Clinico-pathological associations and concomitant mutations of the RAS/RAF pathway in metastatic colorectal cancer.

BACKGROUND: Over the past few years, next-generation sequencing (NGS) has become reliable and cost-effective, and its use in clinical practice has become a reality. A relevant role for NGS is the prediction of response to anti-EGFR agents in metastatic colorectal cancer (mCRC), where multiple exons from KRAS, NRAS, and BRAF must be sequenced simultaneously. METHODS: We optimized a 14-amplicon NGS panel to assess, in a consecutive cohort of 219 patients affected by mCRC, the presence and clinico-pathological associations of mutations in the KRAS, NRAS, BRAF, and PIK3CA genes from formalin-fixed, paraffin-embedded specimens collected for diagnostics and research at the time of diagnosis. RESULTS: We observed a statistically significant association of RAS mutations with sex, young age, and tumor site. We demonstrated that concomitant mutations in the RAS/RAF pathway are not infrequent in mCRC, and as anticipated by whole-genome studies, RAS and PIK3CA tend to be concurrently mutated. We corroborated the association of BRAF mutations in right mCRC tumors with microsatellite instability. We established tumor side as prognostic parameter independently of mutational status. CONCLUSIONS: To our knowledge, this is the first monocentric, consecutively accrued clinical mCRC cancer cohort tested by NGS in a real-world context for KRAS, NRAS, BRAF, and PIK3CA. Our study has highlighted in clinical practice findings such as the concomitance of mutations in the RAS/RAF pathway, the presence of multiple mutations in single gene, the co-occurrence of RAS and PIK3CA mutations, the prognostic value of tumor side and possible associations of sex with specific mutations.

CEA response is associated with tumor response and survival in patients with KRAS exon 2 wild-type and extended RAS wild-type metastatic colorectal cancer receiving first-line FOLFIRI plus cetuximab or bevacizumab (FIRE-3 trial).

BACKGROUND: To examine the relation of carcinoembryonic antigen (CEA) response with tumor response and survival in patients with (K)RAS wild-type metastatic colorectal cancer receiving first-line chemotherapy in the FIRE-3 trial comparing FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab. PATIENTS AND METHODS: CEA response assessed as the percentage of CEA decrease from baseline to nadir was evaluated for its association with tumor response and survival. Receiver operating characteristic analysis revealed an optimal cut-off value of 75% using the maximum of sensitivity and specificity for CEA response to discriminate CEA responders from non-responders. In addition, the time to CEA nadir was calculated. RESULTS: Of 592 patients in the intent-to-treat population, 472 were eligible for analysis of CEA (cetuximab arm: 230 and bevacizumab arm: 242). Maximal relative CEA decrease (%) significantly (P = 0.003) differed between the cetuximab arm (median 83.0%; IQR 40.9%-94.7%) and the bevacizumab arm (median 72.3%; IQR 26.3%-91.0%). In a longitudinal analysis, the CEA decrease occurred faster in the cetuximab arm and was greater than in the bevacizumab arm at all evaluated time points until 56 weeks after treatment start. CEA nadir occurred after 3.3 months (cetuximab arm) and 3.5 months (bevacizumab arm), (P = 0.49). In the cetuximab arm, CEA responders showed a significantly longer progression-free survival [11.8 versus 7.4 months; hazard ratio (HR) 1.53; 95% Cl, 1.15-2.04; P = 0.004] and longer overall survival (36.6 versus 21.3 months; HR 1.73; 95% Cl, 1.24-2.43; P = 0.001) than CEA non-responders. Analysis of extended RAS wild-type patients revealed similar results. CONCLUSION: In the FIRE-3 trial, CEA decrease was significantly faster and greater in the cetuximab arm than in the bevacizumab arm and correlated with the prolonged survival observed in patients receiving FOLFIRI plus cetuximab. CLINICAL TRIALS NUMBER: NCT00433927 (ClinicalTrials.gov); AIO KRK0306 FIRE-3.

PIK3CA Mutated Colorectal Cancers Without KRAS, NRAS and BRAF Mutations Possess Common and Potentially Targetable Mutations in Epigenetic Modifiers and DNA Damage Response Genes.

BACKGROUND/AIM: Despite therapeutic advancements, metastatic colorectal cancer is usually fatal, necessitating novel approaches based on the molecular pathogenesis to improve outcomes. Some colorectal cancers have no mutations in the extended RAS panel (KRAS, NRAS, BRAF) genes and represent a special subset, which deserves particular therapeutic considerations. MATERIALS AND METHODS: The genomic landscape of colorectal cancers from publicly available genomic series was interrogated, using the cBioportal platform. Colorectal cancer cohorts with cancers devoid of KRAS/NRAS or BRAF mutations were evaluated for the presence of mutations in the catalytic sub-unit alpha of kinase PI3K, encoded by the gene PIK3CA. RESULTS: PIK3CA mutations in the absence of KRAS/NRAS/BRAF mutations were observed in 3.7% to 7.6% of colorectal cancers in the different series examined. Patients with all four genes in wildtype configuration (quadruple wild type) represented 32.2% to 39.9% of cases in the different series examined. Compared with quadruple wild type cancers, triple (KRAS/NRAS/BRAF) wild type/PIK3CA mutated cancers had a higher prevalence of high TMB cases and additional mutations in colorectal cancer associated genes except for mutations in TP53. Mutations in genes encoding for epigenetic modifiers and the DNA damage response (DDR) were also more frequent in triple wild type/PIK3CA mutated cancers. The prognosis of the two groups was comparable. CONCLUSION: Colorectal cancers with PIK3CA mutations in the absence of KRAS/NRAS/BRAF mutations have frequently mutations in epigenetic modifiers and DDR response genes, which may provide opportunities for targeting. These mutations are present in a smaller subset of quadruple wild type cancers.

Extended RAS testing in metastatic colorectal cancer-Refining the predictive molecular biomarkers.

Mutations of exon 2 of Kirsten rat sarcoma viral oncogene homologue (KRAS) (exon 2 codons 12/13) lead to constitutive activation of the EGFR (epidermal growth factor receptor) mediated signal transduction pathway and been shown to be a negative predictive biomarker for EGFR-directed monoclonal antibodies among patients with colorectal cancer (CRC). As selection of patients is very important for administration of anti-EGFR therapy, this lone biomarker has proved to be insufficient for selecting the appropriate patients as more patients lacking exon 2 KRAS mutation were resistant to anti-EGFR therapy. The results of various randomized clinical trials have confirmed the presence of other RAS mutation including additional RAS mutations (KRAS exons 3/4 and NRAS exon 1/2/3/4). Extended RAS analysis should be considered before initiating anti-EGFR therapy to patients of metastatic CRC. This can help in proper selection of patients leading to tailored individualistic treatment, decreasing cost of treatment and the adverse effects related to use of monoclonal antibody therapy. The new evidence is supporting the need to make 'Extended RAS' analysis essential before start of treatment with anti-EGFR monoclonal antibody therapy. Prior to this the Extended RAS testing should be standardized.

High-throughput screening of extended RAS mutations based on high-resolution melting analysis for prediction of anti-EGFR treatment efficacy in colorectal carcinoma.

OBJECTIVES: Recent studies have demonstrated that, in advanced colorectal carcinoma (CRC) patients, extended RAS (in KRAS exons 2-4 and NRAS exons 2-4) and BRAF mutations are negative predictors for anti-EGFR treatment efficacy and negative prognostic factor, respectively. Thus, high-throughput and cost-effective methods for identification of the mutation status are required. DESIGN AND METHODS: We developed a PCR-high-resolution melting (HRM)-based method for screening extended RAS and BRAF mutations, and relative frequency of mutations in formalin-fixed paraffin-embedded samples of CRC was analyzed. RESULTS: Among 93 CRC samples, 29 harbored mutations in KRAS exon 2, and 9 harbored mutations in BRAF exon 15. Analysis of 55 KRAS exon 2 and BRAF exon 15 wild-type CRC samples identified the following mutations: 1/55 in exon 3 and 2/55 in exon 4 of KRAS; 1/55 in exon 2, 3/55 in exon 3, and 0/55 in exon 4 of NRAS. CONCLUSIONS: Our PCR-HRM method will enable rapid determination of the extended RAS and BRAF mutation status prior to anti-EGFR treatment in the clinical setting.