Spatio-temporal tumor heterogeneity in metastatic CRC tumors: a mutational-based approach.

It is well known that activating mutations in the KRAS and NRAS genes are associated with poor response to anti-EGFR therapies in patients with metastatic colorectal cancer (mCRC). Approximately half of the patients with wild-type (WT) KRAS colorectal carcinoma do not respond to these therapies. This could be because the treatment decision is determined by the mutational profile of the primary tumor, regardless of the presence of small tumor subclones harboring RAS mutations in lymph nodes or liver metastases. We analyzed the mutational profile of the KRAS, NRAS, BRAF and PI3KCA genes using low-density microarray technology in samples of 26 paired primary tumors, 16 lymph nodes and 34 liver metastases from 26 untreated mCRC patients (n=76 samples). The most frequent mutations found in primary tumors were KRAS (15%) and PI3KCA (15%), followed by NRAS (8%) and BRAF (4%). The distribution of the mutations in the 16 lymph node metastases analyzed was as follows: 4 (25%) in KRAS gene, 3 (19%) in NRAS gene and 1 mutation each in PI3KCA and BRAF genes (6%). As expected, the most prevalent mutation in liver metastasis was in the KRAS gene (35%), followed by PI3KCA (9%) and BRAF (6%). Of the 26 cases studied, 15 (58%) displayed an overall concordance in the mutation status detected in the lymph node metastases and liver metastases compared with primary tumor, suggesting no clonal evolution. In contrast, the mutation profiles differed in the primary tumor and lymph node/metastases samples of the remaining 11 patients (48%), suggesting a spatial and temporal clonal evolution. We confirm the presence of different mutational profiles among primary tumors, lymph node metastases and liver metastases. Our results suggest the need to perform mutational analysis in all available tumor samples of patients before deciding to commence anti-EGFR treatment.

Combined assessment of the TNM stage and BRAF mutational status at diagnosis in sporadic colorectal cancer patients.

The prognostic impact of KRAS mutations and other KRAS-related and non-related genes such as BRAF, NRAS and TP53, on sporadic colorectal cancer (sCRC) remain controversial and/or have not been fully established. Here we investigated the frequency of such mutations in primary sCRC tumors and their impact on patient progression-free survival (PFS) and overall survival (OS). Primary tumor tissues from 87 sCRC patients were analysed using a custom-built next generation sequencing (NGS) panel to assess the hotspot mutated regions of KRAS/NRAS (exons 2, 3 and 4), BRAF (exon 15) and TP53 (all exons). Overall, mutations in these genes were detected in 46/87 sCRC tumors analyzed (53%) with the following frequencies per gene: TP53, 33%; KRAS, 28%; BRAF, 7%; and NRAS, 1%. A significant association was found between KRAS mutations and right side colon tumor location (p=0.05), well-differentiated tumors (p=0.04) and absence of lymphovascular invasion (p=0.05). In turn, BRAF-mutated tumors frequently corresponded to poorly- or moderately-differentiated sCRC (p=0.02) and showed a higher frequency of peritoneal carcinomatosis (p=0.006) and microsatellite instability (p=0.007). From the prognostic point of view, the BRAF mutational status together with the TNM stage were the only variables that showed an independent adverse impact on patient outcome in the multivariate analyses for both PFS and OS. Based on these results a scoring system was built and patients were classified into three prognostic subgroups with different PFS rates at 2 years: 91% vs. 77% vs. 0%, respectively (p<0.0001). Additional prospective studies in larger series of sCRC patients where mutations in genes other than those investigated here are required to validate the utility of the proposed predictive model.

Prognostic impact of a novel gene expression profile classifier for the discrimination between metastatic and non-metastatic primary colorectal cancer tumors.

Despite significant advances have been achieved in the genetic characterization of sporadic colorectal cancer (sCRC), the precise genetic events leading to the development of distant metastasis remain poorly understood. Thus, accurate prediction of metastatic disease in newly-diagnosed sCRC patients remains a challenge. Here, we evaluated the specific genes and molecular pathways associated with the invasive potential of colorectal tumor cells, through the assessment of the gene expression profile (GEP) of coding and non-coding genes in metastatic (MTX) vs. non-metastatic (non-MTX) primary sCRC tumors followed for >5 years. Overall, MTX tumors showed up-regulation of genes associated with tumor progression and metastatic potential while non-MTX cases displayed GEP associated with higher cell proliferation, activation of DNA repair and anti-tumoral immune/inflammatory responses. Based on only 19 genes a specific GEP that classifies sCRC tumors into two MTX-like and non-MTX-like molecular subgroups was defined which shows an independent prognostic impact on patient overall survival, particularly when it is combined with the lymph node status at diagnosis. In summary, we show an association between the global GEP of primary sCRC cells and their metastatic potential and defined a GEP-based classifier that provides the basis for further prognostic stratification of sCRC patients who are at risk of distant metastases.

Genomic characterization of liver metastases from colorectal cancer patients.

Metastatic dissemination is the most frequent cause of death of sporadic colorectal cancer (sCRC) patients. Genomic abnormalities which are potentially characteristic of such advanced stages of the disease are complex and so far, they have been poorly described and only partially understood. We evaluated the molecular heterogeneity of sCRC tumors based on simultaneous assessment of the overall GEP of both coding mRNA and non-coding RNA genes in primary sCRC tumor samples from 23 consecutive patients and their paired liver metastases. Liver metastases from the sCRC patients analyzed, systematically showed deregulated transcripts of those genes identified as also deregulated in their paired primary colorectal carcinomas. However, some transcripts were found to be specifically deregulated in liver metastases (vs. non-tumoral colorectal tissues) while expressed at normal levels in their primary tumors, reflecting either an increased genomic instability of metastatic cells or theiradaption to the liver microenvironment. Newly deregulated metastatic transcripts included overexpression of APOA1, HRG, UGT2B4, RBP4 and ADH4 mRNAS and the miR-3180-3p, miR-3197, miR-3178, miR-4793 and miR-4440 miRNAs, together with decreased expression of the IGKV1-39, IGKC, IGKV1-27, FABP4 and MYLK mRNAS and the miR-363, miR-1, miR-143, miR-27b and miR-28-5p miRNAs. Canonical pathways found to be specifically deregulated in liver metastatic samples included multiple genes related with intercellular adhesion and the metastatic processes (e.g., IGF1R, PIK3CA, PTEN and EGFR), endocytosis (e.g., the PDGFRA, SMAD2, ERBB3, PML and FGFR2), and the cell cycle (e.g., SMAD2, CCND2, E2F5 and MYC). Our results also highlighted the activation of genes associated with the TGFß signaling pathway, -e.g. RHOA, SMAD2, SMAD4, SMAD5, SMAD6, BMPR1A, SMAD7 and MYC-, which thereby emerge as candidate genes to play an important role in CRC tumor metastasis.

Identification and characterization of the gene expression profiles for protein coding and non-coding RNAs of pancreatic ductal adenocarcinomas.

Significant advances have been achieved in recent years in the identification of the genetic and the molecular alterations of pancreatic ductal adenocarcinoma (PDAC). Despite this, at present the understanding of the precise mechanisms involved in the development and malignant transformation of PDAC remain relatively limited. Here, we evaluated for the first time, the molecular heterogeneity of PDAC tumors, through simultaneous assessment of the gene expression profile (GEP) for both coding and non-coding genes of tumor samples from 27 consecutive PDAC patients. Overall, we identified a common GEP for all PDAC tumors, characterized by an increased expression of genes involved in PDAC cell proliferation, local invasion and metastatic capacity, together with a significant alteration of the early steps of the cellular immune response. At the same time, we confirm and extend on previous observations about the genetic complexity of PDAC tumors as revealed by the demonstration of two clearly distinct and unique GEPs (e.g. epithelial-like vs. mesenchymal-like) reflecting the alteration of different signaling pathways involved in the oncogenesis and progression of these tumors. Our results also highlight the potential role of the immune system microenvironment in these tumors, with potential diagnostic and therapeutic implications.