Clinical detection and categorization of uncommon and concomitant mutations involving BRAF

The mitogen-activated protein kinase (MAPK) or RAS/RAF/MEK/ERK signaling pathway regulates cell proliferation, differentiation and apoptosis [1]. This pathway is often dysregulated in human cancers, frequently due to activating mutations of the KRAS, NRAS, or BRAF genes. Selective BRAF inhibitors like vemurafenib and dabrafenib [2], and MEK inhibitors like trametinib have been developed to target BRAF mutant tumors [3]. Since the approval of vemurafenib by the Food and Drug Administration (FDA) of the United States in 2011 for treatment of unresectable or metastatic melanomas with a BRAF p.V600E mutation, clinical detection of the BRAF p.V600E mutation has become the standard of care for patients with metastatic melanoma in order to predict response to vemurafenib, dabrafenib and trametinib [4–7].

The BRAF gene is mutated in approximately 7 % of human cancers overall [8], specifically, 40 % to 60 % of malignant melanomas [9], 10 % to 15 % of colorectal cancers (CRCs) [10], and 1 to 5 % of non-small cell lung cancers (NSCLC) [11, 12]. While p.V600E is the most common mutation detected in many tumor types, more than 100 mutations within exons 11 and 15 of the BRAF gene have been reported in the Catalog of Somatic Mutations in Cancer (COSMIC) database, accessed on 03/10/15. The clinical significance of non-codon 600 mutations is largely unknown. In our previous retrospective study for quality assessment, next generation sequencing (NGS) demonstrated a high analytic sensitivity and a broad reportable range for clinical detection of BRAF mutations. Non-p.V600E mutants constitute a significant portion of BRAF mutations in different tumors: NSCLCs (86 %), melanomas (34 %) and CRCs (23 %) [12]. Discovering the spectrum of non-p.V600E BRAF mutations in different malignancies is a first step toward understanding their clinical significance.

The role of BRAF mutations in the MAPK pathway is complicated not only by the multiplicity of signaling molecular components but also the variation of kinase activity for different BRAF mutants (Fig. 1). Most BRAF mutations, including the most common p.V600E (c.1799 T > A) mutation, cause upregulation of the kinase activity (kinase-activated mutants). Meanwhile, kinase-impaired BRAF mutants have also been reported [11, 13, 14]. While different assay systems have been used to determine the mutant kinase activity, most commonly, basal BRAF kinase activity was determined in vitro by measuring direct MEK phosphorylation [8, 11, 13] or measuring ERK phosphorylation in a kinase cascade assay using purified MEK and ERK proteins [15–17]. Wan et al. defined a high-activity mutant by a basal BRAF kinase activity higher than that of oncogenic RAS-activated wild-type BRAF, an intermediate-activity mutant by a basal BRAF kinase activity between those of wild-type BRAF and oncogenic RAS-activated wild-type BRAF, and a kinase-impaired mutant by a basal BRAF kinase activity lower than that of wild-type BRAF [13]. Demonstration of increased phosphorylation of BRAF and MEK proteins in patient’s tumor cell lysates [18] or inhibition of mutant-induced MEK and/or ERK phosphorylation by BRAF inhibitors in cell culture systems [19, 20] was also used to define kinase-activated mutants. Kinase-impaired mutants were further grouped into reduced-activity mutants, which could still induce MEK and ERK phosphorylation via activation of CRAF in cell culture system (Fig. 1), and silent-activity (or dead) mutant which could not [13, 16, 21]. In the presence of oncogenic RAS, however, the silent-activity mutants could induce MEK and ERK phosphorylation via activation of CRAF (Fig. 1) [22]. Since reduced-activity mutants could still activate MEK/ERK via CRAF [11, 13, 14], demonstration of mutant-induced MEK or ERK phosphorylation in cell culture systems without evidence of inhibition of mutant-induced MEK or ERK phosphorylation by BRAF inhibitors was not sufficient to define a kinase-activated mutant. Kinase-activated mutants and kinase-impaired mutants promote MEK/ERK activation and tumor progression through different mechanisms. Categorization of BRAF mutations according to their kinase activity and the presence of absence of concomitant KRAS or NRAS mutations may shed light on different therapeutic strategies to treat BRAF-mutated tumors.

BRAF mutations show diverse functional consequences and varied response to BRAF inhibitors. In this study, we categorized BRAF mutations detected in NSCLCs, CRCs and melanomas into kinase-activated mutants (75 %), kinase-impaired mutants (15 %) and kinase-unknown mutants (10 %) according to the functional studies reported in the literature. NSCLCs showed a significantly lower incidence of kinase-activated mutants than those of CRCs and melanomas. Mutations at codon 594 accounted for 11 % of BRAF mutations and were the most common kinase-impaired ones. We also demonstrated that concomitant KRAS or NRAS mutations, but not PIK3CA mutations, more likely occur with the kinase-impaired BRAF mutants than the kinase-activated ones.

While some BRAF mutations could be passenger mutations, especially those with impaired or unknown kinase activity, most BRAF mutations with elevated kinase activity are likely involved in oncogenesis and thus could be targetable. Choices of the inhibitors or inhibitor combinations are at least partly made based on BRAF mutation status. In our study, p.V600E (c.1799 T > A) was the most common BRAF mutation, occurring in 27 of 34 (79 %) CRCs, 44 of 68 (65 %) melanomas and 7 of 33 (21 %) NSCLCs. Non-p.V600E codon 600 mutations were seen in 8 of 68 (12 %) BRAF-mutated melanomas. The selective inhibitors of BRAF codon 600 mutants, vemurafenib and dabrafenib, have been shown to improve progression-free and overall survival in metastatic melanoma patients, with either the p.V600E mutation or non-p.V600E mutations at codon 600, such as p.V600K and p.V600R [4, 27–29]. Responding to vemurafenib or dabrafenib has been observed in few NSCLC patients with a BRAF p.V600E mutation [30–33], although the exact benefit of selective BRAF inhibitors for lung cancer patients is currently still under investigation [34]. Whether BRAF inhibitors benefit patients with a kinase-activated BRAF mutation located outside codon 600 remains less clear, although a partial response to vemurafenib has been reported in a melanoma patient with a p.L597R mutation [20]. Responsiveness to MEK inhibitors (TAK-733 and trametinib) in melanoma patients with a codon 597 mutations or p.K601E mutation suggests that future clinical trials of MEK inhibitors in patients with kinase-activated non-codon 600 mutations should be considered [19, 35, 36].

BRAF mutations with reduced kinase activity are unlikely responsive to BRAF inhibitors. These mutants, however, can still drive MAPK pathway through activation of CRAF/MEK/ERK cascade (Fig. 1) [13, 14]. In vitro studies of kinase-reduced mutants such as p.G466V, p.G466E and p.G596R, have also shown that activation of the CRAF/MEK/ERK cascade can be inhibited by MEK inhibitors or sorafenib, an inhibitor for multiple kinase including CRAF. In a previous clinical trial of dasatinib, a tyrosine kinase inhibitor, for metastatic non-small cell lung cancer, a patient with BRAF p.Y472C mutation remained 4-year disease-free after treatment [11]. In vitro studies confirmed the activation of CRAF/MEK/ERK cascade by the kinase-impaired p.Y472C mutant and demonstrated dasatinib-induced senescence and apoptosis in lung cancer cells expressing kinase-impaired p.G466V mutant, but not in cell lines with kinase-activated BRAF mutants.

Mutations with silent/dead kinase activity are unlikely responsive to either BRAF or MEK inhibitors. Mutations with silent/dead kinase activity have been reported in p.D594V, p.D594A and p.K483M mutations of the BRAF gene [13, 16, 22]. Codon 483 encodes the catalytic lysine and the aspartic acid at codon 594 is part of the DFG motif that plays an important role in chelating magnesium and stabilizing ATP binding [22]. Mutations at codon 483 or 594 genes lead to silence of the kinase activity with no direct or indirect activation on the downstream MAPK pathway [13, 16, 22]. Mutations at codon 594, however, have been associated with a higher incidence of co-existent RAS mutation (4 in 34, 12 %) as compared to p.V600E and presumably may also cooperate with mutations within the upstream of RAS or inter-connected pathways [22]. In this study, mutations at codon 594 constituted the second most common BRAF mutations in NSCLCs (7/33, 21 %), CRCs (4/34, 12 %) and melanomas (4/68, 5.9 %). Concomitant activating RAS mutations were observed in 2 of 15 (13 %) codon 594 mutations, but none of 86 codon 600 mutations. In the presence of oncogenic RAS proteins, kinase-silent BRAF forms a complex with CRAF and lead to hyperactivation of the CRAF/MEK/ERK cascade (Fig. 1) [22]. These preclinical studies suggested that MEK inhibitors or CRAF inhibitors may benefit patients with concomitant kinase-silent BRAF mutation and activating RAS mutation.

In contrast to the absence of concomitant BRAF p.V600E mutation and activating RAS mutation, 3 of 7 (43 %) NSCLCs and 3 of 27 (11 %) CRCs with a p.V600E (c.1799 T > A) showed a concomitant mutation in the AKT or PIK3CA genes of the mTOR pathway. In the step-wise genetic alteration model associated with colorectal tumorigenesis, PIK3CA mutations occur after KRAS or BRAF mutations and, in cooperation with other mutations, drive clonal evolution from large adenoma to invasive adenocarcinoma [37]. Thus it is not surprising to see concomitant PIK3CA mutations in 11 % of CRCs with a BRAF p.V600E (c.1799 T > A) mutation, similar to a 16 % of CRCs with an activating KRAS mutation (data not shown) in this cohort. Activating PIK3CA p.H1047R mutation has also been shown to cooperate with BRAF p.V600E mutation to promote progression of benign lung tumors to lung cancers [38]. Mutations in the AKT and PIK3CA genes, however, are uncommon in NSCLCs. PIK3CA mutations have been detected in approximately 2 % of lung adenocarcinomas [39]. The AKT p.E17K mutation was seen in only 2 of 509 NSCLCs [40–43]. In this study, the AKT p.E17K mutation was only detected in two lung adenocarcinomas with a BRAF p.V600E (c.1799 T > A) mutation, suggesting the cooperation between the MAPK and mTOR pathways, similar to that between the KRAS, NRAS or BRAF mutation and PIK3CA mutation.

In general, initiating driving mutations within the same pathway are mutually exclusive. In the setting of clinical diagnosis, caution has to be taken for interpretations of “double initiating mutations” within the same pathway. Mutations in the KRAS, NRAS and PIK3CA genes have been the mechanisms for both innate and acquired resistance to targeted therapeutics with kinase inhibitors or anti-EGFR antibodies [34]. In this study, none of patients with concomitant mutations received kinase inhibitors or anti-EGFR antibodies. In the presence of concomitant PIK3CA or AKT mutations, a combination of BRAF inhibitors or MEK inhibitors with mTOR pathway inhibitors may be more effective [44]. Correlation of the mutant allele frequencies with the estimated tumor frequency may be applied to elucidate if concomitant mutations are present in the same tumor population or only in a subpopulation. The consistency of mutant allele ratios, when testing different random subareas, would further support that concomitant mutations are present in the same population.

In this study, we categorized BRAF mutations according to the reported kinase activity and showed that concomitant KRAS or NRAS mutations more likely occur with the kinase-impaired BRAF mutants than the kinase-activated BRAF mutants. Different therapeutic strategies should be developed based on BRAF mutant kinase activity and the concomitant mutations.