Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany.
Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany; Gerhard-Domagk-Institute of Pathology, University of Münster Medical Center, D-48149 Münster, Germany.
Hum Pathol. 2014 Mar;45(3):573-82. doi: 10.1016/j.humpath.2013.10.025. Epub 2013 Nov 4.
The mutational status of KIT and PDGFRA is highly relevant for prognosis and therapy prediction in gastrointestinal stromal tumors (GIST). PDGFRA exon 18 mutations have direct therapeutic implications since it is crucial to distinguish mutations associated with sensitivity to tyrosine kinase inhibitors from those causing primary resistance, eg, the most common exon 18 mutation p.D842V. In response to a growing demand for reliable, faster and more sensitive methods we established and validated a high-resolution melting (HRM) assay for PDGFRA exon 18. A total of 159 GIST samples were comparatively analyzed by HRM and direct Sanger sequencing. We demonstrate that HRM provides highly reliable mutational results with higher sensitivity and shorter time to diagnosis compared to Sanger sequencing. We determined the sensitivity threshold of HRM at 6% of mutated alleles. PDGFRA exon 18 wild-type status and the most common p.D842V resistance mutation (together representing >90% of the cases) can be detected specifically by HRM. Other rare mutations can be pre-screened by HRM and afterwards determined precisely by DNA sequencing. In this way we detected four novel mutations in PDGFRA exon 18, two of which were associated with an aggressive clinical course. Including these new mutations, we provide a comprehensive overview of all 60 currently known subtypes of PDGFRA exon 18 mutations in GIST. Seven of them (accounting for about 75% of all exon 18-mutated GISTs) are reported to be resistant to imatinib. However, there are at least 10 other mutations which are regarded as sensitive to tyrosine kinase inhibitors.
KIT 和 PDGFRA 的突变状态与胃肠道间质瘤（GIST）的预后和治疗预测密切相关。PDGFRA 外显子 18 突变具有直接的治疗意义，因为区分与酪氨酸激酶抑制剂敏感性相关的突变与导致原发性耐药的突变至关重要，例如最常见的外显子 18 突变 p.D842V。为了满足对可靠、快速和更敏感方法的不断增长的需求，我们建立并验证了 PDGFRA 外显子 18 的高分辨率熔解（HRM）分析。总共对 159 个 GIST 样本进行了 HRM 和直接 Sanger 测序的比较分析。我们证明 HRM 提供了高度可靠的突变结果，与 Sanger 测序相比，具有更高的灵敏度和更短的诊断时间。我们确定 HRM 的灵敏度阈值为 6%的突变等位基因。HRM 可以特异性检测 PDGFRA 外显子 18 野生型状态和最常见的 p.D842V 耐药突变（共同代表超过 90%的病例）。其他罕见的突变可以通过 HRM 进行预筛选，然后通过 DNA 测序精确确定。通过这种方式，我们在 PDGFRA 外显子 18 中检测到了四个新突变，其中两个与侵袭性临床病程相关。包括这些新突变，我们提供了 GIST 中目前已知的所有 60 种 PDGFRA 外显子 18 突变亚型的综合概述。其中 7 种（约占所有外显子 18 突变 GIST 的 75%）被报道对伊马替尼耐药。然而，至少还有 10 种其他突变被认为对酪氨酸激酶抑制剂敏感。
