Routine Multiplex Mutational Profiling of Melanomas Enables Enrollment in Genotype-Driven Therapeutic Trials

PurposeKnowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma. MethodsThe test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5–10% mutant allele frequency) and minimal amounts of DNA (10–20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab. ResultsDirecting this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials. ConclusionWe present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.研究目的：肿瘤突变状态相关知识在癌症治疗中的重要性与日俱增，因针对携带特定肿瘤基因型的患者亚群开发的突变特异性抑制剂正逐步应用于临床。黑色素瘤便是这一治疗范式的近期典型案例。本文报道了一种检测方法的开发、验证与临床应用，该方法可同时检测6个基因（BRAF、NRAS、KIT、GNAQ、GNA11及CTNNB1）中的43种常见体细胞点突变（somatic point mutations），这些突变与黑色素瘤现有及新兴的靶向治疗方案密切相关。 研究方法：本检测采用SNaPshot技术（SNaPshot），包含多重PCR（multiplex PCR）、多重引物延伸（multiplex primer extension）及毛细管电泳（capillary electrophoresis），可快速完成检测，具备高灵敏度（仅需5%~10%的突变等位基因频率（mutant allele frequency）），且样本需求量低，仅需10~20纳克DNA。本检测方法通过细胞系、新鲜冷冻组织及福尔马林固定石蜡包埋组织（formalin-fixed paraffin embedded tissue）进行了验证。随后，我们对范德堡大学分子诊断实验室中完成肿瘤基因分型的前150例黑色素瘤患者的临床特征及其对临床试验入组的影响进行了评估。 研究结果：本检测针对单一疾病（黑色素瘤）开展，150例来自全身各部位的黑色素瘤样本中，有90例（60%）携带本次检测覆盖的突变，其中BRAF基因57例、NRAS基因23例、GNAQ基因6例、KIT基因3例、CTNNB1基因2例。在BRAF V600突变中，V600E、V600K、V600R及V600M分别占79%、12%、5%及4%。携带突变的转移性黑色素瘤患者中，23例（43%，23/54）后续入组了基因型导向的临床试验（genotype-driven trials）。 结论：本研究开发了一种针对黑色素瘤临床相关突变的简易突变谱筛查方法。采用这种基于基因信息的黑色素瘤治疗方案，已对该疾病患者的临床试验入组及治疗优先级排序产生了切实影响。