Ovarian Clear Cell Carcinoma (OCCC) is an aggressive human cancer that is generally resistant to therapy. To explore the genetic origin of OCCC, we determined the exomic sequences of eight tumors after immunoaffinity purification of cancer cells. Through comparative analyses of normal cells from the same patients, we identified four genes that were mutated in at least two tumors. PIK3CA, which encodes a subunit of phosphatidylinositol-3 kinase, and KRAS, which encodes a well known oncoprotein, had previously been implicated in OCCC. The other two mutated genes were novel: PPP2R1A encodes a regulatory subunit of serine/threonine phosphatase 2 and ARID1A encodes AT-rich interactive domain-containing protein 1A, which participates in chromatin remodeling. The nature and pattern of the mutations suggest that PPP2R1A functions as an oncogene and ARID1A as a tumor suppressor gene. In a total of 42 OCCCs, 7% had mutations in PPP2R1A and 57% had mutations in ARID1A. These results suggest that aberrant chromatin remodeling contributes to the pathogenesis of OCCC.

Ovarian clear cell carcinoma (CCC) is one of the most malignant types of ovarian carcinomas, particularly at advanced stages. Unlike the more common type of ovarian cancer, high-grade serous carcinoma, ovarian CCC is often resistant to platinum-based chemotherapy, and therefore an effective treatment for this tumor type at advanced stages is urgently needed. In this study, we analyzed 97 ovarian CCCs for sequence mutations in KRAS, BRAF, PIK3CA, TP53, PTEN, and CTNNB1 as these mutations frequently occur in other major types of ovarian carcinomas. The samples included 18 CCCs for which affinity-purified tumor cells from fresh specimens were available, 69 microdissected tumors from paraffin tissues, and 10 tumor cell lines. Sequence mutations of PIK3CA, TP53, KRAS, PTEN, CTNNB1, and BRAF occurred in 33%, 15%, 7%, 5%, 3%, and 1% of CCC cases, respectively. Sequence analysis of PIK3CA in 28 affinity-purified CCCs and CCC cell lines showed a mutation frequency of 46%. Samples with PIK3CA mutations showed intense phosphorylated AKT immunoreactivity. These findings demonstrate that ovarian CCCs have a high frequency of activating PIK3CA mutations. We therefore suggest that the use of PIK3CA-targeting drugs may offer a more effective therapeutic approach compared with current chemotherapeutic agents for patients with advanced-stage and recurrent CCC.

Gene amplification is one of the common mechanisms that activate oncogenes. In this study, we used single nucleotide polymorphism array to analyze genome-wide DNA copy number alterations in 31 high-grade ovarian serous carcinomas, the most lethal gynecologic neoplastic disease in women. We identified an amplicon at 19p13.12 in 6 of 31 (19.5%) ovarian high-grade serous carcinomas. This amplification was validated by digital karyotyping, quantitative real-time PCR, and dual-color fluorescence in situ hybridization (FISH) analysis. Comprehensive mRNA expression analysis of all 34 genes within the minimal amplicon identified Notch3 as the gene that showed most significant overexpression in amplified tumors compared with nonamplified tumors. Furthermore, Notch3 DNA copy number is positively correlated with Notch3 protein expression based on parallel immunohistochemistry and FISH studies in 111 high-grade tumors. Inactivation of Notch3 by both γ-secretase inhibitor and Notch3-specific small interfering RNA suppressed cell proliferation and induced apoptosis in the cell lines that overexpressed Notch3 but not in those with minimal amount of Notch3 expression. These results indicate that Notch3 is required for proliferation and survival of Notch3-amplified tumors and inactivation of Notch3 can be a potential therapeutic approach for ovarian carcinomas. (Cancer Res 2006; 66(12): 6312-8)

ARID1A is a recently identified tumor suppressor gene that is mutated in approximately 50% of ovarian clear cell and 30% of ovarian endometrioid carcinomas. The mutation is associated with loss of protein expression as assessed by immunohistochemistry. In this study, we evaluated ARID1A immunoreactivity in a wide variety of carcinomas in order to determine the prevalence of ARID1A inactivation in carcinomas; mutational analysis of ARID1A was performed in selected cases. Immunoreactivity was not detected (corresponding to inactivation or mutation of ARID1A) in 36 (3.6%) of 995 tumors. Uterine low-grade endometrioid carcinomas demonstrated a relatively high frequency of loss of ARID1A expression, as 15 (26%) of 58 cases were negative. The other tumor that had a relatively high frequency loss of ARID1A expression was gastric carcinoma (11%). Mutational analysis showed 10 (40%) of 25 uterine endometrioid carcinoma, none of 12 uterine serous carcinomas and none of 56 ovarian serous and mucinous carcinomas harbored somatic ARID1A mutations. All mutations in endometrioid carcinomas were nonsense or insertion/ deletion mutations and tumors with ARID1A mutations demonstrated complete loss or clonal loss of ARID1A expression. In conclusion, this study is the first large-scale analysis of a wide variety of carcinomas showing that uterine low-grade endometrioid carcinoma is the predominant tumor type harboring ARID1A mutations and frequent loss of ARID1A expression. These findings suggest that the molecular pathogenesis of low-grade uterine endometrioid carcinoma is similar to that of ovarian low-grade endometrioid and clear cell carcinoma, tumors that have previously been shown to have a high frequency of loss of expression and mutation of ARID1A.

Dedifferentiated endometrial carcinoma is an aggressive type of endometrial cancer that contains a mix of low grade endometrioid and undifferentiated carcinoma components. We performed targeted sequencing of 8 dedifferentiated endometrial carcinomas and identified somatic frameshift/nonsense mutations in SMARCA4, a core member of the switch/sucrose non-fermenting (SWI/SNF) complex, in the undifferentiated components of 4 tumors. Immunohistochemical analysis confirmed the loss of SMARCA4 in the undifferentiated component of these 4 SMARCA4-mutated cases while the corresponding low grade endometrioid component showed retained SMARCA4 expression. An expanded survey of another member of the SWI/SNF complex showed SMARCB1 loss in the undifferentiated component of 2 SMARCA4-intact tumors. Subsequent immunohistochemical analysis of SMARCA4 and SMARCB1 was done in an additional set of 22 centrally reviewed dedifferentiated endometrial carcinomas and 31 grade 3 endometrioid carcinomas. Combining the results from the index and the expansion set, 15 of 30 (50%) of the dedifferentiated endometrial carcinomas examined showed either SMARCA4 loss (37%) or SMARCB1 loss (13%). The loss of SMARCA4 or SMARCB1 was mutually exclusive and occurred only in the undifferentiated component. All 31 grade 3 endometrioid carcinomas showed intact SMARCA4/SMARCB1 expression. The majority (73%) of the SMARCA4-deficient and half of SMARCB1-deficient undifferentiated component developed in a mismatch repair protein (MMR)-deficient molecular context. The observed spatial association between SMARCA4/SMARCB1 loss and histologic dedifferentiation suggests that loss of these SWI/SNF complex proteins may contribute to the development of dedifferentiated endometrial carcinoma.