{"paper_id":"b269aea9-1b14-402b-9f43-c11d91bb59c0","body_text":"Ovarian clear-cell carcinomas from 18 patients, obtained from the OvCaRe (Ovarian Cancer Research) frozen-tumor bank, and 1 ovarian clear-cell carcinoma–derived cell line (TOV21G) 21  were selected as the discovery cohort for RNA sequencing. Approval from the hospital’s institutional review board was obtained to permit the use of these samples for RNA-sequencing experiments.\nTo determine the frequency of  ARID1A  mutations in ovarian clear-cell carcinoma and other subtypes of ovarian cancer, we performed targeted exon resequencing in the discovery cohort, as well as in a mutation-validation cohort, consisting of 210 samples: samples of ovarian clear-cell carcinoma from 101 patients (independent of the 19 samples used in RNA sequencing for the discovery cohort), samples of endometrioid carcinoma from 33 patients, samples of high-grade serous carcinoma from 76 patients, and the ovarian clear-cell carcinoma–derived cell line ES2. 22  Ten specimens of ovarian clear-cell carcinoma came from Johns Hopkins University, 29 from the Centre Hospitalier de l’Université de Montréal–Hôpital Notre-Dame, and 42 from the Australian Ovarian Cancer Study (AOCS); all others were obtained from the OvCaRe frozen-tumor bank. All patients from both the discovery and mutation-validation cohorts provided written informed consent to have their tumors and germ-line DNA used for research, including genomic studies. Details regarding the consents and other approvals by the institutional review boards are supplied in the  Supplementary Appendix  (available with the full text of this article at NEJM.org).\nAll tumor samples were reviewed independently by a gynecologic pathologist before mutational analysis was performed. In cases in which the review diagnosis differed from the diagnosis at the source institution, the samples were further reviewed by another gynecologic pathologist, who acted as an arbiter. Both review pathologists were unaware of the results of the genomic studies.\nRNA sequencing and analysis were performed as previously described. 19 , 20  For details, see the Methods section in the  Supplementary Appendix .\nGenomic DNA from samples in both the discovery and mutation-validation cohorts were subjected to targeted exon resequencing. Selected  ARID1A  variants (those with truncating changes or radical missense mutations 23  with an allele frequency of >10%) detected by means of exon resequencing were validated in tumor DNA by means of Sanger sequencing. In most cases, germ-line DNA (from formalin-fixed paraffin-embedded sections, blood samples, or cultured fibroblasts) was also analyzed by means of Sanger sequencing (see  Table 3 in the Supplementary Appendix ). Full details are provided in the  Supplementary Appendix .\nImmunohistochemical staining for BAF250a was performed in all samples except 42 ovarian clear-cell carcinoma specimens from the AOCS and 4 from Johns Hopkins University. A total of 455 additional ovarian-carcinoma samples — including 132 ovarian clear-cell carcinomas, 125 endometrioid carcinomas, and 198 high-grade serous carcinomas — from a previously described tissue microarray 6  were used for an immunohistochemical validation cohort and were analyzed for BAF250a expression. All normal gynecologic tissues showed moderate or intense nuclear immunoreactivity for BAF250a. Tumors were scored positive for BAF250a if tumor cells showed definite nuclear staining and negative if tumor nuclei had no immunoreactivity but endothelial and other nontumor cells from the same samples showed immunoreactivity. Cases in which neither normal cells in the stroma nor tumor cells were immunoreactive were considered to be the result of technical failure. Details of the staining protocol are provided in the  Supplementary Appendix . Additional immunohistochemical staining for hepatocyte nuclear factor 1 β  (HNF-1 β ) and estrogen receptor was performed on whole sections for two tumors with contiguous atypical endometriosis, as previously described. 24\nIn two tumors with identified  ARID1A  mutations, sections of atypical (contiguous) and distant endometriosis were identified by a gynecologic pathologist. Laser-capture microdissection was used to isolate endometriotic epithelium. DNA extracted from these cells was analyzed by means of sequencing for the mutations seen in the tumor (see the  Supplementary Appendix ).\n\nThe RNA-sequencing data, including the number of mapped sequencing reads and potential non-synonymous sequence variants, are summarized in  Table 1 in the Supplementary Appendix . RNA sequencing of the 19 samples in the discovery cohort resulted in the detection of the following nucleotide mutations (and corresponding amino acid mutations) (also shown in  Table 1  and  Fig. 1 ): three somatic nonsense mutations — C4201T (Q1401*), C5164T (R1722*), and C1680A (Y560*) (stars denote a stop codon); two somatic indels (insertion–deletion) — 6018-6020delGCT and 5541insG; one somatic missense mutation — T5953C (S1989P) (found in the same sample as the 5541insG mutation); and one gene rearrangement involving  ARID1A  and the neighboring gene  ZDHHC18  (encoding the zinc-finger DHHC domain-containing protein 18). The fusion ends of this rearrangement map to a homozygous deletion involving most of the  ARID1A  gene ( Fig. 1 in the Supplementary Appendix ).\nAll predicted variants of  ARID1A  were validated with the use of Sanger sequencing of DNA from the source tumors, except for the deletion–rearrangement, which was validated with the use of microarray data (Affymetrix SNP 6.0) ( Table 3 in the Supplementary Appendix ). The finding of multiple types of mutations in a single gene,  ARID1A,  in ovarian clear-cell carcinoma led us to further explore  ARID1A  in this cancer type. Since mutations in  PIK3CA  (the phosphoinositide-3-kinase, catalytic, alpha polypeptide gene),  CTNNB1  (the catenin beta-1 gene),  KRAS  (the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue gene), and  TP53  (the tumor protein p53 gene) are recurrent in ovarian clear-cell carcinoma, 25  we analyzed the RNA-sequencing data and performed a polymerase-chain-reaction assay for the presence of variants in these genes ( Table 1 ). Whole-transcriptome sequence data for the 19 samples of the discovery cohort have been deposited at the European Genome–Phenome Archive (accession number, EGAS00000000075).\nARID1A  mutation frequency in ovarian clear-cell carcinomas and other ovarian-cancer subtypes was established through targeted exon resequencing of the mutation-validation cohort of 210 samples of various subtypes of ovarian carcinomas and 1 ovarian clear-cell carcinoma cell line, along with the original discovery cohort of 18 samples of ovarian clear-cell carcinoma and 1 ovarian clear-cell carcinoma cell line.  ARID1A  mutations were identified in 55 of 119 (46%) ovarian clear-cell carcinomas, 10 of the 33 (30%) endometrioid carcinomas, and none of the 76 high-grade serous carcinomas ( Table 2 , and  Table 3 in the Supplementary Appendix ). A total of 17 samples (12 of ovarian clear-cell carcinoma and 5 of endometrioid carcinoma) each had two validated  ARID1A  mutations. In addition, the ovarian clear-cell carcinoma cell line TOV21G had a truncating mutation in  ARID1A  (1645insC).\nWe analyzed germ-line DNA from 55 samples (47 ovarian clear-cell carcinomas and 8 endometrioid carcinomas) in the discovery and mutation- validation cohorts for the presence of 65 truncating mutations (53 found in ovarian clear-cell carcinomas and 12 found in endometrioid carcinomas). In all 55, the mutations were found to be somatic. On this basis, we made the assumption that 12 subsequent truncating mutations (10 in ovarian clear-cell carcinoma and 2 in endometrioid carcinoma) would be somatic (i.e., predicted to be somatic without germ-line DNA testing) ( Table 3 in the Supplementary Appendix ). The presence of  ARID1A  mutations showed a strong association (P<0.001 by Fisher’s exact test) with the two ovarian-cancer subtypes associated with endometriosis (ovarian clear-cell carcinoma and endometrioid carcinoma).\nThe correlation between  ARID1A  mutations and BAF250a expression was evaluated by means of immunohistochemical staining for BAF250a in 182 tumors for which formalin-fixed, paraffin-embedded sections were available in the discovery cohort and the mutation-validation cohort: 73 ovarian clear-cell carcinomas, 33 endometrioid carcinomas, and 76 high-grade serous carcinomas. The presence of mutations was significantly associated with BAF250a loss in endometriosis-associated cancers (P<0.001 by Fisher’s exact test). A total of 27 of 37 samples (73%) and 5 of 10 samples (50%) of ovarian clear-cell carcinoma and endometrioid carcinoma, respectively, with an  ARID1A  mutation showed a loss of BAF250a expression, as compared with 4 of 36 samples (11%) and 2 of 23 samples (9%), respectively, without an  ARID1A  mutation ( Fig. 2A  and  Table 2 ). Loss of BAF250a expression was strongly associated with the endometriosis-related ovarian cancers — with 31 of 73 samples (42%) of ovarian clear-cell carcinoma and 7 of 33 samples (21%) of endometrioid carcinoma showing a loss of expression — as compared with high-grade serous carcinomas, for which 1 of the 76 samples (1%) had loss of expression (P<0.001 by Fisher’s exact test) ( Fig. 2B ).  ARID1A  mutations were not significantly associated with the presence of endometriosis in 86 ovarian clear-cell carcinomas and 33 endometrioid carcinomas ( Table 5 in the Supplementary Appendix ).\nThe immunohistochemical validation cohort was also assessed for BAF250a expression ( Fig. 2B ). This analysis revealed that 55 of the 132 samples (42%) of ovarian clear-cell carcinoma, 39 of the 125 samples (31%) of endometrioid carcinoma, and 12 of the 198 samples (6%) of high-grade serous carcinoma lacked BAF250a expression. These findings are in agreement with the proportions observed in the discovery and mutation-validation cohorts. No significant associations with absence of BAF250a expression were noted on the basis of age of presentation, stage of disease (low or high), or disease-specific survival within any of the cancer subtypes, as assessed by means of Welch’s analysis of variance, Fisher’s exact test, and the log-rank statistic, respectively (P>0.05 for all analyses).\nTwo patients with ovarian clear-cell carcinomas (samples CCC13 and CCC23) carrying  ARID1A  mutations had contiguous atypical endometriosis ( Fig. 3 , and  Fig. 3 in the Supplementary Appendix ). For one of the two patients, the specimen was heterozygous for an  ARID1A  truncating mutation (G6139T [E2047*]) in exon 20. This mutation was also found in 17 of 42 clones derived from atypical endometriosis but in none of 52 clones from a distant endometriotic lesion (P<0.001 by Fisher’s exact test) ( Fig. 3C ). Epithelial samples of both the ovarian clear-cell carcinoma and atypical endometriosis had loss of BAF250a expression, whereas expression was maintained in the distant endometriotic lesion ( Fig. 3B ). HNF-1β was expressed in the ovarian clear-cell carcinoma but not in the contiguous atypical or distant endometriosis, and estrogen receptor was expressed in both the contiguous and distant endometriosis but not in the ovarian clear-cell carcinoma, as was expected. 24  Thus, atypical endometrium could be distinguished from the distant endometrium only on the basis of loss of BAF250a expression, which correlated with the presence of an  ARID1A  mutation.\nFor the other patient, the sample of ovarian clear-cell carcinoma had two somatic mutations in  ARID1A  (and loss of BAF250a expression): both these mutations, along with a  CTNNB1  missense mutation, were present in the tumor and the adjacent atypical endometriosis but not in a distant endometriotic lesion ( Fig. 3B in the Supplementary Appendix ).\n\nOverall, 46% of patients with ovarian clear-cell carcinoma and 30% of those with endometrioid carcinoma had somatic truncating or missense mutations in  ARID1A;  no  ARID1A  mutations were found in any of the 76 specimens of high-grade serous carcinoma analyzed. Loss of  ARID1A  expression was also specific to the subtype of ovarian cancer, with loss of nuclear BAF250a expression seen in 36% of ovarian clear-cell carcinomas and endometrioid carcinomas, but only 1% of high-grade serous carcinomas. Our initial mutation-screening assays involving RNA sequencing in the discovery cohort identified seven somatic mutations in  ARID1A  in the 19 samples; four additional mutations were subsequently identified when these samples were analyzed by means of amplicon-exon resequencing. Most likely, the additional mutations had not been seen in the RNA-sequencing data owing to their transcripts being rapidly targeted for nonsense-mediated decay 26  or the inherently decreased sensitivity of the assay to mutations at the 5 end of transcripts. Thus, although RNA sequencing is a useful tool for discovery, targeted exon resequencing may be more appropriate for the determination of true mutation frequency.\nARID1A  is located at 1p36.11. 27  This chromosomal region is commonly deleted in tumors, and it has been suggested that deletion regions encompassing 1p36 could contain tumor-suppressor genes. 28 , 29  Rearrangements and deletions in  ARID1A  have been identified in a primary breast-cancer cell line and a lung-cancer cell line, respectively, 18  and the loss of BAF250a has also been observed in cervical- and breast-carcinoma cell lines. 30  In a study by Wang and colleagues, 31  the screening of 241 tumors revealed that  ARID1A  transcript levels are decreased in approximately 6% of cancers in general and in 30% of renal carcinomas and 10% of breast carcinomas, specifically; however, none of the 14 ovarian cancers showed loss of expression, probably because they were predominantly the high-grade serous subtype.\nThe  ARID1A  mutations identified in our study were mostly truncating mutations, which were evenly distributed across the gene. The presence of mutations is strongly correlated with the loss of BAF250a protein ( Table 2  and  Fig. 2A ). Loss of BAF250a expression was seen in 73% and 50% of samples of ovarian clear-cell carcinoma and endometrioid carcinoma with an  ARID1A  mutation, respectively, and in only 11% and 9% of samples without an  ARID1A  mutation, respectively. Seventeen of the mutation-positive samples had two  ARID1A  mutations; in all but one of the specimens with two mutations for which immunohistochemical data were available, BA-F250a expression was not detected. That single exception (an endometrioid carcinoma) had both a C-terminal truncating mutation and a mis-sense mutation; either of these changes could produce a detectable protein. A single sample of ovarian clear-cell carcinoma had  ARID1A  loss and rearrangement resulting in the homozygous deletion of the gene. Three other cases of ovarian clear-cell carcinoma also appear to be characterized by loss of heterozygosity, on the basis of the frequency of mutant alleles and wild-type alleles ( Table 3 in the Supplementary Appendix ) and subsequent loss of BAF250a expression. However, the majority of cancers with somatic  ARID1A  mutations and loss of BAF250a expression appear to have a wild-type allele present. Data from exon resequencing and RNA sequencing show excellent agreement between the fraction of mutant and wild-type alleles at both the DNA and RNA levels ( Table 1 ), suggesting that epigenetic silencing is not a significant factor. Post-transcriptional or post-translational regulation or dominant negative effects of the mutations are possible, albeit untested, explanations for the lack of protein expression in these heterozygous cases.\nThe presence of BAF250a immunoreactivity in 15 samples positive for an  ARID1A  mutation (all but 1 of which had truncating mutations) may indicate that haploinsufficiency is pathogenic, as has been reported in mice. 32  Alternatively, immunohistochemical detection of a truncated but nonfunctional BAF250a protein may account for the immunostaining results. The antibody used in the assay targets a region of 111 amino acids (amino acids 1216 through 1326) in the middle of the protein, and 7 of the 15 specimens that were positive for loss of BAF250a expression had mutations that would result in truncation distal to the epitope.\nThe mutations are common in ovarian carcinomas that are associated with endometriosis (ovarian clear-cell carcinoma and endometrioid carcinoma) but not in the unrelated high-grade serous carcinoma. This suggests that the mutations may be pathogenic, rather than random, events. Mutations in the  PTEN  gene (encoding the phosphatase and tensin homologue) have been described in 20% of endometriotic cysts, 33  and conditional expression of either oncogenic  Kras  or deletion of the  Pten  tumor suppressor in the ovarian surface epithelium in mice was found to induce endometriosis. 34  Expression of oncogenic  Kras  accompanied by simultaneous loss of  Pten  resulted in widely metastatic ovarian carcinoma; however,  KRAS  mutations are not seen in human cases of endometriosis and are uncommon in endometriosis-associated ovarian cancers in humans. By comparing ovarian clear-cell carcinomas to their contiguous atypical endometriotic lesions in two patients, we show that the same mutations may be present in the putative precursor lesions and in the tumors. In contrast, the distant endometriotic lesions do not have  ARID1A  mutations. In the case of ovarian clear-cell carcinoma described in  Figure 3 , the mutation (G6139T [E2047*]) was present before the atypical endometriosis resulted in the development of the immunophenotype associated with the cancer (estrogen-receptor–negative, HNF-1β–positive 24 ), suggesting that the mutation is an early event in neoplastic transformation. Taken together, these data suggest that  ARID1A  is a classic tumor-suppressor gene. Unlike  BRCA  or  TP53  mutations, which can be found in the germ-line DNA, all truncating  ARID1A  mutations were somatic. Deletion of  ARID1A  on one allele results in embryonic lethality in mice. 32\nMutations in  ARID1A  and loss of BAF250a expression were seen preferentially in ovarian clear-cell carcinomas and endometrioid carcinomas, cancers that do not feature the chromosomal instability, nearly ubiquitous  TP53  mutations, and frequent abnormalities in  BRCA  (associated with early breast cancer) seen in high-grade serous carcinomas. 5 , 35  It is possible that defects in genes that alter the accessibility of transcription factors to chromatin, such as  ARID1A,  in addition to mutations in the WNT and PI3 kinase pathways, 25  will help to define ovarian clear-cell carcinomas and endometrioid carcinomas. If such a model is correct, other abnormalities affecting the  ARIDIA  locus or dysregulation of other chromatin-remodeling genes may be found in ovarian clear-cell and endometrioid carcinomas that are negative for an  ARID1A  mutation. This idea is supported by the clinical similarities between ovarian clear-cell carcinomas positive for and those negative for an  ARID1A  mutation.\nThe mechanism by which somatic mutations in  ARID1A  enable the progression of benign endometriosis to carcinoma is unclear; however, our findings are consistent with a critical role for  ARID1A  mutations in the genesis of a substantial fraction of ovarian clear-cell and endometrioid carcinomas.","source_license":"CC0","license_restricted":false}