Recent advances in our understanding of the genetic alterations responsible for the development of cancer have been paralleled by an increasing capacity for the development of drugs with selectivity for specific protein targets. Such advances have led to the hope that more effective cancer therapies can be developed that work by selectively inhibiting the specific molecular alterations responsible for cancer initiation and progression. Thus far, this paradigm has been most effective with inhibitors of proteins that are mutationally activated in tumors. Examples include imatinib in patients with CML (ABL), GIST (KIT), and dermatofibrosarcoma protuberans (PDGFR) (34
), and gefitinib/erlotinib in non-small cell lung cancer (EGFR) (4
In CML, ABL
translocations, most commonly BCR-ABL
, are the hallmark of this disease and therefore patient selection beyond traditional pathologic criteria is not needed in order to identify a target population predicted to be sensitive to ABL kinase inhibitors. In contrast, it is now apparent that solid tumors, traditionally classified by tissue of origin and histologic subtype, can have a diversity of mutations that confer similar selective advantage. For example, in non-small cell lung cancers, EGFR, KRAS
mutations are non-overlapping1
and all activate the MAP kinase cascade (36
). In NSCLC, the presence or absence of mutations in the EGFR
genes has been shown to correlate strongly with response to EGFR inhibitors (4
). Whereas an activating mutation in EGFR
is a positive predictor of response to gefitinib and erlotinib, KRAS
mutation confers negative predictive value for the same class of agents (11
). Recent data has also confirmed that KRAS
mutation is sufficient to confer resistance to EGFR-targeting antibodies such as cetuximab and panitumumab (12
). These observations suggest that the response of patients to a particular targeted agent will depend strongly upon the complement of mutations within an individual patient’s tumor and that such predictors (both positive and negative) can be identified. The experience with gefitinib and erlotinib also suggests that it would be valuable to know the tumor genotype of the patients prospectively and to use this information to select the appropriate patients for clinical trial. This is particularly important if the frequency of mutation in the population tested is low.
missense mutations, the vast majority of which are V600E, are the most common kinase domain mutations in human tumors (13
). These mutations, found in approximately 8% of all tumors, are non-overlapping in distribution with RAS mutations. Supporting its classification as an oncogene, V600E
BRAF stimulates ERK signaling, induces proliferation and is capable of promoting transformation and inducing tumors in transgenic mice (41
). To study the biology of BRAF
mutation in NSCLC, we screened a large panel of cell lines for exon 11 and exon 15 BRAF
mutations. We identified five cell lines with known “hot spot” mutations within the BRAF kinase domain, one of which, HCC364, harbors the V600E mutation. We observed that NSCLC with BRAF
mutations were selectively sensitive to MEK inhibition, compared to cell lines expressing EGFR
mutations, the SLC34A2-ROS
fusion, the EML4-ALK
fusion and those with amplification of PDGFRα
. Notably, we did not assess the MEK-dependence of cell lines in which no identifiable driver mutation has been identified. As such cell lines may contain occult mutations in genes such as NF1
) that activate the MAPK pathway, we would expect that a subset of these “BRAF wild-type” cell lines may also be dependent upon MEK for proliferation or survival. Validating this possibility, MEK1
mutations were recently identified with low frequency in NSCLC (43
We observed that MEK inhibition in the V600EBRAF-expressing HCC364 NSCLC cells induced levels of apoptosis comparable to those seen with EGFR inhibition in the EGFR-mutant H3255 model. However, as seen with EGFR inhibitors in EGFR-mutant models, the apoptotic response of BRAF- and KRAS-mutant cell lines to MEK inhibition was variable. These data suggest that additional genetic heterogeneity within the BRAF- and KRAS-mutant classes likely conditions the response of these cells to ERK pathway inhibition. Future studies are underway to determine which genetic and epigenetic alterations commonly co-exist with EGFR, RAS and BRAF mutations in lung cancer tumors and their impact upon MEK/ERK pathway dependence.
Using a cohort of over 900 lung cancer tumors, we identified a total of 17 patients whose lung tumors harbored BRAF
exon 11 or exon 15 mutations, representing 1.9% of the total cases analyzed, an incidence consistent with previous reports (13
). Notably, in contrast to the clinical profile of patients with EGFR kinase domain mutations, the majority of patients with BRAF mutations were current or former smokers. Our observed incidence of V600E mutations (1.2%) was also higher than would have been predicted based upon prior reports. One explanation for the higher observed frequency of V600E mutations may have been the use of mass spectrometry genotyping in the MSKCC series, a technology with greater sensitivity than Sanger sequencing.
Overall, our data suggest that BRAF is a driver mutation in patients in which it is mutated and that targeting MEK may be a useful therapeutic strategy in this subset of patients. Such a targeted strategy, however, has not been pursued to date in the clinic and no ongoing or completed Phase 1 or 2 trial of a MEK-selective inhibitor has yet enriched for NSCLC patients with BRAF mutations. The primary hurdle has been that testing for BRAF mutation is not prospectively performed in patients with NSCLC. Routine testing of NSCLC patients for KRAS mutation, on the other hand, is becoming more widespread, as the presence of a KRAS mutation predicts for de novo resistance to the EGFR inhibitors gefitinib and erlotinib. We now show that BRAF mutant cell lines are also resistant to EGFR inhibition. Based upon these data, we propose clinical studies to determine whether BRAF mutation has similar value in predicting for de novo resistance to EGFR inhibition. If confirmed, we believe that routine clinical testing of all NSCLC for BRAF mutation would be justified. Such an effort would also have the secondary benefit of accelerating the development of BRAF- and MEK-selective inhibitors by aiding in the identification of the minority of lung cancer patients likely to respond to such agents. As demonstrated by our use of the MALDI-TOF mass spectrometry assay to screen simultaneously for EGFR, KRAS and BRAF mutations, we believe that such efforts are now technically feasible. Based upon these results, we have initiated routine prospective genotyping of NSCLC patients for BRAF mutation and have proposed clinical studies of MEK inhibitors with enrollment restricted exclusively to those with an activating BRAF mutation.