BRAFV600E Mutant Colorectal Cancer Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

BRAF Mutations in Non-Metastatic Colorectal Cancer: Current Relevance and Future Implications

BRAF V600E-mutated colorectal cancer (CRC) is a distinct entity that accounts for less than 10 % of all CRC patients and is associated with unique clinical and pathologic features. This biomarker also confers a poor prognostic outcome across all stages of CRC relative to wild-type counterparts. Currently, the landscape of effective therapies, both in terms of adjuvant treatment and in the metastatic setting, remains limited. This review will detail the role of the BRAF V600E mutation as a prognostic biomarker in the management of patients with non-metastatic and metastatic CRC, and highlight recent advances in targeted therapies for this subset of CRC, which may represent the most significant progress made thus far toward improving survival in these patients. The role of mismatch repair status and its relationship with the BRAF V600E mutation in CRC will also be examined.

Abstract 2140: Dual Wnt and EGFR-MAPK dependency of BRAFV600E-mutant colorectal cancer

Abstract Aberrant Wnt pathway activation due to inactivating mutations in the gene encoding RNF43 (an E3 ubiquitin ligase that promotes degradation of the Wnt receptors Frizzled and LRP6) may contribute to the unresponsiveness of BRAFV600E-mutant colorectal cancer (CRC) to BRAF inhibitors. Analysis of The Cancer Genome Atlas (TCGA) CRC data set reveals a striking co-occurrence of the BRAFV600E mutation and truncating mutations in RNF43. RNF43 mutations are likely to be functionally significant, as RNF43 mutations and mutations in the β-catenin destruction complex component APC are almost completely mutually exclusive. The vast majority of BRAFV600E;RNF43-mutant CRCs are hypermutable [microsatellite instability (MSI)-high phenotype]. The mismatch repair deficiency in these tumors may directly contribute to RNF43 mutagenesis, as RNF43 mutations tend to be small insertions/deletions in homopolymeric tracts. To determine if RNF43 mutations confer Wnt dependency in BRAFV600E-mutant CRC, we treated three BRAFV600E;RNF43-mutant CRC patient-derived xenograft (PDX) models with the porcupine inhibitor WNT974 (formerly LGK974), which blocks the palmitoylation and secretion of Wnt ligands. Single agent WNT974 anti-tumor activity was observed in 2/3 PDX models, and correlated with decreased tumor cell proliferation and mucinous differentiation. Single agent anti-tumor activity with the BRAF inhibitor LGX818 was also observed in 2/3 PDX models. No single agent anti-tumor activity was observed with the EGFR inhibitor cetuximab. The double combinations of WNT974+LGX818 and LGX818+cetuximab, and the triple combination of WNT974+LGX818+cetuximab were efficacious in all three BRAFV600E;RNF43-mutant CRC PDX models. In summary, the Wnt pathway and the EGFR-MAPK pathway may jointly promote tumorigenesis of BRAFV600E-mutant CRC, providing a strong rationale to treat patients with BRAFV600E-mutant CRCs harboring upstream Wnt pathway mutations with combinations of WNT974, LGX818 and/or cetuximab. Citation Format: Youzhen Wang, Michael Palmer, Savina Jaeger, Linda Bagdasarian, Shumei Qiu, Steve Woolfenden, Ronald Meyer, Guizhi Yang, John Green, Shifeng Pan, Jun Liu, Hui Gao, Z. Alexander Cao, Andrea Myers, Margaret E. McLaughlin. Dual Wnt and EGFR-MAPK dependency of BRAFV600E-mutant colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2140. doi:10.1158/1538-7445.AM2015-2140

Comparison of the prevalence of KRAS-LCS6 polymorphism (rs61764370) within different tumour types (colorectal, breast, non-small cell lung cancer and brain tumours). A study of the Czech population.

A germline SNP (rs61764370) is located in a let-7 complementary site (LCS6) in the 3'UTR of KRAS oncogene, and it was found to alter the binding capability of the mature let-7 microRNA to the KRAS mRNA. The aim of the study was to evaluate the frequency of the KRAS-LCS6 variant allele in different cancer types that included patients with colorectal cancer (CRC), breast cancer (BC), non-small cell lung cancer (NSCLC) and brain tumour patient subgroups from the Czech Republic. The occurrence of this genetic variant was correlated with the presence of selected somatic mutations representing predictive biomarkers in the respective tumours. DNA of tumour tissues was isolated from 428 colorectal cancer samples, 311 non-small cell lung cancer samples, 195 breast cancer samples and 151 samples with brain tumour. Analysis of SNP (rs61764370) was performed by the PCR+RFLP method and direct sequencing. KRAS, BRAF and EGFR mutation status was assessed using real-time PCR. The status of the HER2 gene was assessed using the FISH method. The KRAS-LCS6 TG genotype has been detected in 16.4% (32/195) of breast cancer cases (in HER2 positive breast cancer 3.3%, in HER2 negative breast cancer 20.1%), in 12.4% (53/428) of CRC cases (KRAS/BRAF wild type CRC in 10.6%, KRAS mutant CRC in 10.1%, BRAF V600E mutant CRC in 18.5%), in 13.2% (41/311) of NSCLC samples, (EGFR mutant NSCLC patients in 8%, EGFR wild type NSCLC in 12.9%), and 17.9% (27/151) of brain tumour cases. The KRAS-LCS6 TG genotype was not significantly different across the studied tumours. In our study, the GG genotype has not been found among the cancer samples. Based on the findings, it is concluded that the occurrence of the KRAS-LCS6 TG genotype was statistically significantly different in association with status of the HER2 gene in breast cancer. Furthermore, significant association between the mutation status of analysed somatic variants in genes of the EGFR signalling pathway (KRAS, BRAF, EGFR) and the KRAS-LCS6 genotype in colorectal cancer and NSCLC has not been established.

IS7-4 - New Promising Agents/Combinations for Colorectal Cancer

Recent advances in the molecular biology research of gastrointestinal cancer have led to successful development and approval of targeted therapies such as cetuximab, panitumumab, bevacizumab, aflibercept and regorafenib in advanced colorectal cancer (CRC). These achievements have encouraged the development of innovative targeted therapies that are currently being tested in clinical trials. The increase in median survival of patients with metastatic CRC observed in the last decade is to some extent related to these new drugs. In addition to the inhibitors of epidermal growth factor receptor (EGFR) in RAS wild-type tumors and vascular endothelial growth factor receptor (VEGFR) in unselected populations, there is plethora of different druggable targets being explored in CRC. Two fundamental achievements will impact in the way we develop new targeted therapies in CRC. First, the recently available large scale databases on the genomic landscape in patients with CRC, like the TCGA consortium1, have provided paramount information on the genes that are frequently disregulated in CRC and has provided some fundamental data to differentiate, at least, two different patient populations with CRC: hypermutated and non-hypermutated tumors. The second approach has been built on the possibility of differentiating subtypes of CRC by gene profiling also in large datasets of patients with this disease2,3. Both approaches are at the very beginning steps but have provided some seminal knowledge to establish different molecular subtypes in CRC. In this regard, we have clearly differentiated the BRAF V600E mutant CRC population that, although it accounts only for 5-8% of patients with CRC, has clear dependence/addiction on this mutated gene/protein. Preclinical studies coming from at least two different groups have shown the dependence of these tumors on BRAF but also on some tyrosine kinase receptors that result activated as a compensatory mechanism of secondary resistance4,5. At this time point several trials are evaluating the combination of BRAF or MEK inhibitors with EGFR inhibitors. Preliminary data of these combination studies has yet been presented with encouraging efficacy6. Following the example of the BRAF mutated tumors population, these diagnostic platforms will provide information on some other populations that may be more dependant on selected gene disregulations. Another important concept that has been established is the plasticity CRC has in terms of clonal selection and clonal evolution. It has been shown by different groups that patients with tumors that have wild-type RAS status, once they are treated with EGFR inhibitors -treatment “pressure”-, they start showing an increase in cells that bear RAS mutations7,8. This tumor plasticity concept is fostering the development of new diagnostic approaches that may help us to provide real-time information on the proportion of different clones that compose the burden of the tumor, such as the “liquid biopsy”9. With the availability of new platforms that detect circulating tumor cells (CTCs) or circulating free DNA (cfDNA) we will have the opportunity to determine with non-invasive techniques the variation in the total tumor burden of the disease as well as the characterization of the most frequent clones that compose the tumor at a selected time-point. The clinical application of predictive biomarkers of response to molecular targeted agents has directed the definition of new paradigms in the development of new agents in oncology. The revolution in the knowledge of the biology and the characterization of different molecular subtypes of CRC as well as the availability of new targeted agents will translate in the next future in more therapeutic options in this disease.1Cancer Genome Atlas Network. Nature 2012. 2Tabernero J et al. Proc ASCO GI 2013. 3Dienstmann R et al. Proc ASCO 2014. 4Corcoran RB et al. Cancer Discov 2012. 5Prahallad A et al. Nature 2012. 6Tabernero J et al. Proc ASCO 2014. 7Misale S, et al. Nature 2012. 8Diaz E, et al. Nature 2012. 9Villar E & Tabernero J. Nature 2012

Phase 1-2 trial of the BRAF inhibitor dabrafenib (D) plus MEK inhibitor trametinib (T) in BRAF V600 mutant colorectal cancer (CRC): Updated efficacy and biomarker analysis.

3517 Background: BRAF V600 mutations occur in 5-15% of metastatic CRC and predict poor prognosis. Although highly effective in BRAF mutant melanoma, BRAF inhibitor monotherapy has shown poor effica...

Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E

BRAF V600E mutation in serrated lesions of the colon has been implicated as an important mutation and as a specific marker for the serrated carcinogenic pathway. Recent findings point to microvesicular hyperplastic polyps that have similar histologic and molecular features to sessile serrated adenomas/polyps, as potential colorectal carcinoma precursors. The aim of this study was to evaluate BRAF V600E mutation status by immunohistochemistry in serrated lesions of the colon with regard to histomorphology. We investigated 194 serrated lesions of the colon, comprising 42 sessile serrated adenomas/polyps, 16 traditional serrated adenomas, 136 hyperplastic polyps and 20 tubular/tubulovillous adenomas (conventional adenomas) with the novel BRAF V600E mutation-specific antibody VE1. In addition, BRAF exon 15 and KRAS exon 2 status was investigated by capillary sequencing in selected cases. All sessile serrated adenomas/polyps (42/42, 100%), 15/16 (94%) traditional serrated adenomas and 84/136 (62%) hyperplastic polyps were VE1+. None of the VE1− serrated lesions showed BRAF V600E mutation. Forty out of 42 (95%) sessile serrated adenomas/polyps displayed areas with microvesicular hyperplastic polyp-like features. In microvesicular hyperplastic polyps, VE1 positivity was significantly associated with nuclear atypia (P=0.003); however, nuclear atypia was also present in VE1− cases. Immunostaining with VE1 allows not only the detection of BRAF V600E mutation but also the correlation with histomorphology on a cellular level in serrated lesions. VE1 enables a subclassification of microvesicular hyperplastic polyps according to the mutation status. This improved classification of serrated lesions including immunohistochemical evaluation of BRAF V600E mutation may be the key to identify lesions with higher potential to progression into sessile serrated adenoma/polyp, and further to BRAF V600E-mutated colorectal cancer.

Pharmacodynamic and efficacy analysis of the BRAF inhibitor dabrafenib (GSK436) in combination with the MEK inhibitor trametinib (GSK212) in patients with BRAFV600 mutant colorectal cancer (CRC).

3507 Background: TheBRAF V600 mutation occurs in 5-10% of metastatic CRC, predicts poor prognosis, and may predict lack of response to standard therapy. The combination of inhibitors of BRAF (dabrafenib; D) and MEK (trametinib; T) has shown significant efficacy in BRAF-mutant melanoma. The safety, efficacy, and pharmacodynamic effects of this combination were studied in BRAF-mutant CRC patients (pts). Methods: BRAF mutant CRC pts were enrolled to an initial efficacy cohort of 26 pts and a subsequent pharmacodynamic (PD) expansion cohort that included biopsies of 15 pts at screening and at steady state. So far, 36 pts have enrolled, including 10 in the PD cohort. Eligible pts had previously-treated BRAFV600E mutant stage IV CRC. Pts were treated with D (150mg BID) and T (2mg QD). Additional analyses were performed on available archival tissues. Results: Data are available for 36 pts: ECOG performance status 0 (58%) or 1 (42%), 81% had received ≥ 2 prior chemotherapy regimens, 36% had received prior EGFR inhibitor treatment, and 83% had ≥ 1 biologic therapy. Among 34 pts with >1 restaging assessment as of November 2012, 1 (3%) achieved a complete response (confirmed, on study >12m), 3 (9%) achieved a partial response (1 confirmed to date), and 18 (53%) had stable disease (SD). Minor responses were seen in 7/18 pts (39%) with SD. Median PFS was 3.5 mo (95% CI: 1.8-4.9); overall duration on study range: 0.03–15.2 mo 7 pts (24%) remained on study for ≥6 cycles with 9 pts still on study. The most frequent AEs, any grade, included pyrexia (67%), nausea (56%), fatigue (53%), chills (47%), vomiting (39%), headache (31%), peripheral edema (31%), anemia (28%), and decreased appetite (28%). 2 pts (6%) discontinued due to AEs. Decreased pERK staining vs pre-dose samples was seen in all post-dose samples leading to absolute (49% ±29%) and relative (69% ±28%, normalized to total ERK) reduction in pERK. Conclusions: Further investigation is needed, as this combination is tolerable at full monotherapy doses of each drug, with manageable toxicities, and has activity in a subset of BRAF mutant pts. Updated safety, efficacy, and correlative data will be presented. Clinical trial information: NCT01072175.

Abstract B14: Inherent resistance to BRAF inhibitors in BRAFV600E colorectal tumors is driven by ligand-dependent activation of the EGFR pathway

Abstract Constitutive activation of the RAS, RAF, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) pathway drives tumor cell proliferation and survival in many cancer types, and activating mutations in KRAS or BRAF occur in over 30% of human tumors. It has been demonstrated that BRAF inhibitors are effective selectively against BRAFV600E tumors and not RAS mutant tumors, despite BRAF functioning as a key effector downstream of RAS and upstream of MEK. Recent Phase I clinical data with RAF inhibitors has demonstrated a significant response rate (81%) in metastatic melanoma patients with BRAFV600E-positive tumors (N Engl J Med. 2010;363:809–19). Interestingly, in contrast to this remarkable response in melanoma, the response rates in BRAFV600E-positive colon tumors have been low in comparison (5.2%) (Kopetz et al., ASCO 2010). It has recently been reported that elevated EGFR levels in a subset of BRAFV600E lines render them resistant to BRAFV600E selective inhibitors (Prahallad et al., Nature. 2012; Corcoran et al., Cancer Discovery. 2012). However, the kinetics of EGFR pathway activation in response to inhibitors and underlying mechanisms of resistance are not entirely clear. In this study, we demonstrate that BRAFV600E/EGFR high tumors show a biphasic response to BRAF inhibitors, whereby the MAPK pathway is effectively blocked upon acute treatment, but subsequently rebounds and becomes resistant to inhibition. Components of the negative feedback loop downstream of the MAPK pathway including Sprouty, MKP3, and CDC25C are downregulated during acute treatment but rebound upon prolonged treatment, coincident with phospho-ERK suppression and subsequent reactivation. We show that this effect is not only dependent on high EGFR levels but also requires EGFR ligand stimulation that can be mediated through autocrine signaling. Resistance to this pathway is associated with increased RAS-GTP levels driving wild-type RAF activation upon extended treatment with B-RAF inhibitors. Importantly, immunohistochemical staining of tissue microarrays representative of both BRAFWT and BRAFV600E mutant CRC tumors with an EGFR antibody illustrates that the BRAFV600E mutation in colorectal cancer is associated with a significantly higher percentage of tumors carrying membrane EGFR compared to BRAFWT CRC tumors, with the median of membrane EGFR positive cells in BRAFV600E tumors 56.5% vs. 15% of cells in BRAFWT tumors, suggesting that activation of both pathways may be required for tumorigenesis in this tissue type. Finally, using both in vitro and in vivo models, we demonstrate that, in BRAFV600E-positive colon tumors with high EGFR pathway activity, inherent resistance to BRAF-selective inhibitors can be overcome by combination treatment with clinically relevant inhibitors of EGFR signaling, providing a potential treatment strategy for BRAFV600E/ EGFR high tumors. In summary, these results provide mechanistic insight into the crosstalk between BRAFV600E and EGFR signaling in CRC, shed light into the low response rate of BRAFV600E mutant colorectal cancer to BRAF inhibitors as single agents, and provide a rationale for the use of BRAF and EGFR inhibitors as combination therapy in this indication.