Detection of phosphorylated insulin receptor in colorectal adenoma and adenocarcinoma: implications for prognosis and clinical outcome.

Colorectal carcinoma remains among the most frequent causes of cancer death. Besides the well-known genetic predisposition, a key role in colorectal adenoma and adenocarcinoma etio-pathogenesis, mainly in sporadic cases, is played by definite risk factors, such as obesity, type 2 diabetes, insulin resistance, hyper-insulinemia, and insulin therapy. These epidemiological data motivated us to determine, by means of immunohistochemistry, the amount of activated (phosphorylated) insulin receptor in archival samples from 22 colorectal adenoma and 117 adenocarcinoma patients, with the objective to estimate the role of this factor in colorectal epithelium transformation and cancer progression. Statistical analysis of the results clearly showed that positive staining for phosphorylated insulin receptor was significantly more frequent in adenomas than adenocarcinomas (P < 0.0001) and, within the adenocarcinoma cohort, it was more frequent in low-grade tumors (P = 0.005). In adenomas, staining was exclusively cytoplasmic, while in adenocarcinomas it was cytoplasmic and/or nuclear (P < 0.0001). Interestingly, disease-free survival in colorectal adenocarcinoma patients pointed out a significantly better prognosis for those bearing a positive staining for phosphorylated insulin receptor (P = 0.02). From these data, we can argue that activated insulin receptor plays a fundamental role at the early stages of tumorigenesis, where late stages could be characterized by a shift toward more active oncogenic drivers. Determining the amount of phosphorylated insulin receptor could thus represent a novel prognostic/predictive tool in colorectal adenocarcinoma patients.

AMBRA1 and SQSTM1 expression pattern in prostate cancer.

Prostate cancer is among the most commonly diagnosed male diseases and a leading cause of cancer mortality in men. There is emerging evidence that autophagy plays an important role in malignant cell survival and offers protection from the anti-cancer drugs in prostate cancer cells. AMBRA1 and the autophagic protein sequestosome-1 (SQSTM1; p62) expression were evaluated by immunohistochemistry and western blot on tissue samples from both benign and malignant prostatic lesions. The data reported in this pilot study demonstrated an increased expression of AMBRA1 and SQSTM1, which were also associated with an accumulation of LC3II in prostate cancer but not in benign lesion. In the present study we found that: (i) at variance with benign lesion, prostate cancer cells underwent SQSTM1 accumulation, i.e., clearly displayed a defective autophagic process but, also, (ii) prostate cancer accumulated AMBRA1 and (iii) this increase positively correlated with the Gleason score. These results underscore a possible implication of autophagy in prostate cancer phenotype and of AMBRA1 as possible cancer progression biomarker in this malignancy.

Endometrial cancer prognosis correlates with the expression of L1CAM and miR34a biomarkers.

BACKGROUND: Patients with endometrial cancer (EC) and presumably with good prognosis may develop a recurrence indicating that the classification of this tumor is still not definitive and that new markers are needed to identify a subgroup at risk of relapse. The cell adhesion molecule L1CAM is highly expressed in several human carcinomas and has recently been described as a new marker for endometrial and ovarian carcinomas. The aim of this study was to determine the relevance of L1CAM in recurrent EC. METHODS: In this work we have analyzed, by immunohistochemical and RT-qPCR analysis, the expression of L1CAM in a cohort of 113 endometrial cancers at different stages, which 50% have relapsed. As a predictor of good outcome, the tumors were also analyzed for the expression of miR-34a, a post-transcriptional regulator of L1CAM. RESULTS: Among metastatic EC, the highest levels (60%) and the median level (24%) of L1CAM in tumors correlate with the progression, suggesting that the expression of this molecule is linked to the tumor component most involved in metastatic processes. We also found an inverse correlation between miR-34a and L1CAM protein expression, suggesting that miR-34a is a positive prognostic marker of EC. CONCLUSIONS: Our results demonstrate the expression of L1CAM and miR-34a in EC as prognostic factors that identify subgroup of patients at high risk of recurrence suggesting for them more aggressive schedules of treatment.

Renal cancer: new models and approach for personalizing therapy.

BACKGROUND: Clear cell RCC (ccRCC) accounts for approximately 75% of the renal cancer cases. Surgery treatment seems to be the best efficacious approach for the majority of patients. However, a consistent fraction (30%) of cases progress after surgery with curative intent. It is currently largely debated the use of adjuvant therapy for high-risk patients and the clinical and molecular parameters for stratifying beneficiary categories. In addition, the treatment of advanced forms lacks reliable driver biomarkers for the appropriated therapeutic choice. Thus, renal cancer patient management urges predictive molecular indicators and models for therapy-decision making. METHODS: Here, we developed and optimized new models and tools for ameliorating renal cancer patient management. We isolated from fresh tumor specimens heterogeneous multi-clonal populations showing epithelial and mesenchymal characteristics coupled to stem cell phenotype. These cells retained long lasting-tumor-propagating capacity provided a therapy monitoring approach in vitro and in vivo while being able to form parental tumors when orthotopically injected and serially transplanted in immunocompromised murine hosts. RESULTS: In line with recent evidence of multiclonal cancer composition, we optimized in vitro cultures enriched of multiple tumor-propagating populations. Orthotopic xenograft masses recapitulated morphology, grading and malignancy of parental cancers. High-grade but not the low-grade neoplasias, resulted in efficient serial transplantation in mice. Engraftment capacity paralleled grading and recurrence frequency advocating for a prognostic value of our developed model system. Therefore, in search of novel molecular indicators for therapy decision-making, we used Reverse-Phase Protein Arrays (RPPA) to analyze a panel of total and phosphorylated proteins in the isolated populations. Tumor-propagating cells showed several deregulated kinase cascades associated with grading, including angiogenesis and m-TOR pathways. CONCLUSIONS: In the era of personalized therapy, the analysis of tumor propagating cells may help improve prediction of disease progression and therapy assignment. The possibility to test pharmacological response of ccRCC stem-like cells in vitro and in orthotopic models may help define a pharmacological profiling for future development of more effective therapies. Likewise, RPPA screening on patient-derived populations offers innovative approach for possible prediction of therapy response.

A cut-off of 2150 cytokeratin 19 mRNA copy number in sentinel lymph node may be a powerful predictor of non-sentinel lymph node status in breast cancer patients.

Since 2007, one-step nucleic acid amplification (OSNA) has been used as a diagnostic system for sentinel lymph node (SLN) examination in patients with breast cancer. This study aimed to define a new clinical cut-off of CK19 mRNA copy number based on the calculation of the risk that an axillary lymph node dissection (ALND) will be positive. We analyzed 1529 SLNs from 1140 patients with the OSNA assay and 318 patients with positive SLNs for micrometastasis (250 copies) and macrometastasis (5000 copies) underwent ALND. Axillary non-SLNs were routinely examined. ROC curves and Youden's index were performed in order to identify a new cut-off value. Logistic regression models were performed in order to compare OSNA categorical variables created on the basis of our and traditional cut-off to better identify patients who really need an axillary dissection. 69% and 31% of OSNA positive patients had a negative and positive ALND, respectively. ROC analysis identified a cut-off of 2150 CK19 mRNA copies with 95% sensitivity and 51% specificity. Positive and negative predictive values of this new cut-off were 47% and 96%, respectively. Logistic regression models indicated that the cut-off of 2150 copies better discriminates patients with node negative or positive in comparison with the conventional OSNA cut-off (p<0.0001). This cut-off identifies false positive and false negative cases and true-positive and true negative cases very efficiently, and therefore better identifies which patients really need an ALND and which patients can avoid one. This is why we suggest that the negative cut-off should be raised from 250 to 2150. Furthermore, we propose that for patients with a copy number that ranges between 2150 and 5000, there should be a multidisciplinary discussion concerning the clinical and bio-morphological features of primary breast cancer before any decision is taken on whether to perform an ALND or not.

Keap1/Nrf2 pathway in kidney cancer: frequent methylation of KEAP1 gene promoter in clear renal cell carcinoma.

The Keap1/Nrf2 pathway is a master regulator of the cellular redox state through the induction of several antioxidant defence genes implicated in chemotherapeutic drugs resistance of tumor cells. An increasing body of evidence supports a key role for Keap1/Nrf2 pathway in kidney diseases and renal cell carcinoma (RCC), but data concerning the molecular basis and the clinical effect of its deregulation remain incomplete.Here we present a molecular profiling of the KEAP1 and NFE2L2 genes in five different Renal Cell Carcinoma histotypes by analysing 89 tumor/normal paired tissues (clear cell Renal Carcinoma, ccRCCs; Oncocytomas; Papillary Renal Cell Carcinoma Type 1, PRCC1; Papillary Renal Cell Carcinoma Type 2, PRCC2; and Chromophobe Cell Carcinoma).A tumor-specific DNA methylation of the KEAP1 gene promoter region was found as a specific feature of the ccRCC subtype (18/37, 48.6%) and a direct correlation with mRNA levels was confirmed by in vitro 5-azacytidine treatment. Analysis of an independent data set of 481 ccRCC and 265 PRCC tumors corroborates our results and multivariate analysis reveals a significant correlation among ccRCCs epigenetic KEAP1 silencing and staging, grading and overall survival.Our molecular results show for the the first time the epigenetic silencing of KEAP1 promoter as the leading mechanism for modulation of KEAP1 expression in ccRCCs and corroborate the driver role of Keap1/Nrf2 axis deregulation with potential new function as independent epigenetic prognostic marker in renal cell carcinoma.