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Aim: Specific MicroRNAs (miRNAs) have been found up- or down-regulated in colorectal cancer, and they are associated with prognosis or response to chemotherapy or targeted drugs. The microenvironment close to the neoplasm plays a leading role in tumor spread and survival. We used microarray technology to profile miRNA expression both in primary tumor and stromal tissue to study differences and clinical implications. Methods: Matched tumor and stroma tissues microdissected from paraffin embedded material of 51 patients with metastatic colorectal cancer, which have been treated with first-line chemotherapy plus bevacizumab, were considered. miRNA expression profile was performed by microarray analysis and confirmed by quantitative real-time Reverse Transcription Polymerase Chain Reaction (qRTPCR). miRNA expression was correlated with: stage at diagnosis (limited vs metastatic), site of primary tumor (right vs left colon vs rectum), first site of metastasis (liver vs lung), progression-free (PFS) and overall survival (OS). Results: We did not find any significant association between miRNA expressions and stage at diagnosis, site of primary tumor, first site of metastasis, PFS or OS. However, 26 miRNAs resulted differentially expressed with at least 2 fold change between tumor tissue and stroma (16 more expressed in the tumor, and 10 more expressed in the stroma). 10/26 were confirmed as differently expressed at qRTPCR: miR-200c-3p, miR-141-3p, miR-200b-3p, miR-200a-3p, miR-1246, miR-92a-3p, miR-194-5p, miR-192-5p, miR-3651-5p, miR-574-3p. Conclusion: colorectal cancer stroma is characterized by specific miRNA expression profile as compared to primary tumor, suggesting that tumor cells and microenvironment may regulate different patterns of tumor behavior. Further studies of genes regulated by these miRNAs may provide details about the role of cancer stroma in the control of tumor’s aggressiveness, preferential site of distant metastases, and prognosis.

Purpose: Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic diseases, with survival rate virtually unchanged for the past 30 years. EOC comprises different histotypes with molecular and clinical heterogeneity, but up till now the present gold standard platinum-based treatment has been conducted without any patient stratification. The aim of the present study is to generate microRNA (miRNA) profiles characteristic of each stage I EOC histotype, to identify subtype-specific biomarkers to improve our understanding underlying the tumor mechanisms. Experimental Design: A collection of 257 snap-frozen stage I EOC tumor biopsies was gathered together from three tumor tissue collections and stratified into independent training (n = 183) and validation sets (n = 74). Microarray and quantitative real-time PCR (qRT-PCR) were used to generate and validate the histotype-specific markers. A novel dedicated resampling inferential strategy was developed and applied to identify the highest reproducible results. mRNA and miRNA profiles were integrated to identify novel regulatory circuits. Results: Robust miRNA markers for clear cell and mucinous histotypes were found. Specifically, the clear cell histotype is characterized by a five-fold (log scale) higher expression of miR-30a and miR-30a*, whereas mucinous histotype has five-fold (log scale) higher levels of miR-192/194. Furthermore, a mucinous-specific regulatory loop involving miR-192/194 cluster and a differential regulation of E2F3 in clear cell histotype were identified. Conclusions: Our findings showed that stage I EOC histotypes have their own characteristic miRNA expression and specific regulatory circuits. Clin Cancer Res; 19(15); 4114–23. ©2013 AACR.

Introduction. Within stage I epithelial ovarian cancer (EOC), the current clinic-pathological parameters, like tumor grade, fail to accurately stratify patient prognosis and it is therefore crucial for optimal treatment that the biological properties of stage I EOCs are further elucidated. We have previously demonstrated miR-200c as a predictor of survival, and a biomarker of relapse (Marchini et al. Lancet Oncology, 2011), suggesting that miRNA profile could be a useful tool to dissect molecular networks in stage I EOC. The aim of the current study is to identify a miRNA signature for each tumor grade, that integrated with clinical variables would be used to improve stage I patients stratification. Experimental procedures. A cohort of 219 snap frozen tumor biopsies, with median follow up of seven years, was gathered together from three independent Italian tumor tissue collections. miRNA landscape was generated with commercially available arrays (Agilent, Palo Alto CA) and analysis performed as recently published (Calura et al., CCR 2013). Signature validation was performed by qRT-PCR using commercially available primers and reagents (Qiagen, Milano, Italy). Results. The entire cohort of patients was stratified by sub-stage, grade and relapse into a training set (n= 151), used for miRNA landscape generation, and a validation set (n= 68) used for qRT-PCR validation. “Resampling score” (RS) strategy (Calura et al., CCR 2013) reported that the largest number of miRNAs found differentially expressed is between grade three and grade one (n= 72), while the comparison between grade one versus borderline tumors showed the lowest number (n= 14). Signature validation in both training and validation set by qRT-PCR of the top seven selected miRNAs with highest RS, confirmed hsa-miR-376c, hsa-miR-377 and hsa-miR-214 as down-regulated in grade three compared to the other grades; hsa-miR-96 expression increases directly from grade one to grade three, while hsa-miR-199a-5p was down-regulated in grade three compared to borderline tumors. No differences were observed for hsa-miR-183 and hsa-miR-29c. miRNA expression profile was correlated to clinical variables in both univariate and multivariate model and only miR-199a-5p, resulted associated to PFS in multivariate Cox proportional hazard model. Conclusions. In the present study we observed that, regardless of tumor histological subtype (Calura et al. CCR 2013), known morphological differences across tumor grades mirror molecular differences in term of miRNA expression profile. To optimize patients stratification and thus improving clinical management of stage I EOC, we are now drawing new miRNA-based networks (i.e. miRNA-gene expression integration) for each tumor grade that will be correlated with known clinical parameters. Citation Format: Enrica Calura, Robert Fruscio, Lara Paracchini, Eliana Bignotti, Paolo Martini, Antonella Ravaggi, Mariacristina Di Marino, Gabriele Sales, Luca Beltrame, Federica Dell'Orto, Romina Baldo, Sergio Pecorelli, Enrico Sartori, laura Zanotti, Dionyssios Katsaros, Germana Tognon, Maurizio D'Incalci, Chiara Romualdi, Sergio Marchini. miRNA landscape analysis of stage I EOC, identifies miR-199a-5p associated to poor prognosis in grade 3 subgroup. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B18.