Tsui Lien Mao

Frequent Mutations of Chromatin Remodeling Gene ARID1A in Ovarian Clear Cell Carcinoma

Remodeling Gone Awry The identification of genes that are mutated at high frequency in human tumors can provide important clues to the molecular pathways that drive tumor growth, which in turn can potentially lead to more effective therapies. Jones et al. (p. 228, published online 9 September; see the cover) looked for such mutations in ovarian clear cell carcinoma, a rare but particularly lethal form of ovarian cancer. Of 42 tumors examined, 57% were found to harbor inactivating mutations in ARID1A, a gene coding for a subunit of the SWI/SNF chromatin remodeling complex, which functions as a master regulator of transcription factor action and gene expression. Thus, proteins associated with the epigenetic control of gene expression can contribute to the development of human cancer. Genetic analysis of a rare but aggressive form of ovarian cancer implicates a chromatin remodeling defect in disease development. 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 previously unknown to be involved in OCCC: PPP2R1A encodes a regulatory subunit of serine/threonine phosphatase 2, and ARID1A encodes adenine-thymine (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.

Frequent Activating Mutations of PIK3CA in Ovarian Clear Cell Carcinoma

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.

Notch3 Gene Amplification in Ovarian Cancer

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 gamma-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.

Mutation and loss of expression of ARID1A in uterine low-grade endometrioid carcinoma.

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 to determine the prevalence of ARID1A inactivation in carcinomas. Mutational analysis of ARID1A was carried out 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 showed a relatively high-frequency 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 carcinomas; 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 showed 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 loss of expression and mutation of ARID1A.

Analysis of DNA Copy Number Alterations in Ovarian Serous Tumors Identifies New Molecular Genetic Changes in Low-Grade and High-Grade Carcinomas

Ovarian serous carcinoma, the most common and lethal type of ovarian cancer, is thought to develop from two distinct molecular pathways. High-grade (HG) serous carcinomas contain frequent TP53 mutations, whereas low-grade (LG) carcinomas arise from serous borderline tumors (SBT) and harbor mutations in KRAS/BRAF/ERBB2 pathway. However, the molecular alterations involved in the progression from SBT to LG carcinoma remain unknown. In addition, the extent of deletion of tumor suppressors in ovarian serous carcinomas has not been well studied. To further address these two issues, we assessed DNA copy number changes among affinity-purified tumor cells from 37 ovarian serous neoplasms including SBT, LG, and HG tumors using high-density 250K single nucleotide polymorphism arrays. Chromosomal instability index as measured by changes in DNA copy number was significantly higher in HG than in LG serous carcinomas. Hemizygous ch1p36 deletion was common in LG serous carcinomas but was rarely seen in SBT. This region contains several candidate tumor suppressors including miR-34a. In contrast, in HG serous carcinomas, significant numbers of amplifications and deletions, including homozygous deletions, were identified. Among homozygous deletions, loci containing Rb1, CDKN2A/B, CSMD1, and DOCK4 were most common, being present in 10.6%, 6.4%, 6.4%, and 4.3%, respectively, in independent 47 affinity-purified HG serous carcinomas. Except for the CDKN2A/B region, these homozygous deletions were not present in either SBT or LG tumors. Our study provides a genome-wide homozygous deletion profile in HG serous carcinomas, which can serve as a molecular foundation to study tumor suppressors in ovarian cancer.

Sinonasal undifferentiated carcinoma and nasopharyngeal-type undifferentiated carcinoma: two clinically, biologically, and histopathologically distinct entities.

Sinonasal undifferentiated carcinoma (SNUC) is a rare aggressive neoplasm arising in the nasal cavity and paranasal sinuses. Primary sinonasal nasopharyngeal-type undifferentiated carcinoma (PSNPC) is an even rarer tumor that has not been adequately reported. Both tumors have been reported to be associated with Epstein–Barr virus (EBV). We studied the clinicopathologic features and EBV status of 36 SNUC and 13 PSNPC patients from Taiwan, an EBV endemic area. The median age of SNUC patients was 53 years (range 20–76 years), with a male/female ratio of approximately 2:1. Five patients had histories of previous nasopharyngeal carcinoma treated with irradiation 6–26 years earlier. The most common locations were nasal cavity and ethmoid sinus. Orbital and intracranial invasion and distant metastasis were frequent findings. The median survival was 10 months. All 36 tumors were negative for EBER-1 by in situ hybridization. The median age of PSNPC patients was 58 years (range 36–75 years), with a male/female ratio of approximately 2:1. The most common location is nasal cavity. Eight patients achieved disease-free survival. Eight tumors had the morphology of lymphoepithelioma, whereas significant inflammatory infiltrate was not detected in the other five tumors. All 13 tumors were positive for EBER-1 by in situ hybridization. Because of the difference in the relation with EBV, prognosis, and response to radiotherapy, SNUC and PSNPC should be considered as two entirely different entities. The most important criteria for PSNPC are vesicular nuclei, syncytial pattern, spindle cells, and absence of necrosis.

Cystic and adenofibromatous clear cell carcinomas of the ovary: distinctive tumors that differ in their pathogenesis and behavior: a clinicopathologic analysis of 122 cases.

Ovarian clear cell carcinomas (CCC) typically present as large adnexal, stage I tumors and are generally considered highly malignant. They are frequently associated with endometriosis and, less often with clear cell adenofibromas. We hypothesized that CCCs are a heterogeneous group of tumors, some arising from a cyst and others from an adenofibroma. To test this hypothesis, 122 cases of CCC were retrieved from the surgical pathology files of National Taiwan University Hospital (74), The Johns Hopkins Hospital (23), and Serei Mikatahara General Hospital (23) (1985 to 2006). Cases were divided into 3 subgroups: (1) cystic, (2) adenofibromatous, and (3) indeterminate. Various features were analyzed including: age, race, laterality, tumor size, architectural pattern (papillary, tubulo-cystic, solid, mixed patterns), grade, mitotic index, association with endometriosis including atypical endometriosis/intraepithelial carcinoma, stage and survival. Nearly 70% of all the patients were diagnosed as stage I. The 2-year and 5-year survival (all stages) was 78% and 68%, respectively. Striking clinicopathologic differences were observed between cystic and adenofibromatous CCCs. Cystic CCC was more frequently diagnosed as stage I compared with adenofibromatous CCC (75% vs. 44%). Conversely, adenofibromatous CCCs were diagnosed more often in advanced stages (stages II-IV) compared with cystic CCCs (56% vs. 18%). Both the cystic and adenofibromatous CCC forms were associated with endometriosis and atypical endometriosis/intraepithelial carcinoma, but the frequency was much higher in the cystic group. Specifically, endometriosis was found in 91% of cystic CCCs and atypical endometriosis/intraepithelial carcinoma in 62% of these cases, whereas endometriosis was found in 44% of adenofibromatous CCCs and atypical endometriosis/intraepithelial carcinoma in 11% of cases. A predominantly papillary pattern was seen in 47% of cystic CCCs, whereas none of the adenofibromatous carcinomas displayed a predominantly papillary pattern. A more favorable outcome was observed for cystic CCCs compared with adenofibromatous CCCs (all stages) which was accounted for by the high proportion of stage I tumors. The 2-year and 5-year survival for the cystic CCCs was 82% and 77% and for the adenofibromatous CCCs (all stages), 62% and 37%, respectively. In summary, subdividing ovarian CCCs into cystic and adenofibromatous CCC reveals differences in a number of clinicopathologic features including their association with endometriosis, histologic patterns, stage distribution, and clinical behavior. Because there were a relatively small number of adenofibromatous CCCs in this series, additional cases must be studied to confirm these findings.

IGF2BP3 (IMP3) expression is a marker of unfavorable prognosis in ovarian carcinoma of clear cell subtype

Clear cell carcinoma is an uncommon subtype of ovarian carcinoma, accounting for 10% of cases. Clear cell carcinoma typically presents with stage I or II disease, and in this setting prognostic markers could aid in management decisions, in particular the decision to treat with adjuvant chemotherapy. We tested whether expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3, also known as IMP3) can serve as a new biomarker to predict outcome for patients with clear cell carcinoma and other subtypes of ovarian carcinoma. The expression of IGF2BP3 was evaluated by immunohistochemistry in 475 ovarian carcinomas of different subtypes and correlated with disease-specific survival. IGF2BP3 antibody specificity was validated by correlation of IGF2BP3 protein with mRNA expression level in a series of 35 ovarian carcinomas (r=0.849, P<0.0001). IGF2BP3 protein expression was an independent marker of reduced disease-specific survival (risk ratio 2.9, 95% confidence interval 1.4–5.8) in the clear cell subtype (N=128), but not in high-grade serous (N=198) or endometrioid (N=121) carcinomas. The prognostic significance of IGF2BP3 expression for reduced disease-specific survival (risk ratio 2.6, 95% confidence interval 1.3–5.0) was confirmed in an independent series of cases (N=150) from three different centers in North America. We conclude that IGF2BP3 is the first biomarker of prognostic significance in ovarian clear cell carcinoma that has been validated in an independent case series.

Loss of ARID1A expression is an early molecular event in tumor progression from ovarian endometriotic cyst to clear cell and endometrioid carcinoma.

Objectives ARID1A is a recently identified tumor suppressor participating in chromatin remodeling. Somatic inactivating mutations of ARID1A and loss of its expression occur frequently in ovarian clear cell and endometrioid carcinomas and in uterine endometrioid carcinomas. Because endometriotic epithelium is thought to be the cell of origin of most ovarian clear cell and endometrioid carcinomas, we undertook an analysis of ARID1A expression of these tumors arising within an endometriotic cyst (endometrioma). Materials and Methods Our immunohistochemical study set consisted of 47 endometriotic cysts containing clear cell carcinoma in 24 cases, well-differentiated ovarian endometrioid carcinoma in 20 cases, and mixed clear cell and endometrioid carcinoma in 3 cases. Results ARID1A loss was observed in 31 (66%) of 47 carcinomas; and therefore, these cases were informative for determining the temporal sequence of loss of ARID1A expression in tumor progression. In 16 of the 47 cases, ARID1A immunoreactivity was retained in both the endometriotic cyst and the carcinoma; and thus, these cases were not informative. All of the 31 informative cases showed loss of ARID1A immunoreactivity in the carcinoma and in the endometriotic cyst epithelium in direct continuity with the carcinoma but not in the cyst epithelium that was not adjacent to the tumor. Conclusions Loss of ARID1A function as shown by loss of expression, presumably due to mutations, is an early molecular event in the development of most ovarian clear cell and endometrioid carcinomas arising in endometriomas.

Clinicopathological Significance of Loss of ARID1A Immunoreactivity in Ovarian Clear Cell Carcinoma

Recent genome-wide analysis has demonstrated that somatic mutations in ARID1A (BAF250) are the most common molecular genetic changes in ovarian clear cell carcinoma (OCCC). ARID1A mutations, which occur in approximately half of OCCC cases, lead to deletion of the encoded protein and inactivation of the putative tumor suppressor. In this study, we determined the significance of loss of ARID1A immunoreactivity with respect to several clinicopathological features in a total of 149 OCCCs. First, we demonstrated that ARID1A immunohistochemistry showed concordance with the mutational status in 91% of cases with 100% sensitivity and 66% specificity. Specifically, among 12 OCCC cases for which ARIDA mutational status was known, ARIDIA immunoreactivity was undetectable in all 9 cases harboring ARID1A mutations and was undetectable in one of 3 cases with wild-type ARID1A. With respect to the entire cohort, ARID1A immunoreactivity was undetectable in 88 (59%) of 149 OCCCs. There was no significant difference between ARID1A negative and positive cases in terms of histopathologic features, age, clinical stage, or overall survival. In conclusion, this study provides further evidence that mutations in ARID1A resulted in loss of ARID1A protein expression in OCCC, although no significant differences between ARID1A positive and negative cases were observed with respect to any clinicopathological features examined.