Steven Sieber

MiR-200c and HuR in ovarian cancer

BackgroundMicroRNAs in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors. This study assessed the role of miR-200c as regulator of class III β-tubulin (TUBB3), a factor associated with drug-resistance and poor prognosis in ovarian cancer.MethodsExpression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Stable overexpression of miR-200c was obtained in A2780 and Hey cell lines. Crosslinking-coupled affinity purification method and ribonucleic-immunoprecipitation assay were used to characterise the complexes between miR-200c, HuR and 3′UTR region of TUBB3 mRNA. Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients.ResultsIn a panel of ovarian adenocarcinoma cell lines, we observed a direct correlation between miR-200c expression and chemoresistance. In A2780 cells miR-200c targeted TUBB3 3′UTR, while a positive correlation was observed between miR-200c and TUBB3 expression in most of the other cell lines. Through the analysis of 3′UTR-associated complexes, we found that the miR-200c can increase the association of the RNA binding protein HuR with TUBB3 mRNA, whereas HuR binding enhanced TUBB3 mRNA translation. Most importantly, in our analysis on 220 ovarian cancer patients we observed that overexpression of miR-200c correlated with poor or good outcome depending on the cellular localization of HuR.ConclusionThis study suggests a model for the combined regulatory activity of miR-200c and HuR on TUBB3 expression in ovarian cancer. When HuR is nuclear, high expression of miR-200c inhibits TUBB3 expression and results in a good prognosis, whereas when HuR occurs in cytoplasm, the same miRNA enhances TUBB3 expression and produces a poor outcome. These findings reveal the usefulness of multidimensional analysis in the investigation of the prognostic role of miRNA expression.

Gender Influences the Class III and V β-Tubulin Ability to Predict Poor Outcome in Colorectal Cancer

Purpose: Colorectal cancer is one of the deadliest diseases in Western countries. To predict the outcome of therapy, we assessed the role of class III (TUBB3) and class V β-tubulin (TUBB6) as predictive biomarkers. Experimental Design: Using immunohistochemistry and nanofluidics, the expression of TUBB3 and TUBB6 was assessed in two cohorts of 180 and 134 patients, respectively. The CYP17A1 RS743572 was genotyped to identify GG carriers with enhanced androgen levels. TUBB3 and TUBB6 were investigated in 22 colorectal cancer cell lines in basal conditions and after serum starvation, the latter serving as activator of this prosurvival pathway. To ascertain the role of androgen receptor (AR) in such regulation, we silenced AR and checked TUBB3 and TUBB6 expression and sensitivity to chemotherapy. Results: There was a link between poor survival, the expression of TUBB3/TUBB6, and AR only in females. Conversely, only in males carriers of the GG phenotype exhibited the worst outcome. Importantly, male cell lines were resistant to serum starvation and exhibited higher levels of TUBB6, thereby suggesting that the pathway is activated by androgens. In female cells this phenomenon was absent. In both genders, AR was the main driver of TUBB3/TUBB6 expression, as constitutive silencing of AR was associated with downregulation of TUBB3/TUBB6 expression and increased sensitivity to oxaliplatin and SN-38. Conclusions: The involvement of androgens in the TUBB3 pathway opens the way for clinical trials to assess the efficacy of antiandrogens for increasing the efficacy of chemotherapy in male colorectal cancer patients. Clin Cancer Res; 18(10); 2964–75. ©2012 AACR.

Ductal carcinoma of male breast with prominent lipid-rich component

&NA; We report a case of mammary duct carcinoma with a prominent lipid‐rich, sebaceous‐like component, occurring in a 55 yr old white male. The patient presented with a painless, subareolar left breast mass and the diagnosis of malignancy was made by fine needle aspiration. Subsequent modified radical mastectomy revealed an infiltrating and in situ ductal carcinoma with dermal invasion and numerous vacuolated sebaceous‐like tumor cells, positive for neutral lipid by Oil Red O stain. We propose that this case represents an unusual variant of lipid‐secreting breast carcinoma. To the best of our knowledge, this subtype of mammary carcinoma is unprecedented in male breast.

Histiocytoid Hemangioma of the Testis: A Case Report

We report a rare case of testicular histiocytoid hemangioma. Pathological features and differential diagnosis of this neoplasm are discussed.

Migrating pleural mesothelial cyst

We herein describe an atypical presentation of a migrating coelomic cyst attached to a pedicle of pericardial fat pad in an asymptomatic 45-year-old woman. A review of the English-language literature revealed only one such case report.

Primary tumor of spleen with morphologic features of malignant fibrous histiocytoma. Immunohistochemical evidence for a macrophage origin.

We report a case of primary splenic malignant fibrous histiocytoma that occurred in a 41-year-old man. Adjacent to the tumor there was a large calcified intrasplenic cyst. Despite splenectomy, postoperative radiation, and systemic chemotherapy, the patient died with metastatic tumor 6 months after diagnosis. Electron microscopic analysis of the tumor demonstrated subpopulations of tumor cells with fibroblastic or histiocytic features. Immunoperoxidase studies of frozen tumor tissue showed positive staining of both spindle and histiocytelike tumor cells with a large panel of monoclonal antibodies against antigens associated with the mononuclear phagocyte system. Expression of these antigens by the tumor supports a mononuclear phagocyte origin.

Integrated multidimensional analysis is required for accurate prognostic biomarkers in colorectal cancer.

CRC cancer is one of the deadliest diseases in Western countries. In order to develop prognostic biomarkers for CRC (colorectal cancer) aggressiveness, we analyzed retrospectively 267 CRC patients via a novel, multidimensional biomarker platform. Using nanofluidic technology for qPCR analysis and quantitative fluorescent immunohistochemistry for protein analysis, we assessed 33 microRNAs, 124 mRNAs and 9 protein antigens. Analysis was conducted in each single dimension (microRNA, gene or protein) using both the multivariate Cox model and Kaplan-Meier method. Thereafter, we simplified the censored survival data into binary response data (aggressive vs. non aggressive cancer). Subsequently, we integrated the data into a diagnostic score using sliced inverse regression for sufficient dimension reduction. Accuracy was assessed using area under the receiver operating characteristic curve (AUC). Single dimension analysis led to the discovery of individual factors that were significant predictors of outcome. These included seven specific microRNAs, four genes, and one protein. When these factors were quantified individually as predictors of aggressive disease, the highest demonstrable area under the curve (AUC) was 0.68. By contrast, when all results from single dimensions were combined into integrated biomarkers, AUCs were dramatically increased with values approaching and even exceeding 0.9. Single dimension analysis generates statistically significant predictors, but their predictive strengths are suboptimal for clinical utility. A novel, multidimensional integrated approach overcomes these deficiencies. Newly derived integrated biomarkers have the potential to meaningfully guide the selection of therapeutic strategies for individual patients while elucidating molecular mechanisms driving disease progression.

Herpes virus microRNA expression and significance in serous ovarian cancer.

Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality.

Abstract 3725: HGF/c-Met axis drives cancer aggressiveness in the neo-adjuvant setting of ovarian cancer

Ovarian cancer is the most lethal gynecologic malignancy. Recently, NACT (Neo Adjuvant Chemotherapy) has been tested as alternative approach for the management of ovarian cancer patients. A biological predictor helpful in selecting patients for NACT would be desirable. This study was aimed at identifying actionable mechanisms of resistance to NACT. Expression of a panel of microRNAs was screened in a discovery set of 85 patients. Analysis of the potential targets was conducted in the same RNAs by calculating significant correlations between microRNAs and genes. Quantitative fluorescent immunohistochemistry was employed in a validation set of 109 patients. MiR-193a-5p was significantly overexpressed in the NACT setting. Analysis of its potential targets demonstrated that this microRNA is also significantly correlated with HGF and MET genes. Analysis of protein expression in samples taken before and after NACT demonstrated that both HGF and c-Met are increased after NACT. Patients who relapse shortly after NACT exhibited the highest relative basal expression of both HGF and c-Met, while the opposite phenomenon was observed in the best responders. Mir-193a-5p, HGF and c-Met expression may help select eligible patients for this modality of treatment. Moreover, inhibitors of this pathway may improve the efficacy of NACT.

Abstract 916: A multidimensional analysis of predictive biomarkers in colorectal cancer

Background: CRC cancer is one of the deadliest diseases in Western countries. In order to develop predictive biomarkers for CRC (colorectal cancer) aggressiveness, we analyzed retrospectively 267 CRC patients via a novel, multidimensional biomarker platform. Methods: Using nanofluidic technology for qPCR analysis and quantitative fluorescent immunohistochemistry for protein analysis, we assessed 33 microRNAs, 124 mRNAs and 9 protein antigens. Analysis was conducted in each single dimension (microRNA, gene or protein) using both the multivariate Cox model and Kaplan-Meier method. Thereafter, we simplified the censored survival data into binary response data (aggressive vs. non aggressive cancer). Subsequently, we combined the data into multidimensional scores using inverse sliced regression for sufficient dimension reduction. Accuracy was assessed using the Area under the Curve (AUC) and the receiver operating characteristic (ROC) method. Results: Single dimension analysis led to the discovery of individual factors that were significant predictors of outcome. These included seven specific microRNAs, four genes, and one protein. When these factors were quantified individually as predictors of aggressive disease, the highest demonstrable area under the curve (AUC) was 0.68. By contrast, when all results from single dimensions were combined into multidimensional biomarkers, AUCs were dramatically increased with values approaching and even exceeding 0.9. Conclusions: Single dimension analysis generates statistically significant predictors, but their predictive strengths are suboptimal for clinical utility. A novel, multidimensional approach overcomes these deficiencies. Newly derived multidimensional biomarkers have the potential to meaningfully guide the selection of therapeutic agents for individual patients while elucidating molecular mechanisms driving disease progression. Citation Format: Marisa Mariani, Shiquan He, Mark McHugh, Mirko Andreoli, Deep Pandya, Steven Sieber, Zheyang Wu, Paul Fiedler, Shohreh Shahabi, Cristiano Ferlini. A multidimensional analysis of predictive biomarkers in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 916. doi:10.1158/1538-7445.AM2014-916