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Figure 1.
Tumor Response in a Patient With a Salivary Acinic Cell Carcinoma Harboring a BRAF Kinase Domain Duplication Treated With Regorafenib
Tumor Response in a Patient With a Salivary Acinic Cell Carcinoma Harboring a BRAF Kinase Domain Duplication Treated With Regorafenib
Figure 2.
Visualization of BRAF Rearrangement Resulting in Kinase Domain Duplication
Visualization of BRAF Rearrangement Resulting in Kinase Domain Duplication

A, BRAF is located on chromosome 7q34. B, The kinase domain spans exons 11 through 18. C, The BRAF kinase domain is duplicated owing to genomic rearrangement. D and E, Graphical representation of the 3′ portion of the BRAF gene from 9 patients with BRAF kinase domain duplication. D, Eight patients harbored a breakpoint in intron 9. E, One patient sample contained a breakpoint in exon 3. All observed events completely encompass the kinase domain spanning exons 11 through 18. Dotted lines show intergenic space, solid lines represent noncoding sequence, black boxes are coding sequences, and shaded blue boxes show duplicated sequences. Arrowheads represent breakpoints.

1.
Davies  H, Bignell  GR, Cox  C,  et al.  Mutations of the BRAF gene in human cancer.  Nature. 2002;417(6892):949-954.PubMedGoogle ScholarCrossref
2.
Rodriguez  FJ, Ligon  AH, Horkayne-Szakaly  I,  et al.  BRAF duplications and MAPK pathway activation are frequent in gliomas of the optic nerve proper.  J Neuropathol Exp Neurol. 2012;71(9):789-794.PubMedGoogle ScholarCrossref
3.
Rajakulendran  T, Sahmi  M, Lefrançois  M, Sicheri  F, Therrien  M.  A dimerization-dependent mechanism drives RAF catalytic activation.  Nature. 2009;461(7263):542-545.PubMedGoogle ScholarCrossref
4.
Poulikakos  PI, Persaud  Y, Janakiraman  M,  et al.  RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E).  Nature. 2011;480(7377):387-390.PubMedGoogle ScholarCrossref
5.
Pfister  S, Janzarik  WG, Remke  M,  et al.  BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas.  J Clin Invest. 2008;118(5):1739-1749.PubMedGoogle ScholarCrossref
6.
Palanisamy  N, Ateeq  B, Kalyana-Sundaram  S,  et al.  Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma.  Nat Med. 2010;16(7):793-798.PubMedGoogle ScholarCrossref
Research Letter
February 2016

Identification of BRAF Kinase Domain Duplications Across Multiple Tumor Types and Response to RAF Inhibitor Therapy

Author Affiliations
  • 1Division of Hematology-Oncology, University of California Irvine, Orange
  • 2Georgia Cancer Specialists, Sandy Springs
  • 3Northside Hospital Cancer Institute, Sandy Springs, Georgia
  • 4Foundation Medicine Inc, Cambridge, Massachusetts
  • 5Swedish Cancer Institute, Seattle, Washington
  • 6Department of Pathology and Laboratory Medicine, Albany Medical Center, Albany, New York
 

Copyright 2016 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.

JAMA Oncol. 2016;2(2):272-274. doi:10.1001/jamaoncol.2015.4437

The Raf family (ARAF, BRAF, CRAF) of serine/threonine kinases are activators of downstream MEK kinases in the Ras-Raf-MEK-ERK signaling pathway. BRAF is the most potent activator of MEK kinases, and alterations in the Ras-Raf-MEK-ERK pathway are observed in nearly 30% of human malignant neoplasms. More than 300 BRAF mutations are described, mostly within exons 11 through 15 of the catalytic kinase domain (CR3), and BRAF V600E accounts for 90% of observed BRAF mutations.1 Alternate mechanisms of BRAF activation including amplification, non-V600 mutations, and chromosomal rearrangements are less well studied. BRAF fusions have been observed infrequently, and BRAF amplification arises more commonly as a resistance mechanism to RAF-directed tyrosine kinase inhibitor therapy.2BRAF kinase domain duplication (BRAF-KD) has only been observed in gliomas.

Report of a Case

A woman in her 30s with an advanced acinic cell tumor of the right parotid gland was initially treated with systemic chemotherapy, followed by empirical erlotinib hydrochloride therapy. Her disease ultimately progressed with neck, liver, and lung lesions (Figure 1A). Progression biopsy of the right parotid gland mass was subjected to comprehensive genomic profiling revealing an intrachromosomal duplication event at 7q34 with a breakpoint at intron 9 resulting in duplication of the entire BRAF kinase domain (Figure 2A-D), and an additional breakpoint in the intergenic space downstream of the BRAF gene (Figure 2E). No other oncogenic driver alterations were observed across the 315 genes assayed (data not shown). She was treated with regorafenib monohydrate and achieved a considerable partial response in all disease sites (Figure 1). As this article went to press, the patient continued to maintain a partial response lasting more than 12 months with regorafenib therapy.

Results

To identify additional BRAF-KD events, we interrogated sequencing data from 50 000 clinical samples in the Foundation Medicine Inc database. Nine samples harbored 3′ duplication of the BRAF gene, with 8 of 9 cases (89%) having a genomic breakpoint at intron 9. In a single sample the BRAF breakpoint occurred at intron 3, resulting in duplication of exons 3 through 18 (Figure 2E). BRAF-KD was mutually exclusive from other tyrosine kinase fusions and established oncogenic alterations. BRAF-KD represented 0.5% of BRAF alterations and was not identified in available Catalogue of Somatic Mutations in Cancer or Cancer Genome Atlas data.

Discussion

Using comprehensive genomic profiling, we identified BRAF-KD across multiple tumor types and demonstrate response to RAF-directed therapy. The tandem duplications observed here lack the N-terminal autoinhibitory domain, and deletion of the N-terminal RAS-binding domain is known to cause constitutive kinase activation.3BRAF-KD is predicted to generate a protein with 2 functional kinase domains, one of which cannot be regulated by the CR1 regulatory domain. Nearly all BRAF-KDs occurred at breakpoints in intron 9 of the BRAF gene, and intron 9 insertions generate a truncated transcript analogous to the BRAF alternate splice form that is an observed resistance mechanism in melanoma.4BRAF duplication results in increased BRAF mRNA and expression of CCND1, a well-established downstream mitogen-activated protein kinase (MAPK) target gene.5 Thus, the BRAF-KD observed here would be expected to activate downstream MAPK signaling. The salivary acinic cell carcinoma contained a BRAF-KD with no other putative driver alterations, suggesting that the response is attributable to BRAF inhibition, and regorafenib has demonstrated efficacy in BRAF fusions retaining the entire BRAF kinase domain in preclinical models.6 However, regorafenib is a multitargeted tyrosine kinase inhibitor and in the absence of functional validation we cannot definitively conclude that BRAF-KD inhibition is the sole mechanism of efficacy.

Increasing clinical incorporation of tumor-profiling technologies is likely to further refine the clinicopathologic features of BRAF-KDs. Overall, this is the first report and largest series examining BRAF-KDs, providing evidence that BRAF-KDs are a clinically important genomic alteration and therapeutic target.

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Article Information

Corresponding Author: Samuel J. Klempner, MD, University of California Irvine, 101 The City Dr S, Orange, CA 92868 (sklempne@uci.edu).

Published Online: November 12, 2015. doi:10.1001/jamaoncol.2015.4437.

Author Contributions: Drs Klempner and Ali had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Klempner, Bordoni, Ou, Ali.

Acquisition, analysis, or interpretation of data: Klempner, Gowen, Kaplan, Stephens, Ou, Ali.

Drafting of the manuscript: Klempner, Ali.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Gowen.

Administrative, technical, or material support: Kaplan, Ali.

Study supervision: Ali.

Conflict of Interest Disclosures: Mr Gowen and Drs Stephens and Ali are employees of and hold equity in Foundation Medicine, Inc. No other disclosures are reported.

Additional Contributions: We thank the patient for granting permission to publish this information.

References
1.
Davies  H, Bignell  GR, Cox  C,  et al.  Mutations of the BRAF gene in human cancer.  Nature. 2002;417(6892):949-954.PubMedGoogle ScholarCrossref
2.
Rodriguez  FJ, Ligon  AH, Horkayne-Szakaly  I,  et al.  BRAF duplications and MAPK pathway activation are frequent in gliomas of the optic nerve proper.  J Neuropathol Exp Neurol. 2012;71(9):789-794.PubMedGoogle ScholarCrossref
3.
Rajakulendran  T, Sahmi  M, Lefrançois  M, Sicheri  F, Therrien  M.  A dimerization-dependent mechanism drives RAF catalytic activation.  Nature. 2009;461(7263):542-545.PubMedGoogle ScholarCrossref
4.
Poulikakos  PI, Persaud  Y, Janakiraman  M,  et al.  RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E).  Nature. 2011;480(7377):387-390.PubMedGoogle ScholarCrossref
5.
Pfister  S, Janzarik  WG, Remke  M,  et al.  BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas.  J Clin Invest. 2008;118(5):1739-1749.PubMedGoogle ScholarCrossref
6.
Palanisamy  N, Ateeq  B, Kalyana-Sundaram  S,  et al.  Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma.  Nat Med. 2010;16(7):793-798.PubMedGoogle ScholarCrossref
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