Targeted next-generation sequencing for molecular diagnosis of endometriosis-associated ovarian cancer

Recent molecular and pathological studies suggest that endometriosis may serve as a precursor of ovarian cancer (endometriosis-associated ovarian cancer, EAOC), especially of the endometrioid and clear cell subtypes. Accordingly, this study had two cardinal aims: first, to obtain mutation profiles of EAOC from Taiwanese patients; and second, to determine whether somatic mutations present in EAOC can be detected in preneoplastic lesions. Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from ten endometriosis patients with malignant transformation. Macrodissection was performed to separate four different types of cells from FFPE sections in six patients. The four types of samples included normal endometrium, ectopic endometriotic lesion, atypical endometriosis, and carcinoma. Ultra-deep (>1000×) targeted sequencing was performed on 409 cancer-related genes to identify pathogenic mutations associated with EAOC. The most frequently mutated genes were PIK3CA (6/10) and ARID1A (5/10). Other recurrently mutated genes included ETS1, MLH1, PRKDC (3/10 each), and AMER1, ARID2, BCL11A, CREBBP, ERBB2, EXT1, FANCD2, MSH6, NF1, NOTCH1, NUMA1, PDE4DIP, PPP2R1A, RNF213, and SYNE1 (2/10 each). Importantly, in five of the six patients, identical somatic mutations were detected in atypical endometriosis and tumor lesions. In two patients, genetic alterations were also detected in ectopic endometriotic lesions, indicating the presence of genetic alterations in preneoplastic lesion. Genetic analysis in preneoplastic lesions may help to identify high-risk patients at early stage of malignant transformation and also shed new light on fundamental aspects of the molecular pathogenesis of EAOC. Key messages - Molecular characterization of endometriosis-associated ovarian cancer genes by targeted NGS. - Candidate genes predictive of malignant transformation were identified. - Chromatin remodeling, PI3K-AKT-mTOR, Notch signaling, and Wnt/β-catenin pathway may promote cell malignant transformation. Similar content being viewed by others

Nezhat F, Datta MS, Hanson V, Pejovic T, Nezhat C, Nezhat C (2008) The relationship of endometriosis and ovarian malignancy: a review. Fertil Steril 90:1559–1570 Rogers PA, D’Hooghe TM, Fazleabas A, Gargett CE, Giudice LC, Montgomery GW, Rombauts L, Salamonsen LA, Zondervan KT (2009) Priorities for endometriosis research: recommendations from an international consensus workshop. Reprod Sci 16:335–346 Worley MJ, Welch WR, Berkowitz RS, Ng SW (2013) Endometriosis-associated ovarian cancer: a review of pathogenesis. Int J Mol Sci 14:5367–5379 Gemmill JA, Stratton P, Cleary SD, Ballweg ML, Sinaii N (2010) Cancers, infections, and endocrine diseases in women with endometriosis. Fertil Steril 94:1627–1631 Kobayashi H, Sumimoto K, Moniwa N, Imai M, Takakura K, Kuromaki T, Morioka E, Arisawa K, Terao T (2007) Risk of developing ovarian cancer among women with ovarian endometrioma: a cohort study in Shizuoka, Japan. Int J Gynecol Cancer 17:37–43 Stern RC, Dash R, Bentley RC, Snyder MJ, Haney AF, Robboy SJ (2001) Malignancy in endometriosis: frequency and comparison of ovarian and extraovarian types. Int J Gynecol Pathol 20:133–139 Wiegand KC, Shah SP, Al-Agha OM, Zhao Y, Tse K, Zeng T, Senz J, McConechy MK, Anglesio MS, Kalloger SE et al (2010) ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Med 363:1532–1543 Yamamoto S, Tsuda H, Takano M, Iwaya K, Tamai S, Matsubara O (2011) PIK3CA mutation is an early event in the development of endometriosis-associated ovarian clear cell adenocarcinoma. J Pathol 225:189–194 Nezhat FR, Pejovic T, Reis FM, Guo SW (2014) The link between endometriosis and ovarian cancer: clinical implications. Int J Gynecol Cancer 24:623–628 Machado-Linde F, Sanchez-Ferrer ML, Cascales P, Torroba A, Orozco R, Silva Sanchez Y, Nieto A, Fiol G (2015) Prevalence of endometriosis in epithelial ovarian cancer. Analysis of the associated clinical features and study on molecular mechanisms involved in the possible causality. Eur J Gynaecol Oncol 36:21–24 Pearce CL, Templeman C, Rossing MA, Lee A, Near AM, Webb PM, Nagle CM, Doherty JA, Cushing-Haugen KL, Wicklund KG et al (2012) Association between endometriosis and risk of histological subtypes of ovarian cancer: a pooled analysis of case-control studies. Lancet Oncol 13:385–394 Chang WH, Wang KC, Lee WL, Huang N, Chou YJ, Feng RC, Yen MS, Huang BS, Guo CY, Wang PH (2014) Endometriosis and the subsequent risk of epithelial ovarian cancer. Taiwan J Obstet Gynecol 53:530–535 Prat J, Oncology FCoG (2015) FIGO’s staging classification for cancer of the ovary, fallopian tube, and peritoneum: abridged republication. J Gynecol Oncol 26:87–89 LaGrenade A, Silverberg SG (1988) Ovarian tumors associated with atypical endometriosis. Hum Pathol 19:1080–1084 Anglesio MS, Bashashati A, Wang YK, Senz J, Ha G, Yang W, Aniba MR, Prentice LM, Farahani H, Li Chang H et al (2015) Multifocal endometriotic lesions associated with cancer are clonal and carry a high mutation burden. J Pathol 236:201–209 Yamamoto S, Tsuda H, Takano M, Tamai S, Matsubara O (2012) Loss of ARID1A protein expression occurs as an early event in ovarian clear-cell carcinoma development and frequently coexists with PIK3CA mutations. Mod Pathol 25:615–624 Patch AM, Christie EL, Etemadmoghadam D, Garsed DW, George J, Fereday S, Nones K, Cowin P, Alsop K, Bailey PJ et al (2015) Whole-genome characterization of chemoresistant ovarian cancer. Nature 521:489–494 Jiang L, Huang J, Morehouse C, Zhu W, Korolevich S, Sui D, Ge X, Lehmann K, Liu Z, Kiefer C et al (2013) Low frequency KRAS mutations in colorectal cancer patients and the presence of multiple mutations in oncogenic drivers in non-small cell lung cancer patients. Cancer Genet 206:330–339 Chen SJ, Liu H, Liao CT, Huang PJ, Huang Y, Hsu A, Tang P, Chang YS, Chen HC, Yen TC (2015) Ultra-deep targeted sequencing of advanced oral squamous cell carcinoma identifies a mutation-based prognostic gene signature. Oncotarget Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin JP, Jarvinen H, Powell SM, Jen J, Hamilton SR et al (1993) Clues to the pathogenesis of familial colorectal cancer. Science 260:812–816 Thibodeau SN, Bren G, Schaid D (1993) Microsatellite instability in cancer of the proximal colon. Science 260:816–819 Gras E, Catasus L, Arguelles R, Moreno-Bueno G, Palacios J, Gamallo C, Matias-Guiu X, Prat J (2001) Microsatellite instability, MLH-1 promoter hypermethylation, and frameshift mutations at coding mononucleotide repeat microsatellites in ovarian tumors. Cancer 92:2829–2836 Roberts SA, Gordenin DA (2014) Hypermutation in human cancer genomes: footprints and mechanisms. Nat Rev Cancer 14:786–800 Hoang LN, McConechy MK, Kobel M, Anglesio M, Senz J, Maassen M, Kommoss S, Meng B, Postovit L, Kelemen LE et al (2015) Polymerase epsilon exonuclease domain mutations in ovarian endometrioid carcinoma. Int J Gynecol Cancer Off J Int Gynecol Cancer Soc 25:1187–1193 Zou Y, Liu FY, Liu H, Wang F, Li W, Huang MZ, Huang Y, Yuan XQ, Xu XY, Huang OP et al (2014) Frequent POLE1 p.S297F mutation in Chinese patients with ovarian endometrioid carcinoma. Mutat Res 761:49–52 Cancer Genome Atlas Research N, Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, Shen H, Robertson AG, Pashtan I, Shen R et al (2013) Integrated genomic characterization of endometrial carcinoma. Nature 497:67–73 Akahane T, Sekizawa A, Purwosunu Y, Nagatsuka M, Okai T (2007) The role of p53 mutation in the carcinomas arising from endometriosis. Int J Gynecol Pathol Off J Int Soc Gynecol Pathol 26:345–351 Bayramoglu H, Duzcan E (2001) Atypical epithelial changes and mutant p53 gene expression in ovarian endometriosis. Pathol Oncol Res POR 7:33–38 Bischoff FZ, Heard M, Simpson JL (2002) Somatic DNA alterations in endometriosis: high frequency of chromosome 17 and p53 loss in late-stage endometriosis. J Reprod Immunol 55:49–64 Kurman RJ, Shih Ie M (2010) The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory. Am J Surg Pathol 34:433–443 Kurman RJ, Shih Ie M (2011) Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer—shifting the paradigm. Hum Pathol 42:918–931 Gamallo C, Palacios J, Moreno G, Calvo de Mora J, Suarez A, Armas A (1999) beta-catenin expression pattern in stage I and II ovarian carcinomas : relationship with beta-catenin gene mutations, clinicopathological features, and clinical outcome. Am J Pathol 155:527–536 Saegusa M, Okayasu I (2001) Frequent nuclear beta-catenin accumulation and associated mutations in endometrioid-type endometrial and ovarian carcinomas with squamous differentiation. J Pathol 194:59–67 McConechy MK, Ding J, Senz J, Yang W, Melnyk N, Tone AA, Prentice LM, Wiegand KC, McAlpine JN, Shah SP et al (2014) Ovarian and endometrial endometrioid carcinomas have distinct CTNNB1 and PTEN mutation profiles. Modern Pathol Off J U S Can Acad Pathol Inc 27:128–134 Zhao C, Wu LS, Barner R (2011) Pathogenesis of ovarian clear cell adenofibroma, atypical proliferative (borderline) tumor, and carcinoma: clinicopathologic features of tumors with endometriosis or adenofibromatous components support two related pathways of tumor development. J Cancer 2:94–106 Gadducci A, Lanfredini N, Tana R (2014) Novel insights on the malignant transformation of endometriosis into ovarian carcinoma. Gynecol Endocrinol 30:612–617 Gui Y, Guo G, Huang Y, Hu X, Tang A, Gao S, Wu R, Chen C, Li X, Zhou L et al (2011) Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nat Genet 43:875–878 Fujimoto A, Totoki Y, Abe T, Boroevich KA, Hosoda F, Nguyen HH, Aoki M, Hosono N, Kubo M, Miya F et al (2012) Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators. Nat Genet 44:760–764 Manceau G, Letouze E, Guichard C, Didelot A, Cazes A, Corte H, Fabre E, Pallier K, Imbeaud S, Le Pimpec-Barthes F et al (2013) Recurrent inactivating mutations of ARID2 in non-small cell lung carcinoma. Int J Cancer J Int Cancer 132:2217–2221 India Project Team of the International Cancer Genome C (2013) Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups. Nat Commun 4:2873 Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, Nickerson E, Auclair D, Li L, Place C et al (2012) A landscape of driver mutations in melanoma. Cell 150:251–263 Shain AH, Pollack JR (2013) The spectrum of SWI/SNF mutations, ubiquitous in human cancers. PLoS ONE 8, e55119. doi:10.1371/journal.pone.0055119 Varela I, Tarpey P, Raine K, Huang D, Ong CK, Stephens P, Davies H, Jones D, Lin ML, Teague J et al (2011) Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469:539–542 Kuo KT, Mao TL, Jones S, Veras E, Ayhan A, Wang TL, Glas R, Slamon D, Velculescu VE, Kuman RJ et al (2009) Frequent activating mutations of PIK3CA in ovarian clear cell carcinoma. Am J Pathol 174:1597–1601 Oishi T, Itamochi H, Kudoh A, Nonaka M, Kato M, Nishimura M, Oumi N, Sato S, Naniwa J, Sato S et al (2014) The PI3K/mTOR dual inhibitor NVP-BEZ235 reduces the growth of ovarian clear cell carcinoma. Oncol Rep 32:553–558 Rudd ML, Price JC, Fogoros S, Godwin AK, Sgroi DC, Merino MJ, Bell DW (2011) A unique spectrum of somatic PIK3CA (p110alpha) mutations within primary endometrial carcinomas. Clin Cancer Res Off J Am Assoc Cancer Res 17:1331–1340 Stewart CJ, Leung Y, Walsh MD, Walters RJ, Young JP, Buchanan DD (2012) KRAS mutations in ovarian low-grade endometrioid adenocarcinoma: association with concurrent endometriosis. Hum Pathol 43:1177–1183 Zannoni GF, Improta G, Chiarello G, Pettinato A, Petrillo M, Scollo P, Scambia G, Fraggetta F (2014) Mutational status of KRAS, NRAS, and BRAF in primary clear cell ovarian carcinoma. Virchows Archiv Int J Pathol 465:193–198 Jones S, Wang TL, Shih Ie M, Mao TL, Nakayama K, Roden R, Glas R, Slamon D, Diaz LA Jr, Vogelstein B et al (2010) Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Science 330:228–231 Shih Ie M, Panuganti PK, Kuo KT, Mao TL, Kuhn E, Jones S, Velculescu VE, Kurman RJ, Wang TL (2011) Somatic mutations of PPP2R1A in ovarian and uterine carcinomas. Am J Pathol 178:1442–1447 Shi Y (2009) Serine/threonine phosphatases: mechanism through structure. Cell 139:468–484 Hemmings BA, Adams-Pearson C, Maurer F, Muller P, Goris J, Merlevede W, Hofsteenge J, Stone SR (1990) alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure. Biochemistry 29:3166–3173 Slupe AM, Merrill RA, Strack S (2011) Determinants for substrate specificity of protein phosphatase 2A. Enzym Res 2011, 398751. doi:10.4061/2011/398751 Chen W, Arroyo JD, Timmons JC, Possemato R, Hahn WC (2005) Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity. Cancer Res 65:8183–8192 Plotnik JP, Budka JA, Ferris MW, Hollenhorst PC (2014) ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells. Nucleic Acids Res 42:11928–11940 Fernandez-Medarde A, Santos E (2011) Ras in cancer and developmental diseases. Genes Cancer 2:344–358 Zhang Y, Yan LX, Wu QN, Du ZM, Chen J, Liao DZ, Huang MY, Hou JH, Wu QL, Zeng MS et al (2011) miR-125b is methylated and functions as a tumor suppressor by regulating the ETS1 proto-oncogene in human invasive breast cancer. Cancer Res 71:3552–3562 Nakayama T, Ito M, Ohtsuru A, Naito S, Sekine I (2001) Expression of the ets-1 proto-oncogene in human colorectal carcinoma. Modern Pathol Off J USA Can Acad Pathol Inc 14:415–422 Groeneweg JW, Foster R, Growdon WB, Verheijen RH, Rueda BR (2014) Notch signaling in serous ovarian cancer. J Ovarian Res 7:95 Crobach S, Ruano D, van Eijk R, Fleuren GJ, Minderhout I, Snowdowne R, Tops C, van Wezel T, Morreau H (2015) Target-enriched next-generation sequencing reveals differences between primary and secondary ovarian tumors in formalin-fixed, paraffin-embedded tissue. J Mol Diagn JMD 17:193–200 Kang H, Jeong JY, Song JY, Kim TH, Kim G, Huh JH, Kwon AY, Jung SG, An HJ (2015) Notch3-specific inhibition using siRNA knockdown or GSI sensitizes paclitaxel-resistant ovarian cancer cells. Mol Carcinog. doi:10.1002/mc.22363 Zhang L, Zhou Y, Cheng C, Cui H, Cheng L, Kong P, Wang J, Li Y, Chen W, Song B et al (2015) Genomic analyses reveal mutational signatures and frequently altered genes in esophageal squamous cell carcinoma. Am J Hum Genet 96:597–611 Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, Kryukov GV, Lawrence MS, Sougnez C, McKenna A et al (2011) The mutational landscape of head and neck squamous cell carcinoma. Science 333:1157–1160 Spencer DH, Sehn JK, Abel HJ, Watson MA, Pfeifer JD, Duncavage EJ (2013) Comparison of clinical targeted next-generation sequence data from formalin-fixed and fresh-frozen tissue specimens. J Mol Diagn JMD 15:623–633 Hedegaard J, Thorsen K, Lund MK, Hein AM, Hamilton-Dutoit SJ, Vang S, Nordentoft I, Birkenkamp-Demtroder K, Kruhoffer M, Hager H et al (2014) Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS ONE 9:e98187 Acknowledgments We thank ACT Genomics Co., Ltd. for their assistance with bioinformatics analysis. We are also grateful to Dr Shu-Jen Chen and Dr Yen-Jung Lu for their expertise in the field of next-generation sequencing and bioinformatics. This study was supported by grants from Kaohsiung Medical University Hospital (KMUH102-M207, KMUH103-3R64, and KMOH103-10V07) and the Ministry of Health and Welfare (MOHW103-TD-B-111-05, MOHW104-TDU-B-212-124-003, and MOHW103-TDU-212-114007). This work was supported by the Ministry of Science and Technology of Taiwan [grant numbers 103-2314-B-037-057, 102-2628-B-037-011-MY3 and 102-2632-B-037-001-MY3] and the Kaohsiung Medical University (Hospital) Research Fund [grant numbers KMU-TP104A02, KMU-TP103G01, KMU-TP103G04, KMUTP103G05, KMU-TP103A15, and KMUH103-10V07]. Author contributions Concept and design of the experiments: TKE, YFS, CCW, CCC, THH, TCL, and EMT. Performance of the experiments: TKE, YFS, CCW, WTC, YTC, and CCC. Data analysis and discussion: TKE, YFS, CCW, CCC, JW, MHV, YTC, TCL, and EMT. Contribution of reagents/materials/analysis tools: TCL, CCW, THH, WTC, YTC, CHS, and EMT. Clinical information: CCW, YTC, CHS, and EMT. Manuscript preparation: TKE, YFS, CCW, CCC, JW, THH, MHV, YTC, TCL, CHS, and EMT. All authors read and approved the final manuscript. Author information Authors and Affiliations Corresponding author Ethics declarations Conflict of interest The authors declare that they have no competing interests. Electronic supplementary material Below is the link to the electronic supplementary material. Table S1 (download DOCX ) (DOCX 56 kb) Supplementary data 1 (download DOCX ) ACTOnco™ CCP target gene list. (DOCX 16 kb) Supplementary data 2 (download XLSX ) Mutation data includes allele frequency. (XLSX 68 kb) Supplementary figures (download PDF ) (PDF 341 kb) Rights and permissions About this article Cite this article Er, TK., Su, YF., Wu, CC. et al. Targeted next-generation sequencing for molecular diagnosis of endometriosis-associated ovarian cancer. J Mol Med 94, 835–847 (2016). https://doi.org/10.1007/s00109-016-1395-2 Received: Revised: Accepted: Published: Issue date: DOI: https://doi.org/10.1007/s00109-016-1395-2