Human malignant melanoma: detection of BRAF- and c-kit-activating mutations by high-resolution amplicon melting analysis
Human malignant melanoma: detection of BRAF- and c-kit-activating mutations by high-resolution amplicon melting analysis
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DOI：
10.1016/j.humpath.2005.03.015
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发表时间：
2005-05-01
期刊：
影响因子：
3.3
通讯作者：
Layfield, LJ
中科院分区：
文献类型：
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作者：
Willmore-Payne, C;Holden, JA;Layfield, LJ
在大量恶性黑色素瘤病例中报告了BRAF激酶的激活突变。这表明用特异性RAF激酶抑制剂的疗法可用于治疗这种疾病。如果对RAF激酶抑制的反应依赖于活化的BRAF蛋白的存在，则有必要评估恶性黑色素瘤病例是否存在BRAF突变。高分辨率扩增子熔解分析能够检测从石蜡包埋的组织切片中分离的DNA中的单个碱基对变化，并且避免了直接DNA测序的需要。结果可在48小时内提供。在这份报告中，我们使用高分辨率扩增子熔解分析来评估90例恶性黑色素瘤的BRAF突变。其中转移性黑色素瘤74例，原发性皮肤黑色素瘤12例，原位黑色素瘤4例。43例（48%）发现BRAF激活突变。其中41个突变位于外显子15。外显子15突变包括V600 E（34例）、V600 K（6例）和V600 R（1例）。在第11外显子发现两个激活突变，G469 V和G469 R。在侵袭性黑色素瘤的交界部分中存在或不存在BRAF突变在侵袭性部分中得以维持。我们还评估了这90例，以及另外10例（共100例）的c-kit表达。大多数侵袭性和转移性恶性黑色素瘤不表达c-kit，尽管所有原位病变和侵袭性病变的交界处成分均呈强阳性。令人惊讶的是，2例转移性恶性黑色素瘤（2%）表现出强烈的和弥漫性的c-kit表达，并含有c-kit激活突变，L576 P，检测到的高分辨率扩增子熔解分析和直接DNA测序证实。这2例c-kit突变阳性病例不含BRAF突变。转移性恶性黑色素瘤中c-kit激活突变的存在表明少数黑色素瘤可能通过c-kit基因的体细胞突变而进展。恶性黑色素瘤中BRAF和c-kit激活突变的存在表明，治疗这种疾病的新方法涉及特定的酪氨酸激酶抑制剂可能是值得的，高分辨率熔解分析的突变分析可能有助于指导治疗。(c)2005年爱思唯尔公司All rights reserved.
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. (c) 2005 Elsevier Inc. All rights reserved.