Genomic heterogeneity in peritoneal implants: A differential analysis of gene expression using nanostring Human Cancer Reference panel identifies a malignant signature.

OBJECTIVES: Peritoneal implants of ovarian borderline serous tumors are diagnostically challenging. Distinguishing invasive from non-invasive cases is crucial for patient management. This study aims to develop a molecular signature to distinguish invasive implants with malignant potential from those with benign. METHODS: Archival formalin-fixed paraffin embedded tissues were retrieved from 3 institutions, with consensus histologic review. Lesions were classified as a non-invasive implant (n = 10), invasive implant (n = 9) or high grade (HG) peritoneal metastasis from HG serous ovarian carcinoma (n = 4). The nCounter® GX Human Cancer Gene Reference Assay was used to profile expression of 230 cancer genes and 6 control genes. The DEGs in HG peritoneal metastases compared to non-invasive implants were identified using T-tests performed in the NanoString Diff package, then used to cluster cases using the Eisen cluster 3.0 package. Lasso in glmnet package was used to select the subset of genes that most strongly correlate with a malignant potential. RESULTS: 37 genes were downregulated and 16 genes were upregulated in HG peritoneal metastases. Using this 53-gene signature, one of nine of the invasive implants clustered with the HG peritoneal metastasis. Expression of ABCB1, CDC2, CDKN1A, FAT1, MMP9, MSH2, NQO1 and TOP2A were sufficient to indicate malignant potential of implants. The HG peritoneal metastasis and one invasive implant exhibited a high malignant likelihood (>92%) whereas the non-invasive implants and eight invasive implants displayed a low malignant likelihood (≤0.1%). CONCLUSIONS: Invasive implants are heterogenous and often morphologically indistinguishable lesions with transcriptomes that may be classified as malignant or not. Additional research is needed to determine the importance of these genes as drivers and/or surrogates of malignant potential, and their utility for triaging invasive implants.

Trefoil factor family 3 (TFF3) expression and its interaction with estrogen receptor (ER) in endometrial adenocarcinoma.

OBJECTIVES: TFF3 has been found to be up-regulated at the gene and protein levels in endometrioid adenocarcinoma (EAC) when compared to uterine serous carcinoma (USC) and normal endometrium. In addition, TFF3 has been proven to be an estrogen-responsive gene and its expression level positively correlated to estrogen-receptor (ER) status in breast cancer cell culture. The aims of this study are to determine the expression and the prognostic value of TFF3 in a large series of human endometrial cancer and its relation with ER. METHODS: We evaluated 328 endometrial carcinomas using TFF3 and ER antibody on paraffin-embedded tissue. 74% were type I (EAC), and 26% were type II (USC, CCC and carcinosarcoma). RESULTS: In type I carcinomas, TFF3(+) expression was associated with no lympho-vascular invasion (p=0.0131), disease status (p=0.0132), recurrence-free survival (p=0.0424) and overall survival (p=0.0018). There was a positive association between TFF3 and ER (p<.0001). The combination of TFF3(+)/ER(+) was associated with low FIGO grade (p=.0122), early FIGO stage (p=.0062), absence of recurrence (p=.0037), absence of LVI (p=.0011), no lymph node involvement (p=.0116) and disease status (p=.0107). TFF3 appeared to be an independent prognostic marker in predicting recurrences (p=.046). In type II carcinomas, TFF3 failed to have a prognostic value. CONCLUSION: 1-TFF3 seems to be a novel pathway in the pathogenesis of type I endometrial carcinomas. 2-The strong association of TFF3 and ER in the estrogen-dependent endometrioid carcinoma could explain the reason for its frequent expression by this tumor type. 3-TFF3(+) seems to forecast a good prognosis in type I endometrial carcinomas. Based on our data, TFF3 expression in endometrial cancer deserves further investigation.

Pair Box 8 (PAX8) protein expression in high grade, late stage (stages III and IV) ovarian serous carcinoma.

OBJECTIVES: Pair-Box 8 (PAX8) is a transcription factor which has been found to be overexpressed in ovarian serous carcinoma (OSC). Silencing PAX8 by using shRNA led to a drop in cell viability in ovarian cancer cell lines, suggesting its use as a targeted therapeutic agent. The prognostic value of PAX8 in OSC is still widely unknown. The aim of this study was to evaluate PAX8 as a prognostic biomarker in patients with advanced stage OSC. METHODS: PAX8 was evaluated using immunohistochemistry on a tissue microarray of 148 OSC and the expression was correlated to the following clinico-pathologic variables; age of diagnosis, tumor stage, optimal debulking, recurrence free survival (RFS) and overall survival (OS). RESULTS: We found that PAX8 was expressed in 61% of cases. There was no association between PAX8 and tumor stage, optimal debulking and disease recurrence. In addition, PAX8 failed to have a predictive value in disease outcome. CONCLUSION: Despite showing that PAX8 protein is not a useful predictive marker in patients with high grade, advanced stage OSC, its overexpression in a large number of these cases makes the inhibition of PAX8 a very attractive targeted therapy.

Clinical Implications of Marker Expression of Carcinoma-Associated Fibroblasts (CAFs) in Patients with Epithelial Ovarian Carcinoma After Treatment with Neoadjuvant Chemotherapy.

Cancer-associated fibroblasts (CAFs) play an important role in tumor initiation and progression. The aim of this study is to explore the role of 2 CAF markers, fibroblast activated protein (FAP) and α-smooth muscle actin (αSMA), in patients with epithelial ovarian cancer (EOC) post-neoadjuvant chemotherapy. Sixty-six patients with the diagnosis of EOC treated with debulking surgery after neoadjuvant therapy were retrieved from the archives. Immunohistochemistry for FAP and αSMA antibodies were performed on paraffin-embedded tissue. Fisher's exact test was performed to test the association between FAP and αSMA expression and disease status. Kaplan-Meier method with log-rank test was used to check the survival difference between different FAP tumor/stroma expressions. FAP stroma (pos) . expression was strongly associated with higher recurrences rate [OR: 15.95; 95 % CI: 1.521-835.206; p = 0.0072]. Cases with combined FAP stroma (pos) and FAP tumor (neg) had higher death rate [OR: 4.845; 95 % CI: 1.53-16.61; p = 0.0046] and higher recurrence rate [OR: 5.12; 95 % CI: 0.91-54.42; p = 0.0487] compared to all the others. Cases with combined FAP stroma (neg) and FAP tumor (neg) were more likely to have lower recurrence rates [OR: 0.086; 95 % CI: 0.001-0.997; p = 0.0248]. αSMA was expressed by tumor-associated stroma in 95 % of cases and by tumor cells in 9 % of cases. No statistical power was found for αSMA and disease status. Our data indicate that FAP plays an important role in predicting tumor aggressiveness in patients with EOC post-neoadjuvant therapy, and its frequent expression in this malignancy implicates that FAP targeted therapy could be a very attractive strategy.

Histologic parameters predictive of disease outcome in women with advanced stage ovarian carcinoma treated with neoadjuvant chemotherapy.

The use of neoadjuvant chemotherapy followed by tumor reduction surgery, also called interval debulking surgery (IDS), is considered an alternative therapeutic regimen for selected patients with advanced stage epithelial ovarian cancer (EOC). Although minimal residual disease has been proven to be a prognostic factor in traditional cytoreduction for advanced stage EOC, predictive factors after IDS still remain unexplored. The aim of this study was to determine the prognostic value of post-neoadjuvant histologic changes with clinical outcome. Three pathologists evaluated 67 cases for the following parameters: fibrosis, necrosis, residual tumor, and inflammation. The Cohen's kappa statistic was used to measure agreement among pathologists. Univariate and multivariate Cox proportional hazards models were used to determine the association between histologic parameters and recurrence-free survival (RFS) and overall survival (OS). There was substantial to almost perfect agreement among the three pathologists in all four histologic parameters (k ranged from 0.65 to 0.97). Fibrosis was associated with longer RFS (P = 0.0257) with a median of 20 months for tumors with fibrosis (3+) versus 12 months for tumors with fibrosis (1+, 2+) and longer OS (P = 0.0249) with a median of 51 months for tumors with fibrosis (3+) versus 32 months for tumors with fibrosis (1+, 2+). Our results revealed that patients with tumors exhibiting fibrosis (1+, 2+), as well as necrosis (0, 1+), had significant shorter RFS and OS (P = 0.059 and P = 0.0234, respectively). We suggest that the assessment of fibrosis and necrosis should be implemented in pathologic evaluation and prospectively validated in future studies.

The role of hypoxic-inducible factor (HIF1α) and aldolaseC protein in endometrial carcinogenesis: a retrospective study of 279 patients.

OBJECTIVES: Hypoxia-inducible factor (HIF1α) plays an integral role in response to hypoxia, controlling dozens of target genes including aldolaseC (ALDC), an important enzyme in the glycolytic pathway. It also induces angiogenesis, allowing survival and proliferation of cancer cells. The aims of our study were to evaluate the expressions of HIF1α and ALDC in patients with endometrial cancer (EC) and define their association with disease outcome and to determine the existence of an association between HIF1α and ALDC proteins. DESIGN: This is a population-based retrospective cohort study using the gynaecological-oncology database. The authors identified all women with EC with adequate follow-up. Immunohistochemistry using antibodies to ALDC and HIF1α was performed on paraffin-embedded tissue from 279 patients. To test the association between ALDC /HIF1α protein using immunohistochemistry (IHC) (positive and negative) and the clinical parameters, Fisher's exact test was performed for categorical parameters and the logistic regression model was used for continuous ones. Pearson correlation was used to check the association of IHC between ALDC and HIF1α. SETTING: Academic referral centre. PARTICIPANTS: Women with EC from 2000 to 2010 obtained from the gynaecological-oncology database. OUTCOME MEASURES: The disease outcome is defined by alive with no evidence of disease versus all other outcomes. RESULTS: ALDC and HIF1α were overexpressed in the vast majority of EC cases (78% and 76%, respectively). There was a strong positive association between HIF1α and ALDC (p=0.0017). There was a significant association between ALDC and depth of myometrial invasion (p=0.0438), and between HIF1α and tumour grade (p=0.0231) and tumour subtype (p=0.018). However, there was no association between neither ALDC nor HIF1α and disease status. CONCLUSIONS: ALDC and HIF1α play an important role in endometrial carcinogenesis. Their expression by the majority of EC makes inhibition of HIF1α a very attractive therapeutic option for treating patients with EC and we suggest that it will be prospectively validated in future studies.