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Case Report/Case Series |

Identification of a Novel Complex BRAF Mutation Associated With Major Clinical Response to Vemurafenib in a Patient With Metastatic Melanoma FREE

Benoit Busser, PharmD, PhD1; Marie Therese Leccia, MD, PhD1; Guillaume Gras-Combe, MD2; Ivan Bricault, MD, PhD3; Isabelle Templier, MD4; Antoine Claeys, MD4; Marie Jeanne Richard, PharmD, PhD5; Florence de Fraipont, PharmD, PhD5; Julie Charles, MD, PhD1
[+] Author Affiliations
1Institut Albert Bonniot INSERM/UJF U823, Grenoble, France
2Department of Neurosurgery, CHRU Grenoble University Hospital, Grenoble, France
3Department of Medical Imaging, CHRU Grenoble University Hospital, Grenoble, France
4Department of Dermatology, CHRU Grenoble University Hospital, Grenoble, France
5Department of Cancer Clinical Chemistry, CHRU Grenoble University Hospital, Grenoble, France
JAMA Dermatol. 2013;149(12):1403-1406. doi:10.1001/jamadermatol.2013.8198.
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Importance  There is an increasing interest in BRAF V600 mutations in melanomas and their associated sensitivity to vemurafenib, a BRAF inhibitor. However, physicians cannot find information in the literature about vemurafenib response for rare and/or atypical BRAF mutations.

Observations  We describe the identification of a novel complex BRAF mutation associated with major clinical response to vemurafenib in a patient with metastatic melanoma. Using a pyrosequencing method, we determined that the tumor positive for mutated BRAF, uncovering a novel c.1799_1803delinsAT; p.V600-K601>D variant. We uncovered this atypical BRAF mutation with 2 different sequencing methods, both in the primary lesion and in 1 metastasis. The patient was immediately treated with vemurafenib as monotherapy and achieved a prolonged (5.5-month) positive response.

Conclusions and Relevance  We analyzed the consequences of the BRAF V600-K601>D mutation in terms of amino acids. We referred to the published data and databases to screen chemical properties of well-known BRAF V600 mutations and other complex BRAF mutations to find common features of activated BRAF mutations. Importantly, we highlighted that both the site of the mutation and the involved amino acids are important to predict vemurafenib response. Our conclusion is that complex BRAF mutation surrounding codon 600 could also be sensitive to BRAF inhibitors.

Figures in this Article

The median survival time for patients with unresectable melanoma remains dramatically short, only 6 to 9 months from the time of diagnosis to death, with only 10% to 15% of patients living 3 years. Approximately 40% to 60% of cutaneous melanomas carry mutations in the BRAF gene, which is the most frequently mutated protein kinase in human cancers.1 These activating mutations induce a constitutive BRAF-mediated signaling, leading to subsequent activation of the downstream mitogen-activated protein kinase (MAPK) pathway. These findings have led to the clinical development of specific and potent BRAF-mutated inhibitors.

Approximately 90% of BRAF mutations in melanoma cells occur at exon 15, codon 600, and result in the substitution of glutamic acid for valine (BRAF V600E), although other activating mutations are described at the same codon (eg, BRAF V600K, V600R, and V600D). The COSMIC database (Catalogue of Somatic Mutations in Cancer)2 also describes other complex BRAF mutations situated between codons 587 and 602.

Vemurafenib (PLX4032) acts as an adenosine triphosphate–competitive inhibitor and has a marked antitumor effect against BRAF V600 mutated cancer cells but not against cells with wild-type BRAF.3 Nevertheless, response to vemurafenib is frequently unknown for rare or complex BRAF mutations.

We report the case of a patient with a metastatic melanoma harboring a novel complex BRAF mutation who experienced a positive response 5.5 months’ duration under vemurafenib therapy.

A man in his late 50s presented to our dermatology clinic for the resection of a cutaneous pigmented lesion on the left lumbar region. A local surgical resection was performed, and the subsequent histopathologic examination of the excised melanocytic lesion displayed features of ulcerated malignant melanoma, Clark level III, with Breslow thickness of 3.9 mm and a mitotic rate of 1/mm2. After sentinel lymph node dissection, the patient was staged as AJCC IIIc4 and discharged home for quarterly clinical follow-up and bi-annual body computed tomography (CT) imaging evaluation, according to French standards of care.

Months later, the patient presented for his follow-up consultation and complained of axillary and inguinal lymphadenopathies, confirmed by physical examination, which revealed 6 subcutaneous nodules. A CT scan of the chest, abdomen, and pelvis was performed and confirmed the presence of multiple ipsilateral subcutaneous nodules (2 in the pectoral region, 5 in the axillary region, and 2 in the inguinal region). The CT scan of the brain showed no abnormalities. One pectoral nodule was excised for pathologic analysis, which confirmed melanoma metastasis.

Tumor DNA was screened for the oncogenic mutations in melanoma (ie, BRAF, NRAS, and KIT). Using a pyrosequencing method developed and clinically used for KRAS and EGFR testing,5,6 we determined that the specimen was wild type for NRAS and wild type for KIT. However, the pyrogram was positive for mutated BRAF, uncovering a novel c.1799_1803delinsAT;p.V600-K601>D variant (Figure 1) which denotes the replacement of 5 consecutive nucleotides 1799 to1803 (TGAAA), by 2 other nucleotides (AT).

Place holder to copy figure label and caption
Figure 1.
BRAF Pyrograms After Reverse-Strand Pyrosequencing

The pyrograms correspond to the reference BRAF wild type (WT), BRAF V600E mutation, and the novel BRAF V600-K601>D mutation. Black arrowheads and open arrowheads indicate lower base incorporation and new/higher base incorporation, respectively, compared with the expected ratio.

Graphic Jump Location

This mutation leads to the replacement of both the valine (V600) and the lysine (K601) by an aspartate (D600) in the mutated BRAF protein. Moreover, a retrospective pyrosequencing of the primary lesion revealed the same mutation, which was confirmed by classical Sanger sequencing of BRAF exon 15 (Figure 2). The experimental details for BRAF sequencing are listed in the Table.

Place holder to copy figure label and caption
Figure 2.
BRAF Electropherograms After Sanger Sequencing

Both the wild type (WT) and the novel V600-K601>D mutant DNA were sequenced on the reverse strand. The corresponding amino acids, number of codons, and forward sequences are indicated in the boxes. Ala indicates, alanine; Asp, aspartate; Leu, leucine; Lys, lysine; Ser, serine; Thr, threonine.

Graphic Jump Location
Table Graphic Jump LocationTable.  Primer Sequences and Nucleotides Dispensation Order Used for the BRAF Sequencing

To our knowledge, this complex BRAF V600-K601>D mutation has never been previously reported and was not described in the COSMIC database of the Wellcome Trust Sanger Institute.7 Accordingly, we could not find any published information regarding whether such a BRAF-mutated melanoma should be treated with vemurafenib.

After multidisciplinary consultation, the patient began treatment with vemurafenib, 960 mg orally, twice per day. One month after the initiation of vemurafenib treatment, the patient maintained a good performance status but complained of nausea, photosensitivity, and insomnia. Physical examination revealed multiple cutaneous lesions, and pathologic analysis identified 5 keratoacanthomas and 1 squamous cell carcinoma (SCC), which are frequently reported as adverse effects of anti-BRAF therapies.8 Palpation showed a noticeable decrease in the size of the known nodules, already suggesting tumor response. To minimize nausea, we decreased the vemurafenib dose to 720 mg orally, twice per day.

After 3 monthly cycles of vemurafenib treatment, the patient’s Eastern Cooperative Oncology Group (ECOG) performance status was 0, and CT scan documented the decrease in the overall sizes of both axillary (Figure 3A) and pectoral subcutaneous nodules (Figure 3B) from before vemurafenib treatment to after the third treatment cycle was completed.

Place holder to copy figure label and caption
Figure 3.
Computed Tomographic (CT) Scans of the Patient Showing the Positive Response After 3 Months of Vemurafenib Treatment

Axillary (A) and pectoral (B) lesions (arrowheads) seen on CT scans provide evidence for tumor shrinkage after 3 months of vemurafenib treatment.

Graphic Jump Location

Three months later, the patient presented with complaints of headaches, dizziness, and asthenia. However, neurologic examination findings were normal. The body CT imaging confirmed response to vemurafenib, but the CT scan of the brain demonstrated a solitary bleeding nodule in the right parietal region suggestive of a new metastasis. Unfortunately, the patient’s neurologic function rapidly worsened leading to coma and subsequent death.

In melanoma research, the finding that BRAF V600E mutation was strongly associated with antitumor effect of vemurafenib1 prompted European and US agencies to approve vemurafenib after accelerated review. Therefore, BRAF testing is now mandatory, and vemurafenib is indicated as monotherapy for the treatment of adult patients with BRAF V600 mutation–positive unresectable or metastatic melanoma.

The database of patients with BRAF mutations surrounding codon V600 contains hundreds of point mutation cases,2 eg, V600E, V600K, and V600R substitutions, associated with good response to vemurafenib.3,9 However, almost no available data about vemurafenib response for tumors with complex BRAF mutations can be found in the literature, probably because of their low incidence rates.

BRAF V600E mutations respond to vemurafenib and are likely to result in 500-fold increased BRAF activity.10 A similar but complex BRAF mutation pV600-K601>E identified in melanoma11 and papillary thyroid carcinoma12 was also found to induce an increased kinase activity.13 Effectively, the negatively charged glutamate (E) residue mimics the structure of the phosphorylated loop of activated wild-type BRAF.14 Similarly in V600D mutations, the replacement of valine by another negatively charged residue such as aspartate (D) is also associated with a 700-fold increase in BRAF activity,10 and bioinformatic modeling of another BRAF complex in-frame mutation involving aspartate D600 confirmed that aspartate (D) replacement also leads to high kinase activity.15

Thus, it is easily conceivable that the novel in-frame complex BRAF V600-K601>D mutation observed in our patient’s tumor has the same effect, since it leads to the replacement of the valine and lysine at positions 600 and 601 by a single aspartate (D600) in the mutated BRAF protein. Altogether, these data suggest that this new mutation is likely to induce a constitutively activated BRAF protein and should respond to BRAF inhibitors in the same way as the classic V600E mutation.

This hypothesis is in accordance with the good response of our patient to vemurafenib. This treatment rapidly and successfully induced an objective clinical response. The patient had a progression-free survival (PFS) of 5.5 months after beginning vemurafenib monotherapy, which precisely corresponds to the median PFS of 5.3 months shown in the phase III registration study of vemurafenib1 for patients with metastatic melanoma.

In summary, given the fact that our patient achieved a prolonged response, vemurafenib treatment could also be evaluated for tumors with complex BRAF mutations surrounding exon 15, codon 600. Analysis of the location as well as the physical and chemical properties of the amino acids generated by the BRAF mutation might improve the predictability of vemurafenib sensitivity.

Corresponding Author: Benoit Busser, PharmD, PhD, Biochimie des Cancers et Biothérapies, Institut de Biologie et Pathologie du CHU de Grenoble, 38 043 Grenoble, Cedex 9, France (bbusser@chu-grenoble.fr).

Accepted for Publication: September 10, 2013.

Published Online: October 9, 2013. doi:10.1001/jamadermatol.2013.8198.

Author Contributions: Drs Busser and Charles had full access to all 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: Busser, Charles.

Acquisition of data: Busser, Gras-Combe, Bricault, Templier, Claeys, de Fraipont, Charles.

Analysis and interpretation of data: Busser, Leccia, Richard, de Fraipont, Charles.

Drafting of the manuscript: Busser, Gras-Combe, Bricault, Templier, Claeys, Charles.

Critical revision of the manuscript for important intellectual content: Busser, Leccia, Richard, de Fraipont, Charles.

Obtained funding: de Fraipont.

Administrative, technical, or material support: Gras-Combe, Bricault, Templier, Claeys, de Fraipont.

Study supervision: Busser, Leccia, Richard, Charles.

Conflict of Interest Disclosures: Dr de Fraipont has received honoraria from Roche. No other disclosures are reported.

Funding/Support: This work was supported by the 2010 grant “Innovations diagnostiques et thérapeutiques” from Grenoble University Hospital.

Role of the Sponsor: The sponsor had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We thank Violaine Tolsma and Perrine Lévêque, MD, for the clinical data collection. The expert technical support for molecular analysis was performed by Mylène Bargues, Emilie Morel, Stephanie Dumas, Céline Gagnardot, Valérie Konik-Mathevet, and Odile Vermeulen. We also thank Stratton Beatrous, for manuscript revision.

Chapman  PB, Hauschild  A, Robert  C,  et al; BRIM-3 Study Group.  Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364(26):2507-2516.
PubMed   |  Link to Article
Wellcome Trust Sanger Institute. Catalogue of Somatic Mutations in Cancer. http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/. Accessed September 10, 2013.
Rubinstein  JC, Sznol  M, Pavlick  AC,  et al.  Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Transl Med. 2010;8:67.
PubMed   |  Link to Article
Balch  CM, Gershenwald  JE, Soong  SJ,  et al.  Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol. 2009;27(36):6199-6206.
PubMed   |  Link to Article
Dufort  S, Richard  MJ, de Fraipont  F.  Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues. Anal Biochem. 2009;391(2):166-168.
PubMed   |  Link to Article
Dufort  S, Richard  MJ, Lantuejoul  S, de Fraipont  F.  Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC. J Exp Clin Cancer Res. 2011;30:57.
PubMed   |  Link to Article
Forbes  SA, Bindal  N, Bamford  S,  et al.  COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011;39(Database issue):D945-D950.
PubMed   |  Link to Article
Sinha  R, Edmonds  K, Newton-Bishop  JA, Gore  ME, Larkin  J, Fearfield  L.  Cutaneous adverse events associated with vemurafenib in patients with metastatic melanoma: practical advice on diagnosis, prevention and management of the main treatment-related skin toxicities. Br J Dermatol. 2012;167(5):987-994.
PubMed   |  Link to Article
Bollag  G, Hirth  P, Tsai  J,  et al.  Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature. 2010;467(7315):596-599.
PubMed   |  Link to Article
Wan  PT, Garnett  MJ, Roe  SM,  et al; Cancer Genome Project.  Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004;116(6):855-867.
PubMed   |  Link to Article
Reifenberger  J, Knobbe  CB, Sterzinger  AA,  et al.  Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas. Int J Cancer. 2004;109(3):377-384.
PubMed   |  Link to Article
Trovisco  V, Soares  P, Soares  R, Magalhães  J, Sá-Couto  P, Sobrinho-Simões  M.  A new BRAF gene mutation detected in a case of a solid variant of papillary thyroid carcinoma. Hum Pathol. 2005;36(6):694-697.
PubMed   |  Link to Article
Hou  P, Liu  D, Xing  M.  Functional characterization of the T1799-1801del and A1799-1816ins BRAF mutations in papillary thyroid cancer. Cell Cycle. 2007;6(3):377-379.
PubMed   |  Link to Article
Dhillon  AS, Kolch  W.  Oncogenic B-Raf mutations: crystal clear at last. Cancer Cell. 2004;5(4):303-304.
PubMed   |  Link to Article
Barzon  L, Masi  G, Boschin  IM,  et al.  Characterization of a novel complex BRAF mutation in a follicular variant papillary thyroid carcinoma. Eur J Endocrinol. 2008;159(1):77-80.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.
BRAF Pyrograms After Reverse-Strand Pyrosequencing

The pyrograms correspond to the reference BRAF wild type (WT), BRAF V600E mutation, and the novel BRAF V600-K601>D mutation. Black arrowheads and open arrowheads indicate lower base incorporation and new/higher base incorporation, respectively, compared with the expected ratio.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
BRAF Electropherograms After Sanger Sequencing

Both the wild type (WT) and the novel V600-K601>D mutant DNA were sequenced on the reverse strand. The corresponding amino acids, number of codons, and forward sequences are indicated in the boxes. Ala indicates, alanine; Asp, aspartate; Leu, leucine; Lys, lysine; Ser, serine; Thr, threonine.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.
Computed Tomographic (CT) Scans of the Patient Showing the Positive Response After 3 Months of Vemurafenib Treatment

Axillary (A) and pectoral (B) lesions (arrowheads) seen on CT scans provide evidence for tumor shrinkage after 3 months of vemurafenib treatment.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable.  Primer Sequences and Nucleotides Dispensation Order Used for the BRAF Sequencing

References

Chapman  PB, Hauschild  A, Robert  C,  et al; BRIM-3 Study Group.  Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364(26):2507-2516.
PubMed   |  Link to Article
Wellcome Trust Sanger Institute. Catalogue of Somatic Mutations in Cancer. http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/. Accessed September 10, 2013.
Rubinstein  JC, Sznol  M, Pavlick  AC,  et al.  Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Transl Med. 2010;8:67.
PubMed   |  Link to Article
Balch  CM, Gershenwald  JE, Soong  SJ,  et al.  Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol. 2009;27(36):6199-6206.
PubMed   |  Link to Article
Dufort  S, Richard  MJ, de Fraipont  F.  Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues. Anal Biochem. 2009;391(2):166-168.
PubMed   |  Link to Article
Dufort  S, Richard  MJ, Lantuejoul  S, de Fraipont  F.  Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC. J Exp Clin Cancer Res. 2011;30:57.
PubMed   |  Link to Article
Forbes  SA, Bindal  N, Bamford  S,  et al.  COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011;39(Database issue):D945-D950.
PubMed   |  Link to Article
Sinha  R, Edmonds  K, Newton-Bishop  JA, Gore  ME, Larkin  J, Fearfield  L.  Cutaneous adverse events associated with vemurafenib in patients with metastatic melanoma: practical advice on diagnosis, prevention and management of the main treatment-related skin toxicities. Br J Dermatol. 2012;167(5):987-994.
PubMed   |  Link to Article
Bollag  G, Hirth  P, Tsai  J,  et al.  Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature. 2010;467(7315):596-599.
PubMed   |  Link to Article
Wan  PT, Garnett  MJ, Roe  SM,  et al; Cancer Genome Project.  Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004;116(6):855-867.
PubMed   |  Link to Article
Reifenberger  J, Knobbe  CB, Sterzinger  AA,  et al.  Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas. Int J Cancer. 2004;109(3):377-384.
PubMed   |  Link to Article
Trovisco  V, Soares  P, Soares  R, Magalhães  J, Sá-Couto  P, Sobrinho-Simões  M.  A new BRAF gene mutation detected in a case of a solid variant of papillary thyroid carcinoma. Hum Pathol. 2005;36(6):694-697.
PubMed   |  Link to Article
Hou  P, Liu  D, Xing  M.  Functional characterization of the T1799-1801del and A1799-1816ins BRAF mutations in papillary thyroid cancer. Cell Cycle. 2007;6(3):377-379.
PubMed   |  Link to Article
Dhillon  AS, Kolch  W.  Oncogenic B-Raf mutations: crystal clear at last. Cancer Cell. 2004;5(4):303-304.
PubMed   |  Link to Article
Barzon  L, Masi  G, Boschin  IM,  et al.  Characterization of a novel complex BRAF mutation in a follicular variant papillary thyroid carcinoma. Eur J Endocrinol. 2008;159(1):77-80.
PubMed   |  Link to Article

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