Mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial-mesenchymal transition.

Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial-mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.

Trop-2 protein overexpression is an independent marker for predicting disease recurrence in endometrioid endometrial carcinoma.

BACKGROUND: Endometrial cancer is the most common gynecologic malignancy in developed countries. Trop-2 is a glycoprotein involved in cellular signal transduction and is differentially overexpressed relative to normal tissue in a variety of human adenocarcinomas, including endometrioid endometrial carcinomas (EEC). Trop-2 overexpression has been proposed as a marker for biologically aggressive tumor phenotypes. METHODS: Trop-2 protein expression was quantified using tissue microarrays consisting of formalin-fixed paraffin-embedded specimens from 118 patients who underwent surgical staging from 2001-9 by laparotomy for EEC. Clinicopathologic characteristics including age, stage, grade, lymphovascular space invasion, and medical comorbidities were correlated with immunostaining score. Univariate and multivariate analyses were performed for overall survival, disease-free survival, and progression-free survival in relation to clinical parameters and Trop-2 protein expression. RESULTS: Clinical outcome data were available for 103 patients. Strong Trop-2 immunostaining was significantly associated with higher tumor grade (p=0.02) and cervical involvement (p<0.01). Univariate analyses showed a significant association with reduced disease-free survival (DFS) (p=0.01), and a trend towards significance for overall and progression-free survival (p=0.06 and p=0.05, respectively). Multivariate analyses revealed Trop-2 overexpression and advanced FIGO stage to be independent prognostic factors for poor DFS (p=0.04 and p <0.001, respectively). CONCLUSIONS: Trop-2 protein overexpression is significantly associated with higher tumor grade and serves as an independent prognostic factor for DFS in endometrioid endometrial cancer.

Trop-2 overexpression in poorly differentiated endometrial endometrioid carcinoma: implications for immunotherapy with hRS7, a humanized anti-trop-2 monoclonal antibody.

OBJECTIVE: We evaluated the expression of human trophoblast cell surface marker (Trop-2) in endometrial endometrioid carcinoma (EEC) and the potential application of hRS7, a humanized monoclonal anti-Trop-2 antibody, as a therapeutic agent against poorly differentiated EEC. METHODS: Trop-2 expression was evaluated by immunohistochemistry in 131 EEC with different degrees of differentiation and 32 normal endometrial controls (NEC). Trop-2 expression was also evaluated by quantitative real-time polymerase chain reaction and flow cytometry in 3 primary EEC cell lines derived from patients harboring poorly differentiated EEC. Finally, the sensitivity of grade 3 EEC cell lines to hRS7 antibody-dependent cellular cytotoxicity was tested in standard 5-hour Cr release assays. RESULTS: Trop-2 expression was detected in 126 (96.2%) of 131 EEC samples. Tumor tissues showed markedly increased Trop-2 positivity compared with NEC (P = 0.001). Trop-2 expression was significantly higher in all grades of EEC versus NEC. Grade 3 tumors displayed significantly stronger Trop-2 immunostaining compared with grade 1 EEC (P = 0.01). High Trop-2 expression by quantitative real-time polymerase chain reaction and flow cytometry was found in 1 grade 3 EEC primary cell line (EEC-ARK-1). Unlike Trop-2-negative EEC cell lines, EEC-ARK-1 was found highly sensitive to hRS7-mediated antibody-dependent cellular cytotoxicity in vitro (range of killing, 33.9%-50.6%; P = 0.004). Human serum did not significantly inhibit hRS7-mediated cytotoxicity against EEC-ARK-1 (P = 0.773). CONCLUSIONS: Trop-2 is highly expressed in EEC, and its expression is significantly higher in poorly differentiated EEC when compared with well-differentiated EEC. Primary grade 3 EECs overexpressing Trop-2 are highly sensitive to hRS7-mediated cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for the treatment of high-grade EEC refractory to standard treatment modalities.

Mammaglobin B is an independent prognostic marker in epithelial ovarian cancer and its expression is associated with reduced risk of disease recurrence.

BACKGROUND: Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients. METHODS: MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome. RESULTS: MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08-0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17-0.99, p = 0.048) had an independent prognostic value for disease-free survival. CONCLUSION: This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.

Claudin 4 identifies a wide spectrum of epithelial neoplasms and represents a very useful marker for carcinoma versus mesothelioma diagnosis in pleural and peritoneal biopsies and effusions.

We evaluated the usefulness of the tight-junction associated protein Claudin 4 (CL-4) in the diagnosis of mesothelioma and mimickers, analyzing biopsies from 454 tumors, including 82 mesotheliomas, 336 carcinomas of different origin (278 primary, 58 metastatic to serosae), 36 nonepithelial spindle cell neoplasms, as well as 97 cytological samples from reactive effusions (12), mesothelioma (23) and metastatic carcinomas (62). CL-4 was consistently negative in normal and reactive mesothelium, as well as in all 82 mesotheliomas. In contrast, strong reactivity was found in 57/58 serosal metastasis, and in 245/278 primary carcinomas, with uppermost expression (150/153) in those most frequently involved in the differential with mesothelioma (lung, breast, gastrointestinal tract, pancreas, ovary, primary serous papillary carcinoma of peritoneum). On effusions, reactive and neoplastic mesothelial cells were regularly negative, while metastatic tumor cells stained positively in 60/62 (96.8%) cases. Among spindle cell neoplasms, only 2/9 biphasic synovial sarcomas and 4/4 follicular dendritic cell sarcomas stained positively. Results indicate that CL-4 reacts with the majority of epithelial neoplasms that often metastasize to serous membranes, representing a pancarcinoma marker with extremely high sensitivity and specificity. CL-4 may be considered a primary immunohistochemical reagent to rule out the diagnosis of mesothelioma.

Placental morphology in pregnancies associated with pregravid obesity.

OBJECTIVE: Pre-gravid obesity is associated with increased morbidity and mortality for both mother and offspring. We aimed to investigate the changes in placental structure and to characterize the placental inflammatory status in obese women. METHODS: Placentas from 10 women with pregravid body mass index ≥ 30 kg/m(2) were analyzed histopathologically and compared with those from 10 normal weight women. The effect of pregravid obesity on placental structure was investigated by examining placental maturity, angiogenesis, inflammatory response, and vascular abnormalities. Hematoxylin and eosin stain, immunohistochemical analysis, and morphometric analysis were performed. RESULTS: Immaturity of the villous tree was demonstrated more often in the pregravid obesity group (p = 0.005). The villous tree was mainly represented by villi of larger diameter (p = 0.001) and lower number (p = 0.023) compared with controls. By contrast, the number of capillaries per villus was increased in the obese group (p = 0.001), while the global number of capillaries per surface unit did not differ in the studied groups (p = 1). There was no difference between the pre-gravid obese group and controls in inflammatory status and vascular pathology. CONCLUSIONS: Pregravid obesity is associated with focal immaturity of the villous tree and angiogenetic abnormalities of the placenta.

Identical TP53 mutations in pelvic carcinosarcomas and associated serous tubal intraepithelial carcinomas provide evidence of their clonal relationship.

Pelvic carcinosarcomas (PCSs) are rare aggressive biphasic tumors that localize in the ovary, fallopian tube, or peritoneum and present frequently as bilateral disease. We undertook a morphological, p53 immunohistochemical and TP53 gene mutational analysis study in a single institution cohort of 16 PCSs in order to investigate the nature of bilateral tumors and to shed light on their origin and pathogenesis. Of the 16 patients, 10 presented with bilateral disease, 6 with a carcinosarcoma in both adnexa, and the remaining cases with a carcinosarcoma in one adnexum and a carcinoma in the opposite. The carcinoma component showed high-grade serous features in 13/16 of cases (81 %). In 10 patients (63 %), a serous tubal intraepithelial carcinoma (STIC) was found, in one case bilateral, making a total of 11 STICs. STIC was found only in cases with a carcinoma component with high-grade serous features. All 10 bilateral tumors and all 11 PCS-associated STICs showed a similar p53 immunostaining pattern. At mutation analysis of the TP53 gene, all five bilateral PCS contained an identical mutation in both localizations. Furthermore, a TP53 mutation was found in 8 of 10 STICs, with an identical mutation in the associated PCS. The finding of similar p53 immunostaining in all bilateral cases and identical TP53 mutations in most PCS-associated STIC provides evidence for a clonal relation between these neoplastic lesions, supporting a metastatic nature of bilateral PCS and suggesting that they have an extraovarian origin in a STIC.

Trop-2 overexpression as an independent marker for poor overall survival in ovarian carcinoma patients.

BACKGROUND: Prognostic factors currently available are insufficient to predict the clinical course of epithelial ovarian cancer (EOC). In a previous microarray study we identified the human trophoblast cell surface antigen Trop-2 as one of the top differentially expressed genes in serous papillary EOCs compared to normal human ovarian surface epithelial (HOSE) short-term cultures. The aim of the present investigation was to analyse Trop-2 expression at mRNA and protein level and to assess its prognostic significance in EOC. METHODS: Using quantitative real-time PCR we tested a total of 104 fresh-frozen EOC tissues and 24 HOSE for Trop-2 mRNA expression. Trop-2 protein expression was then examined by immunohistochemistry in matched formalin-fixed paraffin-embedded EOC samples and in 13 normal ovaries. Finally, we correlated Trop-2 expression to EOC conventional clinicopathological features and patient outcomes. RESULTS: We found a significant Trop-2 mRNA and protein upregulation in EOCs compared to normal controls (p<0.001). Trop-2 protein overexpression was significantly associated with the presence of ascites (p=0.04) and lymph node metastases (p=0.04). By univariate survival analysis, Trop-2 protein overexpression was significantly associated with decreased progression-free (p=0.02) and overall survival (p=0.01). Importantly, Trop-2 protein overexpression was an independent prognostic marker for shortened survival time in multivariate Cox regression analysis (p=0.04, HR=2.35, CI(95%)=1.03-5.34). CONCLUSIONS: Our results indicate, for the first time, that Trop-2 protein overexpression correlates with an aggressive malignant phenotype and may constitute a novel prognostic factor for EOC. The targeting of Trop-2 overexpression by immunotherapeutic strategies may represent an attractive and potentially effective approach in patients harbouring EOC.

Overexpression of mammaglobin B in epithelial ovarian carcinomas.

OBJECTIVE: Mammaglobin B is a uteroglobin gene family member recently found highly differentially expressed in serous papillary ovarian cancer by gene expression profiling. In order to evaluate its potential as a novel ovarian cancer biomarker, in this study we quantified and compared Mammaglobin B expression in various histologic types of epithelial ovarian carcinomas (EOC). METHODS: Mammaglobin B expression was evaluated by real-time PCR and/or immunohistochemistry in fresh-frozen biopsies and paraffin-embedded tissues derived from a total of 137 patients including 69 primary EOC with different histologies, 28 serous papillary omental metastasis, 8 borderline tumors, 26 benign cystadenomas and 14 normal ovaries. RESULTS: High levels of Mammaglobin B gene expression were detected in 100% (68 out of 68) of the ovarian cancer biopsies tested by real-time PCR. In contrast, normal human ovarian surface epithelium (HOSE) expressed negligible levels of Mammaglobin B mRNA (EOC versus HOSE, p<0.01). Although Mammaglobin B gene expression levels were higher in endometrioid, mucinous and undifferentiated tumors when compared to serous papillary tumors, clear cell tumors and those with mixed histology, these differences were not statistically significant. In agreement with real-time PCR results, EOC were found to express significantly higher levels of Mammaglobin B protein when compared to normal ovaries and benign cystadenomas (p<0.01). However, only 29 out of 68 (42%) of the EOC samples found positive for Mammaglobin B by real-time PCR showed immunoreactivity by IHC. CONCLUSIONS: Mammaglobin B gene is highly expressed in EOC and may represent a novel molecular marker for multiple histological types of ovarian cancer. Additional studies to evaluate the clinical utility of Mammaglobin B as a diagnostic and/or therapeutic target in ovarian cancer are warranted.

Differential gene expression profiles between tumor biopsies and short-term primary cultures of ovarian serous carcinomas: identification of novel molecular biomarkers for early diagnosis and therapy.

OBJECTIVE: To identify novel molecular biomarkers useful for the early diagnosis and therapy of ovarian cancer by gene expression profiling. To compare the genetic fingerprints of flash-frozen ovarian serous carcinomas to those of matched highly purified primary tumor cell cultures. METHODS: Gene expression profiles of 19 flash-frozen ovarian serous papillary carcinoma (OSPC) were analyzed and compared to 15 controls (highly purified human ovarian surface epithelium short-term cultures, HOSE) using oligonucleotide microarrays complementary to >14,500 human genes. In addition, gene expression profiling of 5 highly purified primary OSPC cultured in vitro for less than 2 weeks was compared to flash-frozen ovarian carcinoma biopsies obtained from matched samples. Quantitative RT-PCR and IHC staining techniques were used to validate microarray data at RNA and protein levels for some of the differentially expressed genes. RESULTS: Unsupervised analysis of gene expression data readily distinguished normal tissue from flash-frozen OSPC and identified 901 and 557 genes that exhibited >3-fold up-regulation or down-regulation, respectively, in OSPC when compared to HOSE. Mammaglobin 2, an ovarian secreted protein, was identified as the top differentially expressed gene in OSPC (19 out 19 OSPC versus 0 out of 15 HOSE) with over 827-fold up-regulation relative to HOSE. The claudin and kallikrein family of proteins including the clostridium perfringens enterotoxin receptors claudin 3 and 4, kallikreins 6, 7, 8, 10, 11 and the immunomodulatory molecule B7-H4 were found among the most highly overexpressed genes in OSPC when compared to HOSE. Genetic fingerprints of flash-frozen OSPC were found to have high correlation with those of purified primary OSPC short-term in vitro cultures with only 31 out of 8,637 genes (0.35%) differentially expressed between the two groups. CONCLUSIONS: Short-term in vitro culture of primary ovarian carcinomas may greatly increase the purity of ovarian tumor RNA available for gene expression profiling without causing major alteration in OSPC fingerprints. Mammaglobin 2, kallikreins 6, 7, 8, 10, 11, claudin 3 and 4 and B7-H4 gene expression products represent candidate biomarkers endowed with great potential for early screening and therapy of OSPC patients.