Elsevier

Human Pathology

Volume 36, Issue 5, May 2005, Pages 486-493
Human Pathology

Original contribution
Human malignant melanoma: detection of BRAF- and c-kit–activating mutations by high-resolution amplicon melting analysis

https://doi.org/10.1016/j.humpath.2005.03.015Get rights and content

Summary

Activating mutations in the BRAF kinase have been reported in a large number of cases of malignant melanoma. This suggests that therapy with specific RAF kinase inhibitors may find use in treating this disease. If the response to RAF kinase inhibition is dependent on the presence of an activated BRAF protein, it will be necessary to evaluate cases of malignant melanoma for the presence or absence of BRAF mutations. High-resolution amplicon melting analysis is able to detect single base-pair changes in DNA isolated from paraffin-embedded tissue sections and obviates the need for direct DNA sequencing. Results can be available within 48 hours. In this report, we used high-resolution amplicon melting analysis to evaluate 90 cases of malignant melanoma for BRAF mutations. Of these 90 cases, 74 were metastatic melanomas, 12 were primary cutaneous melanomas, and 4 were in situ melanomas. BRAF activation mutations were found in 43 cases (48%). Forty-one of these mutations were in exon 15. The mutations in exon 15 included V600E (34 cases), V600K (6 cases), and V600R (1 case). Two activating mutations were found in exon 11, G469V and G469R. The presence or absence of a BRAF mutation in the junctional component of an invasive melanoma was maintained in the invasive component. We also evaluated these 90 cases, as well as an additional 10 cases (total of 100) for the expression of c-kit. The majority of invasive and metastatic malignant melanomas did not express c-kit, although all in situ lesions and the junctional components of invasive lesions were strongly c-kit positive. Surprisingly, 2 cases of metastatic malignant melanoma (2%) showed strong and diffuse c-kit expression and contained a c-kit–activating mutation, L576P, as detected by high-resolution amplicon melting analysis and confirmed by direct DNA sequencing. These 2 c-kit mutation–positive cases did not contain BRAF mutations. The presence of a c-kit–activating mutation in metastatic malignant melanoma suggests that a small number of melanomas may progress by a somatic mutation of the c-kit gene. The presence of BRAF- and c-kit–activating mutations in malignant melanoma suggests new approaches to treating this disease involving specific tyrosine kinase inhibitors may prove worthwhile and that mutation analysis by high-resolution melting analysis might help guide therapy.

Introduction

Protein kinases are members of a large family of proteins that play important roles in cellular signaling [1], [2]. It has become apparent that several types of human malignancies contain specific mutations in genes coding for protein kinases and that these mutations probably represent primary oncogenic events [3], [4]. The mutations result in the production of an activated protein kinase that is no longer sensitive to normal inhibitory signals. Constant signaling by such mutationally activated proteins stimulates cell proliferation, activates anti-apoptotic pathways, and drives oncogenesis [5], [6], [7]. Several new types of anticancer drugs are available, which are inhibitory against the activated protein kinases [8]. Because these activated protein kinases are unique to the tumor cell, the drugs that inhibit them are far less toxic than standard anticancer drugs as they target only the malignant cell. This type of targeted molecular therapy is highlighted by the successful treatment of gastrointestinal stromal tumors (GISTs), chronic myelogenous leukemia, and adenocarcinomas of the lung with tyrosine kinase inhibitors such as imatinib and gefitinib [9], [10], [11], [12], [13]. These successes suggest that other hard-to-treat malignancies may be characterized by specific mutations that result in activated proteins sensitive to targeted anticancer drugs.

Malignant melanoma is a difficult-to-treat malignancy. Immune-based therapy has received the widest attention. However, treatment with immune system modifiers such as interleukin 2 and interferon α has not been shown to provide convincing survival benefit and may lead to severe toxicity [14]. Melanoma vaccines have met with limited success [15]. Chemotherapeutic drugs such as dacarbazine and newer combinations of drugs such as temozolomide and thalidomide are active against the disease, but response rates are only around 20% [16]. Clearly, progress in treating melanoma has been slow, and significant advances may require new insights into melanoma biology.

Recently, it has been reported that a high percentage of malignant melanomas contain activating mutations in the BRAF protein kinase [17], [18]. BRAF is a cytosolic protein kinase. It has a RAS-binding site and is activated by membrane-bound RAS. When BRAF is activated, it, in turn, activates a signaling cascade involving proteins in the mitogen-activated protein kinase system. The result is cell proliferation [19], [20]. Mutations in the BRAF gene present in malignant melanoma lead to a constitutively activated BRAF kinase [21]. The constitutively activated kinase is thought to be responsible for melanoma cell proliferation and transformation [22], [23]. The importance of BRAF mutations in a high proportion of melanomas is underscored by the discovery of a RAF kinase inhibitor (BAY 43-9006) that targets the activated BRAF kinase [24] and, at least in cell culture systems [21], leads to melanoma cell death. In addition to BRAF, melanomas have also been reported to overexpress c-kit [25], [26], the target of imatinib, a drug that has been used so successfully in treating GISTs and other neoplasms. However, no c-kit mutations, which probably are a prerequisite for imatinib response [9], have been described in melanoma.

We have recently reported on the use of high-resolution amplicon melting analysis to characterize the c-kit–activating mutations in a series of GISTs [27]. This analysis can be expanded to evaluate mutations in any gene of interest and is readily performed on DNA isolated from paraffin-embedded tissue sections. Results are available in 48 hours. In this report, we used high-resolution melting analysis to search for BRAF- and c-kit–activating mutations to molecularly characterize a series of malignant melanomas. In agreement with others, we found that a high percentage of malignant melanomas contain activating BRAF mutations. Surprisingly, we also found 2 cases of melanoma that showed strong expression of the c-kit protein, and each case contained an identical c-kit exon 11–activating mutation. These results suggest that targeted molecular therapy with specific protein kinase inhibitors may usher in a new paradigm for treating malignant melanoma. If so, the ability to rapidly characterize the tumor with respect to BRAF or c-kit mutations by high-resolution amplicon analysis will be invaluable.

Section snippets

Sources of tissue and immunohistochemistry

Paraffin blocks from 100 cases diagnosed as malignant melanoma were retrieved from the surgical pathology files at the University of Utah Hospital. Of these 100 cases, 84 were metastatic melanomas, 12 were primary cutaneous melanomas, and 4 were in situ melanomas. For the 12 primary cutaneous melanomas, the average Breslow depth was 5.5 mm (range, 1-14 mm). No primary mucosal melanomas were evaluated. The cases spanned the years from 1995 to 2004 and were chosen by sequential dates in the

High-resolution amplicon melting analysis for exons 15 and 11 BRAF-activating mutations

Activating mutations in the BRAF gene have been reported most commonly in exon 15, which codes for the activation loop of the kinase, and less so in exon 11, which codes for the glycine-rich conserved nucleotide binding site [17]. Specific primers as indicated in Table 1 were designed to amplify the corresponding BRAF exons. After PCR, the presence or absence of a mutation is easily detected by high-resolution melting analysis of the amplified products. Although it may not be necessary for

Discussion

The recent identification of activating BRAF mutations in a high percentage of malignant melanomas has led way to optimism that therapy with specific RAF kinase inhibitors might be beneficial in this disease, which, at least for advanced stages, has limited therapeutic options. If the response to RAF kinase inhibitors in malignant melanoma is dependent on BRAF-activating mutations in the tumor, as some laboratory studies suggest [21], then it will be important to be able to detect these

Acknowledgments

The authors thank Trena Held for preparing the tables. We also thank Dr Carl Wittwer for his support and help with high-resolution melting analysis. This work was supported by the Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology.

References (37)

  • C. Garcia-Echeverria et al.

    Therapeutically targeted anticancer agents: inhibitors of receptor tyrosine kinases

    Mini Rev Med Chem

    (2004)
  • M.C. Heinrich et al.

    Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor

    J Clin Oncol

    (2003)
  • H. Kantarjian et al.

    Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia

    New Engl J Med

    (2002)
  • J.G. Paez et al.

    EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy

    Science

    (2004)
  • T.J. Lynch et al.

    Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib

    New Engl J Med

    (2004)
  • W. Pao et al.

    EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib

    Proc Natl Acad Sci U S A

    (2004)
  • A. Hauschild et al.

    Prospective randomized trial of interferon alfa-2b and interleukin-2 as adjuvant treatment of resected intermediate- and high-risk primary melanoma without clinically detectable node metastasis

    J Clin Oncol

    (2003)
  • J.A. Sosman et al.

    When will melanoma vaccines be proven effective?

    J Clin Oncol

    (2004)
  • Cited by (199)

    • Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem

      2018, Nanomedicine: Nanotechnology, Biology, and Medicine
      Citation Excerpt :

      More importantly, the construct provided a synergistic mechanism, via the laser-induced LSPR of the GNR platform, to increase the overall tumor-cytotoxicity of the nano-carrier. BRAF mutations and overexpression occur in about 40 to 70% of malignant melanoma.4-6 Inhibiting the kinase BRAF results in rapid tumor regression and prolonged survival in patients.10-13

    View all citing articles on Scopus
    View full text