Influence of KRAS mutations on clinical outcome in patients with curatively resected stage III colon cancer treated with adjuvant chemotherapy.

OBJECTIVE: To profile and correlate KRAS mutations with outcome in stage III colon cancer (CC) patients who underwent adjuvant chemotherapy following curative resection surgery. PATIENTS AND METHODS: In this retrospective study, eligible patients were those with resected stage III CC who underwent 6-months adjuvant chemotherapy, either with fluoropyrimidine monotherapy (FP) or with oxaliplatin-based regimens (O-FP). Disease-free survival (DFS) and overall survival (OS) were analyzed and computed using the Kaplan-Meier method and the log-rank test. RESULTS: The study population included 148 patients (n=65 FP and n=83 O-FP). We identified KRAS mutations in 41/148 (27%) patients, of which 18 (44%) received FP and 23 (56%) O-FP. Five-year DFS and OS were significantly higher in patients with KRAS wild-type vs. mutant [DFS: 78 vs. 56%, HR: 0.47 (95% CI: 0.25; 0.87), p=0.01; OS: 73 vs. 68%, HR: 0.44 (95% CI: 0.21; 0.88), p=0.01]. In patients treated with FP, the 5-year DFS and OS was significantly improved in the KRAS wild-type vs. mutant group, respectively [DFS: 80 vs. 43%, HR: 2.88 (95% CI: 0.67; 3.76), p=0.014; OS: 85 vs. 68%, HR: 0.27 (95% CI: 0.10; 0.73), p=0.005]. Conversely, 5-year DFS and OS were not statistically different for patients with KRAS wild-type vs. mutations treated with O-FP, respectively [DFS: 78 vs. 65%, HR: 1.59 (95% CI: 0.67; 3.76), p=0.281; OS: 80 vs. 75%, HR: 0.73 (95% CI: 0.55; 2.12), p=0.57)]. CONCLUSIONS: Our results suggest that curatively resected stage III CC patients exhibiting wild-type KRAS status might benefit from FP alone. Conversely, an oxaliplatin-containing regimen should be recommended in KRAS mutated patients.

Universal screening to identify Lynch syndrome: two years of experience in a Northern Italian Center.

Lynch syndrome is caused by germline mutations of genes affecting the mismatch repair proteins MLH1, MSH2, MSH6 or PMS2. Identification of Lynch syndrome patients using germline molecular testing in colorectal cancer (CRC) affected patients and in their healthy relatives is a cost-effective model of cancer prevention. Several studies demonstrate that universal tumor testing using immunohistochemical (IHC) analysis of CRC samples is the most efficient approach to identifying patients affected by Lynch syndrome. We studied a cohort of 352 consecutive CRCs for MSH2, MLH1, MSH6 and PMS2 protein expression using universal IHC screening. IHC mismatch repair (MMR) defects were identified in 70 out of 352 cases (19.8%) including six CRCs MSH2/MSH6 defective, two CRCs, respectively, MSH6 and PMS2 defective, 58 CRCs MLH1/PMS2 defective and four CRCs showing atypical MMR pattern. MLH1 promoter methylation and V600E BRAF mutation analysis were investigated on 61 CRCs. Cancer genetic counseling was offered to all 68 patients affected by MMR defective CRCs and 25 patients opted in to this service (36.8% compliance). Pathogenetic variants of MSH2 genes were identified in two cases (55 and 79 years old). Universal screening based on an IHC approach showed a Lynch syndrome incidence of 1/173. The protocol recommended by regional law improved patient compliance. This study demonstrates that the IHC approach for both MMR deficiency and V600E BRAF mutation detections is the most efficient approach for Lynch syndrome screening in the Italian population.

Inherited cancer syndromes in 220 Italian ovarian cancer patients.

BACKGROUND: A subsets of ovarian carcinomas (OCs) are related to inherited conditions including Hereditary Breast and Ovarian Cancers (HBOC) and Lynch Syndrome (LS). The identification of inherited conditions using genetic testing might be a strategic model for cancer prevention that include benefits for the ovarian cancer patients and for their family members. METHODS: We describe a retrospective Italian experience for the identification of inherited conditions in 232 patients affected by OCs using both somatic and germline analyses. RESULTS: Immunohistochemical and microsatellite analyses performed on OCs identified 20 out of 101 MMR defective cancers and 15 of these were from patients carriers of the MMR germline pathogenetic variants. BRCA1 and BRCA2 testing offered to 198 OC patients revealed 67 (34%) pathogenetic variant carriers of BRCA1/2 genes. Interestingly LS patients revealed a mean age of OC onset of 45.4 years, which was significantly lower than the mean age of OCs onset of HBOC patients. CONCLUSIONS: Somatic and germline analyses offered to OC patients has proved to be an efficient strategy for the identification of inherited conditions involving OC also in absence of suggestive family histories. The identification of LS and HBOC syndromes through OC patients is an effective tool for OC prevention.

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells.

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.