Daichi Maeda

Integrated molecular analysis of clear-cell renal cell carcinoma

Clear-cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer and its molecular pathogenesis is incompletely understood. Here we report an integrated molecular study of ccRCC in which ≥100 ccRCC cases were fully analyzed by whole-genome and/or whole-exome and RNA sequencing as well as by array-based gene expression, copy number and/or methylation analyses. We identified a full spectrum of genetic lesions and analyzed gene expression and DNA methylation signatures and determined their impact on tumor behavior. Defective VHL-mediated proteolysis was a common feature of ccRCC, which was caused not only by VHL inactivation but also by new hotspot TCEB1 mutations, which abolished Elongin C–VHL binding, leading to HIF accumulation. Other newly identified pathways and components recurrently mutated in ccRCC included PI3K-AKT-mTOR signaling, the KEAP1-NRF2-CUL3 apparatus, DNA methylation, p53-related pathways and mRNA processing. This integrated molecular analysis unmasked new correlations between DNA methylation, gene mutation and/or gene expression and copy number profiles, enabling the stratification of clinical risks for patients with ccRCC.

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.

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.

Glypican-3 expression in clear cell adenocarcinoma of the ovary

Glypican-3 is a heparan sulfate proteoglycan that is overexpressed in various neoplasms such as hepatocellular carcinoma, malignant melanoma, and testicular yolk sac tumor. Glypican-3 is currently regarded as a tumor marker and potential target for immunotherapy. To clarify the significance of glypican-3 expression in ovarian clear cell adenocarcinoma, we evaluated glypican-3 expression by immunohistochemistry in nonneoplastic and neoplastic ovaries, and other Müllerian duct derivatives including endometrium in different menstrual phases. Among the benign lesions examined, glypican-3 expression was identified exclusively in the endometrial epithelium in the gestational period. A total of 213 cases of ovarian adenocarcinoma, including 94 clear cell adenocarcinomas, were studied. Glypican-3 expression was observed in 44% of clear cell adenocarcinomas, whereas it was rarely observed in other histological subtypes: mucinous (4%), endometrioid (5%), and serous (11%; P<0.0001). All six ovarian yolk sac tumors showed diffuse immunoreactivity for glypican-3. In cases of clear cell adenocarcinoma, no correlations were found between glypican-3 expression and clinicopathological factors, such as tumor stage, lymph node metastasis, peritoneal dissemination, and death rate. However, glypican-3 expression was significantly associated with poor overall survival in stage III/IV clear cell adenocarcinoma cases. Our results suggest that overexpression of glypican-3 may be related to the development and aggressive behavior of ovarian clear cell adenocarcinoma.

Pathogenesis and the role of ARID1A mutation in endometriosis-related ovarian neoplasms.

Endometriosis-related ovarian neoplasms (ERONs) are a unique group of tumors as they are associated with endometriosis, especially endometriosis presenting as an ovarian endometriotic cyst (endometrioma). ERONs include clear cell carcinoma, endometrioid carcinoma, and seromucinous borderline tumor. A growing body of evidence from both clinicopathologic and molecular studies suggests that most, if not all, ERONs develop from endometriotic cyst epithelium through different stages of tumor progression. The endometriotic cyst contains abundant iron-induced reactive oxygen species that are thought to be mutagenic, and chronic exposure of cystic epithelium to this microenvironment facilitates the accumulation of somatic mutations that ultimately result in tumor development. Molecular analyses of ERONs, including genome-wide screens, have identified several molecular genetic alterations that lead to aberrant activation or inactivation of pathways involving ARID1A, PI3K, Wnt, and PP2A. Among all molecular genetic changes identified to date, inactivating mutations of the ARID1A tumor suppressor gene are the most common in ERON. Understanding the molecular changes and pathogenesis involved in the development of ERON is fundamental for future translational studies aimed at designing new diagnostic tests for early detection and identifying critical molecular features for targeted therapeutics.

Frequent somatic mutations of the telomerase reverse transcriptase promoter in ovarian clear cell carcinoma but not in other major types of gynaecological malignancy.

Up‐regulated expression of telomerase reverse transcriptase (TERT) and subsequent maintenance of telomere length are essential in tumour development. Recent studies have implicated somatic gain‐of‐function mutations at the TERT promoter as one of the mechanisms that promote transcriptional activation of TERT; however, it remains unclear whether this genetic abnormality is prevalent in gynaecological neoplasms. We performed mutational analysis in a total of 525 gynaecological cancers, and correlated TERT promoter mutations with clinicopathological features. With the exception of ovarian clear cell carcinomas, in which mutations were found in 37 (15.9%) of 233 cases, the majority of gynaecological malignancies were wild‐type. TERT promoter mutation does not appear to be an early event during oncogenesis, as it was not detected in the contiguous endometriosis associated with ovarian clear cell carcinoma. Ovarian clear cell carcinoma cell lines with TERT promoter mutations exhibited higher TERT mRNA expression than those with wild‐type sequences (p = 0.0238). TERT promoter mutation tended to be mutually exclusive with loss of ARID1A protein expression (p = 4.4 × 10–9) and PIK3CA mutation (p = 0.0019) in ovarian clear cell carcinomas. No associations with disease‐specific survival were observed for ovarian clear cell carcinoma. The above results, in conjunction with our previous report showing longer telomeres in ovarian clear cell carcinomas relative to other types of ovarian cancer, suggests that aberrations in telomere biology may play an important role in the pathogenesis of ovarian clear cell carcinoma. Copyright

Idiopathic retroperitoneal fibrosis, inflammatory aortic aneurysm, and inflammatory pericarditis—-Retrospective analysis of 11 case histories

Retroperitoneal fibrosis, inflammatory aortic aneurysm, and pericardial and mediastinal fibrosis are characterized by infiltration of immuno-inflammatory cells and deposition of thickened fibrous tissues. Several recent studies suggested that an immunoglobulin-G4 (IgG4)-related immunological mechanism may play a role in these diseases. By searching the clinical database of patients admitted to our department between 2000 and 2010, we summarized the clinical data of 11 patients who were diagnosed to have these disorders. The diagnoses were idiopathic retroperitoneal fibrosis (8 cases), mediastinal and/or pericardial fibrosis (4 cases), inflammatory abdominal aneurysm (2 cases), and inflammatory coronary periarteritis (1 case). Hypertension, diabetes, and dyslipidemia were found in 45%, 36%, and 55%, respectively, in these patients, and they were all either current or former smokers. Two patients with pericardial involvement showed a rushed clinical course, resulting in in-hospital death. Serum levels of IgG were elevated in 67%, and soluble interleukin-2 receptor was elevated in 75%, when measured. Immunohistochemical analysis showed marked infiltration of IgG4-positive plasma cells in the pericardium in patients who died of constrictive pericarditis. Our data support the notion that immune-inflammatory mechanism, which might be IgG4-related sometimes, may play a role in idiopathic retroperitoneal fibrosis, inflammatory aortic aneurysm, and mediastinal/pericardial fibrosis, although clinical course may differ substantially.

β-catenin (CTNNB1) S33C mutation in ovarian microcystic stromal tumors.

Microcystic stromal tumor (MCST) is a recently described subtype of ovarian tumor characterized by prominent microcystic histologic pattern and diffuse immunoreactivity for CD10 and vimentin. However, its pathobiology, particularly its histogenesis, remains largely unclear. Here, we report 2 cases of ovarian MCST, in which we have performed extensive histologic, immunohistochemical, and genetic investigations to determine its distinct nature among ovarian neoplasms. The patients were 32 and 41 years of age. Both tumors were solid and cystic masses involving the right ovary. Microscopically, tumor cells with generally bland, round-to-ovoid nuclei grew in microcystic, macrocystic, and solid patterns. Intervening thick fibrous stroma was observed. Immunohistochemically, tumor cells were diffusely and strongly positive for CD10, vimentin, and Wilms tumor 1. Furthermore, we detected aberrant nuclear expression of &bgr;-catenin protein in both cases. Of interest, mutation analyses revealed the presence of an identical point mutation, c.98C>G, in exon 3 of &bgr;-catenin (CTNNB1) in both tumors. This is an oncogenic mutation that causes replacement of serine with cysteine at codon 33, leading to the loss of a phosphorylation site in the &bgr;-catenin protein. The results of this study strongly suggest that dysregulation of the Wnt/&bgr;-catenin pathway plays a fundamental role in the pathogenesis of ovarian MCST. Finally, by comparing the immunophenotype of MCST with its histologic mimics and other ovarian sex cord-stromal tumors, we were able to identify unique features of MCST and a panel of markers useful in differential diagnosis.

Ribonucleotide reductase M2 subunit is a novel diagnostic marker and a potential therapeutic target in bladder cancer

Morikawa T, Maeda D, Kume H, Homma Y & Fukayama M
(2010) Histopathology57, 885–892

Hunner-Type (Classic) Interstitial Cystitis: A Distinct Inflammatory Disorder Characterized by Pancystitis, with Frequent Expansion of Clonal B-Cells and Epithelial Denudation

Interstitial cystitis (IC) is a chronic bladder disease with urinary frequency, bladder discomfort or bladder pain of unknown etiology. Based on cystoscopic findings, patients with IC are classified as either Hunner-type/classic IC (HIC), presenting with a specific Hunner lesion, or non-Hunner-type IC (NHIC), presenting with no Hunner lesion, but post-hydrodistension mucosal bleeding. Inflammatory cell infiltration, composed predominantly of lymphocytes, plasma cells and epithelial denudation, has in the past been documented as a major pathological IC finding. However, the significance of the pathological evaluation of IC, especially with regard to the difference between HIC and NHIC, has been downplayed in recent years. In this study, we performed immunohistochemical quantification of infiltrating T-lymphocytes, B-lymphocytes and plasma cells, and measured the amount of residual epithelium in urinary bladder biopsy specimens taken from patients with HIC and NHIC, and those with no IC, using image analysis software. In addition, in situ hybridization of the light chains was performed to examine clonal B-cell expansion. Lymphoplasmacytic infiltration was significantly more severe in HIC specimens than in NHIC specimens (P <0.0001). Substantial lymphoplasmacytic inflammation (≥200 cells/mm2) was observed in 93% of HIC specimens, whereas only 8% of NHIC specimens were inflamed. Plasmacytic infiltration was more prominent in HIC specimens compared with NHIC and non-IC cystitis specimens (P <0.005). Furthermore, expansion of light-chain-restricted B-cells was observed in 31% of cases of HIC. The amount of residual epithelium was decreased in HIC specimens compared with NHIC specimens and non-IC cystitis specimens (P <0.0001). These results suggest that NHIC and HIC are distinct pathological entities, with the latter characterized by pancystitis, frequent clonal B-cell expansion and epithelial denudation. An abnormality in the B-cell population may be involved in the pathogenesis of HIC.