Background: KRAS codons 12 and 13 mutations predict resistance to anti-EGFR monoclonal antibodies (moAbs) in metastatic colorectal cancer. Also, BRAF V600E mutation has been associated with resistance. Additional KRAS mutations are described in CRC. Methods: We investigated the role of KRAS codons 61 and 146 and BRAF V600E mutations in predicting resistance to cetuximab plus irinotecan in a cohort of KRAS codons 12 and 13 wild-type patients. Results: Among 87 KRAS codons 12 and 13 wild-type patients, KRAS codons 61 and 146 were mutated in 7 and 1 case, respectively. None of mutated patients responded vs 22 of 68 wild type (P=0.096). Eleven patients were not evaluable. KRAS mutations were associated with shorter progression-free survival (PFS, HR: 0.46, P=0.028). None of 13 BRAF-mutated patients responded vs 24 of 74 BRAF wild type (P=0.016). BRAF mutation was associated with a trend towards shorter PFS (HR: 0.59, P=0.073). In the subgroup of BRAF wild-type patients, KRAS codons 61/146 mutations determined a lower response rate (0 vs 37%, P=0.047) and worse PFS (HR: 0.45, P=0.023). Patients bearing KRAS or BRAF mutations had poorer response rate (0 vs 37%, P=0.0005) and PFS (HR: 0.51, P=0.006) compared with KRAS and BRAF wild-type patients. Conclusion: Assessing KRAS codons 61/146 and BRAF V600E mutations might help optimising the selection of the candidate patients to receive anti-EGFR moAbs.

Purpose: To investigate whether prognosis of patients with high-risk gastric cancer may depend on MET copy number gain (CNG) or an activating truncation within a deoxyadenosine tract element (DATE) in the promoter region of the MET ligand HGF. Patients and Methods: A single-institution cohort of 230 patients with stage II/III gastric cancer was studied. Formalin-fixed paraffin-embedded tumor specimens were used for DNA extraction. Quantitative polymerase chain reaction (qPCR) for MET CNG and sequencing for HGF DATE truncation (< 25 deoxyadenosines instead of 30) were used. Results were analyzed for association with disease-free survival (DFS) and overall survival (OS). To assess the reliability of the qPCR measurement, a random sample of cases was reanalyzed using an alternative assay (fluorescent in situ hybridization [FISH]) with calculation of the intracorrelation coefficient (ICC). Results: In 216 assessable patients, MET CNG five or more copies and homozygous HGF-truncated DATE occurred in 21 patients (10%) and 30 patients (13%), respectively. Patients with MET CNG five or more copies (MET-positive) showed significantly worse prognosis with multivariate hazard ratio (HR) of 3.02 (95% CI, 1.71 to 5.33; P < .001) for DFS and multivariate HR of 2.91 (95% CI, 1.65 to 5.11; P < .001) for OS. The agreement between qPCR and FISH was high, with ICC = 0.9% (95% CI, 0.81% to 0.95%; the closer the ICC is to 1, the greater is the agreement). HGF-truncated DATE did not show relevant prognostic effect. Conclusion: In this study, qPCR revealed approximately 10% of white patients with gastric cancer harboring MET CNG of five or more copies. This marker was significantly associated with unfavorable prognosis. This information is relevant to the current clinical development of anti-MET compounds.

There is increasing evidence that the Let-7 microRNA (miRNA) exerts an effect as a tumor suppressor by targeting the KRAS mRNA. The Let-7 complementary site (LCS6) T>G variant in the KRAS 3′-untranslated region weakens Let-7 binding. We analyzed whether the LCS6 variant may be clinically relevant to patients with metastatic colorectal cancer (MCRC) treated with anti-epidermal growth factor receptor (EGFR) therapy. LCS6 genotypes and KRAS/BRAF mutations were determined in the tumor DNA of 134 patients with MCRC who underwent salvage cetuximab–irinotecan therapy. There were 34 G-allele (T/G+G/G) carriers (25%) and 100 T/T genotype carriers (75%). G-allele carriers were significantly more frequent in the KRAS mutation group than in patients with KRAS wild type (P=0.004). In the 121 patients without BRAF V600E mutation, overall survival (OS) and progression-free survival (PFS) times were compared between carriers of the LCS6 G-allele genotypes and carriers of the wild-type T/T genotype. LCS6 G-allele carriers showed worse OS (P=0.001) and PFS (P=0.004) than T/T genotype carriers (confirmed in the multivariate model including the KRAS status). In the exploratory analysis of the 55 unresponsive patients with KRAS mutation, LCS6 G-allele carriers showed adverse OS and PFS times. These findings deserve additional investigations as they may open novel perspectives for the treatment of patients with MCRC.

Molecular predictors of bevacizumab efficacy in colorectal cancer have not been identified yet. Specific VEGF polymorphisms may affect gene transcription and therefore indirectly influence the efficacy of bevacizumab. Genomic DNA of 111 consecutive metastatic colorectal cancer patients treated with first-line FOLFIRI plus bevacizumab was obtained from blood samples. VEGF -2578 C/A, -1498 C/T, + 405 C/G, + 936 C/T polymorphisms were analyzed by means of PCR-RFLP. DNA samples from 107 patients treated with FOLFIRI alone served as historical control group. The relation of VEGF polymorphisms with PFS, evaluated through Kaplan-Meier method and log-rank test, was the primary end-point. An interaction test with a Cox model has been performed in order to demonstrate the heterogeneity of the effect of VEGF -1498 C/T polymorphism between bevacizumab-and control group. In the bevacizumab-group median PFS and OS of patients carrying VEGF -1498 C/C, C/T and T/T allelic variants were, respectively, 12.8, 10.5, 7.5 months (p = 0.0046, log-rank test) and 27.3, 20.5, 18.6 months (p = 0.038, log-rank test). VEGF -1498 T/T genotype was associated with shorter PFS (HR = 2.13, [1.41-5.10], p = 0.0027). In the control group no significant association of VEGF -1498 C/T allelic variants and PFS or OS was found. Interaction between VEGF -1498 C/T variants and treatment effect suggested that the relation of VEGF -1498 T/T genotype with shorter PFS was caused by the effect of bevacizumab (p = 0.011). Other investigated polymorphisms did not affect the outcome. These data suggest a possible role for VEGF -1498 C/T variants in predicting the efficacy of bevacizumab in the up-front treatment of metastatic colorectal cancer patients. A molecular tool for selecting subjects candidate to benefit from the anti-VEGF could be important for clinical practice. The retrospective and exploratory design of the present study, coupled with the non-randomized nature of the comparison between treated and untreated patients, imply that these results should be considered as hypothesis generators. A prospective validating trial is currently ongoing.

Preclinical and experimental data in vivo indicate that Lethal-7 (Let-7) microRNA downregulates KRAS with antitumor effects in the presence of activating KRAS mutations. We quantified the Let-7a isoform in KRAS-mutated colorectal carcinomas from patients who received salvage cetuximab plus irinotecan. The study population was retrospectively identified among metastatic colorectal cancer patients who underwent third-line therapy with cetuximab plus irinotecan in a period when only epidermal growth factor receptor (EGFR) expression was required for anti-EGFR therapy. In 59 patients harboring KRAS mutations, Let-7a levels were analyzed for association with overall survival (OS) and progression-free survival (PFS) times. An exploratory subgroup analysis was performed using the rs61764370 (LCS6 T>G) polymorphism that experimentally impairs Let-7 binding to KRAS mRNA. In the whole group, higher Let-7a levels were significantly associated with better survival outcomes. For the primary OS endpoint, the multivariate hazard ratio was 0.82 (95% confidence interval, 0.73–0.91; p = .01). The same findings with an accentuated positive effect of high Let-7a levels on both OS and PFS times were observed in an exploratory analysis of the 45 wild-type LCS6 patients (excluding 14 carriers of the LCS6 G allele variant). All survival associations were confirmed after excluding patients with KRAS codon 13 mutations. Among the clinicopathologic features, high Let-7a levels were associated with grade 2–3 skin toxicity (p = .002). In patients with KRAS mutations, Let-7a analysis may serve to identify subgroups of patients who may still benefit from EGFR inhibition and this may open up new perspectives for alternative treatment strategies.

In metastatic colorectal cancer (mCRC) patients, the efficacy of anti-epidermal growth factor receptor (EGFR) antibodies, cetuximab and panitumumab, is limited to patients with KRAS wild-type tumours [1]. However, not all mCRC patients with KRAS wild-type tumours respond to this treatment. Therefore, there is a need for additional predictive markers. A mutation in B-type Raf kinase (BRAF), located downstream of KRAS, has been implicated in the response to anti-EGFR treatment [2]. The V600E is the most common oncogenic mutation of BRAF in cancer and is mutated in ∼5%–10% of mCRC patients. Moreover, BRAF mutational status also appears to have prognostic value in patients with mCRC independently from anti-EGFR therapies [3]. Although different studies have demonstrated a high concordance rate between primary tumour and metastasis in the KRAS gene status, only limited data on BRAF status are available for this concordance. Italiano et al. [4] reviewed the literature and reported an identical mutational pattern of BRAF in primary tumours and matching metastases in all but 2 (3%) of 95 patients, mostly with synchronous metastases.