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Landscape of Somatic Single-Nucleotide and Copy-Number Mutations in Uterine Serous Carcinoma

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Landscape of Somatic Single-Nucleotide and Copy-Number Mutations in Uterine Serous Carcinoma Landscape of somatic single-nucleotide and copy-number mutations in uterine serous carcinoma Siming Zhaoa, Murim Choia, John D. Overtona, Stefania Belloneb, Dana M. Roqueb, Emiliano Coccob, Federica Guzzob, Diana P. Englishb, Joyce Varugheseb, Sara Gasparrinib, Ileana Bortolomaib, Natalia Buzac, Pei Huic, Maysa Abu-Khalafd, Antonella Ravaggie, Eliana Bignottie, Elisabetta Bandierae, Chiara Romanie, Paola Todeschinie, Renata Tassie, Laura Zanottie, Luisa Carrarae, Sergio Pecorellie, Dan-Arin Silasib, Elena Ratnerb, Masoud Azodib, Peter E. Schwartzb, Thomas J. Rutherfordb, Amy L. Stieglerf, Shrikant Manea, Titus J. Boggonf, Joseph Schlessingerf, Richard P. Liftona,1, and Alessandro D. Santinb aDepartment of Genetics, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510; bDepartments of Obstetrics, Gynecology, and Reproductive Sciences, cPathology, dInternal Medicine and Oncology, and fPharmacology, Yale University School of Medicine, New Haven, CT 06510; and eDepartment of Obstetrics, and Gynecology, “Angelo Nocivelli” Institute of Molecular Medicine, University of Brescia, 25123 Brescia, Italy Contributed by Richard P. Lifton, December 27, 2012 (sent for review December 12, 2012) Uterine serous carcinoma (USC) is a biologically aggressive subtype and the recurrence rate after surgery is extremely high (50–80%) of endometrial cancer. We analyzed the mutational landscape of (3–5). This poor prognosis motivates determination of the molec- USC by whole-exome sequencing of 57 cancers, most of which were ular basis of this tumor’s aggressive behavior in hope of developing matched to normal DNA from the same patients. The distribution of new effective treatment modalities. the number of protein-altering somatic mutations revealed that 52 To address these issues, we have sequenced the exomes of USC tumors had fewer than 100 (median 36), whereas 5 had more a large cohort of USC and have identified genes with increased than 3,000 somatic mutations. The mutations in these latter tumors numbers of somatic single-nucleotide and copy-number variants. showed hallmarks of defects in DNA mismatch repair. Among the The results identify cancer genes and define the pathways in- remainder, we found a significantly increased burden of mutation in volved in USC. 14 genes. In addition to well-known cancer genes (i.e., TP53, PIK3CA, GENETICS PPP2R1A, KRAS, FBXW7), there were frequent mutations in CHD4/ Results Mi2b, a member of the NuRD–chromatin-remodeling complex, and Exome Sequencing of USC. Fifty-seven patients with uterine serous TAF1, an element of the core TFIID transcriptional machinery. Addi- carcinoma were studied. Their clinical features are presented in SI tionally, somatic copy-number variation was found to play an im- Appendix, Table S1. Upon surgical removal of tumors, primary cell portant role in USC, with 13 copy-number gains and 12 copy-number lines were prepared (15 tumors) or tumors were frozen (42 losses that occurred more often than expected by chance. In addi- tumors). Exome sequencing was performed on all tumors; for 34 of tion to loss of TP53, we found frequent deletion of a small segment these, DNA samples from normal tissue were available and se- of chromosome 19 containing MBD3, also a member of the NuRD– quenced. Exome sequencing was performed using the NimbleGen/ chromatin-modification complex, and frequent amplification of Roche capture reagent followed by 74 base paired-end DNA se- chromosome segments containing PIK3CA, ERBB2 (an upstream ac- quencing on the Illumina HiSeq platform (6). By design, tumor tivator of PIK3CA), and CCNE1 (a target of FBXW7-mediated ubiq- samples were sequenced to greater depth of coverage to permit uitination). These findings identify frequent mutation of DNA detection of somatic mutations in tumors despite admixture of damage, chromatin remodeling, cell cycle, and cell proliferation normal and tumor cells in these samples. For tumors and normal pathways in USC and suggest potential targets for treatment of this DNA, each targeted base was sequenced by a mean of 187 and 100 lethal variant of endometrial cancer. independent reads, respectively (SI Appendix, Table S2). Of all targeted bases in tumors, 94.5% were read by 20 or more in- endometrial carcinoma | uterine serous papillary cancer | cancer genomics dependent reads; mean per-base per read error rates were 0.42% for normal DNA and 0.48% for tumor DNA. Segments of loss of ndometrial cancer is the most prevalent gynecologic tumor in heterozygosity (LOH) were called from the difference in B-allele Ewomen, with an annual incidence of 47,130 new cases and frequency between tumor-normal pairs (SI Appendix,Fig.S1), 8,010 deaths in 2012 in the United States (1). On the basis of allowing estimates of tumor purity, which were above 60% for clinical and histopathological features, endometrial cancer is frozen tumors and higher for primary cell lines. Somatic mutations fi fi classified into type I and type II disease groups (2). Type I tumors, were identi ed by nding variant reads in tumors that were sig- fi Materials and which constitute the majority of cases, are generally diagnosed at ni cantly more frequent than expected by chance ( Methods fi an early stage, are low grade and endometrioid in histology, are ). At the coverage levels studied, there was no signi cant associated with a history of hyperestrogenism, and typically have relationship between tumor purity and the number of somatic fi a good prognosis. In contrast, type II cancers are poorly differ- variants detected, consistent with suf cient depth of coverage entiated, often with serous papillary [uterine serous carcinoma having been achieved to identify the vast majority of somatic (USC)] or clear cell histology. Although these tumors account for mutations. Variants in genes implicated in the pathogenesis of a minority of endometrial cancers, the majority of relapses and deaths occur in this group of patients (2). Among type II cancers, USC represents the most biologically Author contributions: J.S., R.P.L., and A.D.S. designed research; J.D.O., S.B., D.M.R., E.C., F.G., S.G., I.B., N.B., P.H., A.R., E. Bignotti, E. Bandiera, C.R., P.T., R.T., L.Z., L.C., A.L.S., S.M., aggressive subtype (3, 4). Classically, the neoplastic epithelium is and T.J.B. performed research; D.P.E., J.V., M.A.-K., S.P., D.-A.S., E.R., M.A., P.E.S., and characterized by serous differentiation with psammoma bodies and T.J.R. contributed new reagents/analytic tools; S.Z., M.C., and R.P.L. analyzed data; and a predominantly papillary architecture (3). Pleomorphic cytology S.Z., A.L.S., T.J.B., J.S., R.P.L., and A.D.S. wrote the paper. with nuclear atypia, prominent nucleoli, a vescicular chromatin The authors declare no conflict of interest. pattern, and high mitotic activity are seen. Clinically, USC has Freely available online through the PNAS open access option. a propensity for early intra-abdominal and lymphatic spread (3) 1To whom correspondence should be addressed. E-mail: [email protected]. – and is more commonly diagnosed in women of African ancestry (3 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 5). The overall 5-y survival of USC is only 30 ± 9% for all stages, 1073/pnas.1222577110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1222577110 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 USC were verified by direct Sanger sequencing and were found to mutated tumors showed a paucity of T:A > A:T or C:G > A:T be expressed in all available USC cell lines. transversions (SI Appendix,Fig.S2) (9). Examination of the POLE and MMR genes showed no germ-line mutations; however, so- Tumors with Hypermutator Phenotype. The number of protein-al- matic mutations in these genes were highly prevalent in these tering somatic mutations per tumor markedly deviated from tumors (mean of 4 per tumor, including 4 premature termination a normal distribution (Fig. 1A). In the discovery set of 34 USC with mutations for MMR genes and a mean of 4.5 per tumor for POLE) − matched normal DNA, 30 tumors had fewer than 100 protein- and more frequent than expected by chance (P = 2.23 × 10 3)(SI altering somatic mutations (median 36), whereas 4 had more than Appendix, Table S3). Among the cancers without matched normal 3,000 somatic mutations each. Only one of these tumors was from DNA, one showed a similarly high prevalence of rare protein- a cell line (with limited propagation), and none came from patients altering variants (>3,000) and a skewed distribution of rare pro- who had received chemotherapy before sample acquisition. These tein-altering transversions. Thus, 9% of USC in this cohort have tumors with high mutation burden were also notable for having no a hypermutator phenotype. Because of the skewing effect of the LOH segments or copy-number variants (CNVs), a feature found large number of mutations in these tumors, they were not included in only five other tumors. These features suggest a hypermutator in subsequent analyses of mutation burden. phenotype due to deficiency of mismatch repair (MMR) or poly- e POLE Analysis of Single-Nucleotide Variants. Among somatic mutations in merase ( ) genes (7, 8). Consistent with this, these hyper- the 30 remaining matched tumors, we identified recurrences of somatic mutations at the same positions. Accounting for the rate of − protein-altering somatic mutations in these tumors (1.1 × 10 6)and the size of the exome, the likelihood of seeing the mutation twice by − chance at any position among these tumors is <10 3.Weidentified six genes with recurrent somatic mutations (Table 1). These in- cluded well-established activating mutations in PIK3CA (10), the catalytic subunit of phosphoinositide-3 kinase (five tumors); the well-established G12V mutation in KRAS (three tumors) (11); and a mutation at R465 in FBXW7 in four tumors (12).
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