KRAS, NRAS and BRAF mutations detected by next generation sequencing, and differential clinical outcome in metastatic colorectal cancer (MCRC) patients treated with first line FIr-B/FOx adding bevacizumab (BEV) to triplet chemotherapy

Background First line triplet chemotherapy/BEV significantly improved clinical outcome of MCRC. KRAS/NRAS/BRAF mutations were evaluated by next generation sequencing (NGS) in MCRC patients treated with first line FIr-B/FOx. Methods KRAS exons 2-4 (KRAS2-4), NRAS2-4, BRAF15 were evaluated in 67 tumours by ION Torrent platform. Mutation detection criteria: >500×sequence coverage (cov); >1% mutant allelic fraction (AF). Clinical outcomes were compared by log-rank. Results In 63 samples, KRAS2-4/NRAS2-4/BRAF15 wild-type (wt) were 14 (22.2%), mutant (mut) 49 (77.8%): KRAS2-4 42 (66.7%); NRAS2-4 11 (16.4%); BRAF15 5 (7.5%). Sixty mutations were detected, range 1-3 mut: 43 (71.7%) >1000×cov/>5% AF; 9 (15%) >500×cov/>5% AF; 8 (13.3%) >1000×cov/<5% AF. Mut distribution in KRAS2-4/NRAS2-4/BRAF15: 40 (63.5%) >1000×cov/>5% AF, 8 (12.7%) >500×cov/>5% AF, 1 (1.6%) >1000×cov/<5% AF; BRAF15 1 (1.5%) >500×cov/>5% AF, 4 (6%) >1000×cov/<5% AF. Prevalence of ≥2 mut samples: KRAS2-4/NRAS2-4/BRAF15 8 (12.7%); KRAS2-4 7 (11.1%); NRAS2-4 5 (7.5%). BRAF15 mutant were all ≥2 mut (7.5%), atypical and associated to KRAS and/or NRAS mut: c.1405 G>A; c.1406 G>C; c.1756 G>A, 2 samples; c.1796 C>T. At 21 months (m) follow-up, clinical outcome wt compared to mut was not significantly different: in KRAS2-4/NRAS2-4/BRAF15, progression-free survival (PFS) 18/12 m, overall survival (OS) 28/22 m; 1/≥2 mutations, PFS 14/11, OS 37/22. PFS was trendy worse in RAS/BRAF wt vs ≥2 mut genes (P 0.059). Conclusions Most MCRC harboured KRAS2-4/NRAS2-4/BRAF15 mutations by NGS, often multiple and affecting few tumoral clones; 22% were triple wt. Clinical outcome is not significantly affected by KRAS2-4/NRAS2-4/BRAF15 genotype, trendy different in triple wt, compared with KRAS2-4/NRAS2-4/BRAF15 ≥2 mut.


INTRODUCTION
Gain-of-function mutations of KRAS, NRAS, BRAF, PIK3CA genes, or loss of tumor suppressor function of PTEN, resulting in continuous activation of RAS-mitogenactivated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) pathways, characterize most colorectal cancers (CRC) [1]. More, 12-15% CRC show high mutational load and microsatellite instability [2]. Specific mutations of the different genes involved in MAPK/PI3K pathway may confer different biological aggressiveness and effectiveness of treatment strategies.

DISCUSSION
The present study evaluated the prevalence and individual distribution of KRAS 2-4 /NRAS 2-4 /BRAF 15 mutations by NGS in MCRC patients treated with intensive first line treatment adding BEV to triplet chemotherapy according to previously reported FIr-B/FOx schedule [5] 15 mut, as recently reported [13,14]. One MCRC patient was only detected at 500-1000×cov and <5%AF (1.6%), and harbored NRAS c.182 A>G (Q61R) associated to BRAF c.1796 C>T (T599I) mutations. Thus, massive parallel sequencing by Ion torrent platform can increase mutation detection by increasing diagnostic accuracy, if >1% mutant AF with >1000×cov is included to specifically detect clonal heterogeneity involving KRAS, NRAS and atypical BRAF mutations, thus increasing the detection of multiple genes mutations in individual MCRC patients. To this aim, NGS is able to detect mutant alleles at the 5% level [13,14].
Specific mutations of different genes involved in the same signalling pathway (BRAF and RAS mutations) can confer different biological aggressiveness and effectiveness of treatment strategies. Preliminary analysis of differential clinical outcome in overall MCRC patients treated with FIr-B/FOx intensive first line treatment according to KRAS 2 genotype confirmed previously reported median PFS 13 months and OS 27 months, a trendy worse OS 21 months in KRAS 2 mut, and PFS 8 months and OS 14 months in the prevalent KRAS 2 c.35G > A mut MCRC patients [11]. Clinical outcome was not significantly different in KRAS [2][3][4] , NRAS 2-4 , BRAF 15 mut and wt MCRC patients. The 5 BRAF 15 mut, all atypical and associated to other KRAS 2-4 and/or NRAS 2-4 mut, compared to wt MCRC patients showed trendy worse, not significantly different, PFS 8 months and OS 11 months, even if treated with BEV added to triplet chemotherapy. Worse prognosis was previously shown by the prevalent BRAF 15 c.1799 T > A (V600E) mutation, characterizing 4.7-8.7% CRC, in MCRC patients treated with doublet chemotherapy alone or added to cetuximab, BEV, and cetuximab plus BEV, with median PFS 5.6-8 months and median OS 10.3-15.9 months [24][25][26]. The favourable predictive effect of cetuximab or BEV addiction to chemotherapy in KRAS exon 2 wild-type patients was not significantly confirmed in BRAF mutant MCRC patients [20,24,25]. Mutations in BRAF gene occur in two regions of the BRAF kinase domain, exon 15, the activation segment (which protects the substrate binding site), and, less commonly, exon 11, the G loop (which mediates ATP-binding). Less common BRAF mutations at codons 594 and 596 correlated with longer OS when compared with BRAF V600E mutations (62 vs 12.6 months, P = 0.002) [27], and trendy longer compared to BRAF wild-type (35.9 months) in MCRC patients treated with FOLFOXIRI/BEV [3].  Prospective studies should be developed to better evaluate differential clinical outcome in MCRC patients harbouring KRAS c.35 G > A (G12D), BRAF c.1799 T > A (V600E) and atypical, less common mutations, as well as in KRAS 2-4 /NRAS 2-4 /BRAF 15 wt and in patients harbouring ≥2 mutant genes.
More, high sensitive KRAS 2-4 /NRAS 2-4 /BRAF 15 multigenic analysis performed in metastatic tissues and/ or liquid biopsies could dynamically be helpful to monitor the evolution of mutant genes spectrum, more closely evaluate prognostic implications and individually predict targeted treatment.

Patients and samples
Eighty-seven consecutive, unselected, MCRC patients were enrolled in previously reported phase II study and the expanded clinical program proposing FIr-B/FOx as first line treatment [5,9]. KRAS 2-4 /NRAS 2-4 / BRAF 15 genotype was evaluated in tumoral samples of 67 (77%) patients (Table 1), specifically primary tumours or metastatic sites, pre-or post-treatment.
Study was approved by Local Ethical Committee (Comitato Etico, Azienda Sanitaria Locale n.4 L'Aquila, Regione Abruzzo, Italia) and conducted in accordance with Declaration of Helsinki. All patients provided written, informed consent.

Study design
A retrospective analysis has been planned to evaluate prognostic relevance of KRAS 2-4 /NRAS 2-4 /BRAF 15 genotype on clinical outcome of MCRC patients treated with first line FIr-B/FOx regimen. Clinical criteria of efficacy were PFS, OS, evaluated using Kaplan and Meier method [22]. Patients were evaluated according to involved metastatic sites, classified as L-L and O/MM. Log-rank test was used to compare PFS and OS in different subgroups [23]. PFS was defined as length of time between beginning of treatment and disease progression or death (resulting from any cause) or to last contact; OS as length of time between beginning of treatment and death or to last contact.

CONCLUSIONS
Next generation sequencing of multiple genes shows that most MCRC harbour KRAS 2-4 /NRAS 2-4 /BRAF 15 mutations, prevalently as single gene mut, and frequently multiple gene mutations, at increased sensitivity due to clonal heterogeneity.
Clinical outcome of MCRC patients treated with intensive first line FIr-B/FOx regimen was not significantly affected by KRAS 2-4 /NRAS 2-4 /BRAF 15 genotype status.
Differential prognosis and predictive effect of VEGF-inhibitors added to chemotherapy in MCRC patients harbouring KRAS 2 c.35 G > A and BRAF 15 mut, or KRAS 2-4 /NRAS 2-4 /BRAF 15 wt should be prospectively evaluated by massive parallel sequencing, also according to other mutations differentially activating the downstream RAS-MAPK or PI3K pathways.