An amino-terminal BRAF deletion accounting for acquired resistance to RAF/EGFR inhibition in colorectal cancer.

Although combination therapy with RAF and EGFR inhibitors has improved the survival outcomes of patients with BRAF-mutated colorectal cancer (CRC), acquired resistance invariably develops. The mechanisms of acquired resistance to RAF inhibitors have been largely attributed to activating mutations in RASgenes, MAP2K mutations, and amplifications in BRAF, RAS genes, and EGFR In this report, we describe a patient with BRAF-mutated CRC who acquired an amino-terminal BRAF deletion involving the Ras-binding domain (RBD) after treatment with RAF/EGFR inhibitor therapy. Amino-terminal BRAF deletions involving the RBD are a rare mechanism of acquired resistance to RAF inhibitors, particularly in CRC for which there is only one prior report in the literature.

INTRODUCTION

Colorectal cancer (CRC) is one of the leading causes of cancer worldwide with an estimated 1.8 million new cases in 2018. Despite a greater understanding of disease mechanisms and advances in treatment options over the last several decades, some patients with CRC still have a relatively poor prognosis and show a limited response to conventional therapy. In particular, CRCs harboring BRAF mutations have a poor response to standard therapies and ultimately develop resistance to currently available targeted therapies.

BRAF is part of the RAF family of serine/threonine kinases and is a component of the mitogen-activated protein kinase (MAPK) signaling pathway. The BRAF protein has two main functional domains: a kinase domain that serves to phosphorylate and activate downstream MEK/ERK signaling and an autoinhibitory domain, where RAS-GTP binds, that serves to inhibit RAF kinase activity. Upstream receptor tyrosine kinase (RTK) activity ultimately leads to Ras-mediated disruption of the autoinhibitory region via recruitment of RAF dimers and phosphorylation of RAF. BRAF mutations are found in 7%–10% of patients with metastatic CRC and can confer a poor prognosis (Samowitz et al. 2005; Tol et al. 2009; Roth et al. 2010; Tie et al. 2011). Specifically, the V600E point mutation accounts for nearly 90% of BRAF mutations seen in CRC and results in a constitutively active kinase domain and subsequent MAPK activation (Cancer Genome Atlas Network 2012). The median survival of patients with metastatic CRC harboring BRAFV600E mutations is 1 yr compared to 2–3 yr in patients with wild-type BRAF. Non-V600E BRAF mutations have been associated with a more favorable prognosis in metastatic CRC, exhibiting different responses to targeted therapy depending on the functional class of the mutation (Jones et al. 2017; Yaeger et al. 2019).

BRAF inhibitors such as vemurafenib and dabrafenib have been utilized as targeted therapies for V600E-mutated CRC with the initial expectation that they would be as effective as they are with BRAFV600E-mutated melanomas. Unfortunately, vemurafenib monotherapy has produced limited response rates in patients with V600E-mutant metastatic CRC (5% overall response rate compared to 50%–80% in melanoma) as a result of reactivation of EGFR and MAPK signaling (Chapman et al. 2011; Sosman et al. 2012; Corcoran 2015; Kopetz et al. 2015). Combination therapy with EGFR and BRAF inhibitors has therefore been investigated and has shown improved response rates ranging from 4% to 23% in various early clinical trials (Geel et al. 2014; Hyman et al. 2015; Yaeger et al. 2015; Tabernero et al. 2016; Desai et al. 2017). Despite these promising results, patients who initially responded to therapy ultimately acquire resistance and develop disease progression. A greater understanding of the mechanisms of acquired resistance is therefore crucial to developing strategies to overcome resistance and prolong the overall survival of patients.

Although a limited number of studies have investigated the resistance mechanisms of BRAF-mutated CRC, several in vitro studies have demonstrated that MAPK signaling reactivation is a common feature seen in resistant cell lines. Oddo et al. (2016) demonstrated that MAPK signaling reactivation is achieved via different mechanisms, including amplification of EGFR, KRAS, and BRAF genes as well as acquired mutations in KRAS, EGFR, and MAP2K1. Ahronian et al. (2015) similarly identified various MAPK pathway alterations (KRAS amplification, BRAF amplification MEK1 mutations) as mechanisms of resistance in vitro and in clinical samples. Yaeger et al. (2017) also found various genetic alterations in the MAPK pathway in CRC tumor samples that had progressed on RAF and EGFR inhibitors, with RAS amplification being the most recurrent mechanism of resistance. Although other genomic alterations (e.g., BRAF fusions, splice variants, internal deletions, kinase domain duplications) associated with acquired resistance to BRAF inhibitors have been reported for melanomas and various other tumors (Poulikakos et al. 2011; Rizos et al. 2014; Shi et al. 2014), published reports for CRC are rare. Here, we describe a case in which a large amino-terminal deletion in BRAF was detected in a patient with CRC that had acquired resistance to BRAF and EGFR inhibitors (Fig. 1).

Figure 1.

Timeline of events.

CASE PRESENTATION

The patient is a 40-yr-old woman who initially presented to the emergency department with a partial large bowel obstruction. Colonoscopy revealed an obstructing mass in the transverse colon with biopsy-proven adenocarcinoma. She underwent partial colectomy, and her pathology showed a well to moderately differentiated invasive adenocarcinoma (pT4a N1a–AJCC eighth edition) with proficient mismatch repair status. She was treated with two cycles of capecitabine and oxaliplatin followed by eight cycles of leucovorin, fluorouracil, and oxaliplatin (switched because of poor tolerance), but CEA levels rose 7 mo later and imaging showed a new liver nodule. Then, she underwent partial hepatectomy for a 2-cm lesion consistent with metastatic adenocarcinoma by histopathology. DNA analysis of the liver lesion by a 324-gene next-generation sequencing (NGS) assay revealed BRAFV600E, TP53C176F, PIK3CAC420R, PTEN loss, and a SMAD4 exons 2-3 duplication (Table 1). CEA levels continued to rise over the next 2 mo and imaging revealed new peritoneal, abdominal wall, portacaval lymph node, and ovarian metastases. Patient transferred care to Stanford and was started on irinotecan and cetuximab initially in early 2018 with vemurafenib added several weeks later given the BRAF mutation detected in the liver specimen. The patient had rapid clinical improvement with notable shrinkage in palpable abdominal wall nodules and improvement in pain. Surveillance imaging showed a partial response until 6 mo later when enlarging abdominal wall masses were identified. Computed tomography (CT)-guided biopsy of one of the masses confirmed metastasis, and a new BRAF exon 2–10 deletion and CUL4A amplification was detected by DNA analysis using the same 324-gene NGS assay (Table 1). Irinotecan, cetuximab, and vemurafenib treatment was continued while the patient received palliative radiation therapy to the abdominal wall. Following recommendation from the molecular tumor board, cobimetinib, a MEK inhibitor, was added to her treatment 2 mo later through compassionate use. However, given disease progression, she was transitioned to hospice care soon after.

Table 1.

Pathogenic and likely pathogenic somatic variants identified in the tumors

Gene	Chr	HGVS DNA reference	HGVS protein reference	VAF (%)	Coverage	Predicted effect
Pretreatment liver biopsy (50% tumor content); median exon coverage: 963×
BRAF	7	NM_004333.4:c.1799T>A	p.(Val600Glu)	24.0	1000	Nonsynonymous SNV
PIK3CA	3	NM_006218.2:c.1258T>C	p.(Cys420Arg)	27.7	877	Nonsynonymous SNV
TP53	17	NM_000546.5:c.527G>T	p.(Cys176Phe)	32.8	922	Nonsynonymous SNV
SMAD4	18	NG_013013.2(NM_005359.6): c.(−129+1_−128−1)_(424+1_425−1)dup	p.(Asp142fs)	n/a	n/a	Duplication exon 2–3
PTEN	10	n/a	n/a	n/a	n/a	Whole gene deletion
Post-treatment abdominal wall biopsy (30% tumor content); median exon coverage: 393×
BRAF	7	NM_004333.4:c.1799T>A	p.(Val600Glu)	20.0	365	Nonsynonymous SNV
PIK3CA	3	NM_006218.2:c.1258T>C	p.(Cys420Arg)	19.5	334	Nonsynonymous SNV
TP53	17	NM_000546.5:c.527G>T	p.(Cys176Phe)	29.6	449	Nonsynonymous SNV
SMAD4	18	NG_013013.2(NM_005359.6): c.(−129+1_−128−1)_(424+1_425−1)dup	p.(Asp142fs)	n/a	n/a	Duplication exon 2–3
CUL4A	13	n/a	n/a	n/a	n/a	Amplification
BRAF	7	NG_007873.3(NM_004333.4):c.(138+1_139−1)_(1314+1_1315−1)del	p.(Val47_Met438del)	n/a	n/a	Deletion exon 2–10

(HGVS) Human Genome Variation Society, (VAF) variant allele fraction, (SNV) single-nucleotide variant; (n/a) not available.

DISCUSSION

The BRAF exon 2–10 deletion identified in the peritoneal mass after treatment with vemurafenib involves deletion of the amino-terminal autoinhibitory region (CR1), which includes the Ras-binding domain (RBD) (Fig. 2B). Amino-terminal BRAF deletions are uncommon alterations and have been mostly identified in metastatic melanoma, with fewer reports in other tumor types such as multiple myeloma, glioblastoma, and adenocarcinoma (lung, pancreas, and prostate) (Rizos et al. 2014; Johnson et al. 2018). Although there is currently no in vitro experimental evidence determining the precise effect of amino-terminal BRAF deletions removing the RBD, they are predicted to drive acquired resistance to BRAF inhibitors via Ras-independent constitutive dimerization and activation of the truncated RAF. This mechanism is predicted to be analogous to the loss of RBD by alternative splicing (Poulikakos et al. 2011) or gene fusions (Palanisamy et al. 2010; Hutchinson et al. 2013; Ross et al. 2016; Kulkarni et al. 2017), which are better studied mechanisms.

Figure 2.

Schematic of wild-type BRAF gene (A) and BRAF exon 2–8 deletion (B). Exon numbers are shown above their corresponding protein products, with relevant functional domains labeled. (RBD) Ras-binding domain, (CR) conserved region.

The mechanisms of resistance to RAF inhibitors in CRC have been largely attributed to mutant BRAF amplifications, RAS amplifications, EGFR amplifications, activating RAS mutations, and MAP2K1 mutations (Oddo et al. 2016; Yaeger et al. 2017). The case presented herein provides supporting evidence that resistance to RAF inhibition can also result from amino-terminal BRAF deletions. To the best of our knowledge, there has only been one other previously reported amino-terminal BRAF deletion in CRC. Yaeger et al. reported one patient with metastatic BRAF mutant CRC who eventually progressed on encorafenib, cetuximab, and alpelisib (Yaeger et al. 2017). The patient had a partial response to this combination therapy (62% response by RECIST) for 24 wk before acquiring a BRAF exon 2–8 deletion in a peritoneal metastasis and NRAS mutation in a liver metastasis.

Acquired resistance to combination therapy has been long thought to result from multiple mechanisms working in concert. In one study, amino-terminal BRAF deletions involving the RBD have been described with concurrent BRAFV600E mutations in the majority (90%) of melanomas, but less so (44%) with other tumor types (Johnson et al. 2018). Interestingly, in CRC, both the case described in this report and the case reported by Yaeger et al. harbored a BRAFV600E along with the amino-terminal BRAF deletion. The patient also developed a CUL4A amplification in her post-BRAF inhibitor treatment specimen, which has been shown to promote proliferation and metastasis of CRC cells in vitro (Sui et al. 2017). Finally, the patient's preexisting PTEN loss and PI3K mutations may have also primed the patient for developing RAF inhibitor resistance as these alterations have been associated with resistance in preclinical models (Paraiso et al. 2011; Xing et al. 2012) and decreased response duration in patients with melanoma (Nathanson et al. 2013).

The approach to treating CRC is rapidly evolving as our understanding of resistance mechanisms to targeted therapy continues to grow. Clinical trials investigating combination therapies targeting EGFR, RAF, and MEK are ongoing and have shown encouraging results (Hong et al. 2016; Corcoran et al. 2018; Ursem et al. 2018; Kopetz et al. 2019). With recent evidence supporting the notion that RAF dimerization may be a common feature of the mechanism of acquired resistance to RAF inhibitors in CRC (Yaeger et al. 2017), novel RAF dimer inhibitors have also been evaluated in this context and have been shown to be effective in inhibiting growth of RAF/EGFR inhibitor resistant cells in vitro (Yaeger et al. 2017; Yao et al. 2019). Although it may be safe to assume that amino-terminal BRAF deletions represent class II mutations that function as RAS-independent activated dimers, further characterization of this rare alteration is needed in order to guide effective targeted therapy strategies in the future.

METHODS

The specimens obtained from our patient were sequenced by the FoundationOne CDx (F1CDx) assay. An amount of 50–1000 ng of extracted DNA is subjected to whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-related genes, one promoter region, one noncoding RNA (ncRNA), and select intronic regions from 34 commonly rearranged genes, 21 of which also include the coding exons. In total, the assay detects alterations in a total of 324 genomic regions. Using the Illumina HiSeq 4000 platform, hybrid capture–selected libraries are sequenced to high uniform depth (targeting >500× median coverage with >99% of exons at coverage >100×). Sequence data is then processed using a customized analysis pipeline designed to detect all classes of genomic alterations, including base substitutions, indels, copy-number alterations (amplifications and homozygous gene deletions), and selected genomic rearrangements (e.g., gene fusions). Additionally, genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) are reported.

ADDITIONAL INFORMATION

Data Deposition and Access

The interpreted variants have been deposited to the Catalog of Somatic Mutations in Cancer (COSMIC) database (https://cancer.sanger.ac.uk/cosmic) under the identifier COSP48265. Consent could not be obtained to make the raw sequence data publicly available.

Ethics Statement

The patient information has been appropriately de-identified in accordance with regulatory requirements.

Acknowledgments

We are grateful to the patient who allowed us to participate in her care.

Author Contributions

All authors contributed to conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript. S.H. provided the patient's information.

Competing Interest Statement

The authors have declared no competing interest.