Abnormal Uterine Bleeding,” The Journal of Reproductive Medicine, Vol. 48, No. 11, 2003, pp. 882-888

[42]   N. Bansal, V. Yendluri and R. M. Wenham, “The Molecular Biology of Endometrial Cancers and the Implications for Pathogenesis, Classification, and Targeted Therapies,” Cancer Control, Vol. 16, No. 1, 2009, pp. 8-13.

[43]   S. J. Froelich-Ammon and N. Osheroff, “Topoisomerase Poisons: Harnessing the Dark Side of Enzyme Mechanism,” The Journal of Biological Chemistry, Vol. 270, No. 37, 1995, pp. 21429-21432. doi:10.1074/jbc.270.37.21429

[44]   K. Tanabe, H. Utsunomiya, M. Tamura, H. Niikura, T. Takano, K. Yoshinaga, et al., “Expression of Retinoic Acid Receptors in Human Endometrial Carcinoma,” Cancer Science, Vol. 99, No. 2, 2008, pp. 267-271. doi:10.1111/j.1349-7006.2007.00684.x

[45]   Y. H. Cheng, H. Utsunomiya, M. E. Pavone, P. Yin and S. E. Bulun, “Retinoic Acid Inhibits Endometrial Cancer Cell Growth via Multiple Genomic Mechanisms,” Journal of Molecular Endocrinology, Vol. 46, No. 2, 2011, pp. 139-153. doi:10.1530/JME-10-0064