采用实时定量逆转录聚合酶链反应检测方法对存档的非小细胞肺癌肿瘤标本中 EML4-ALK 融合变体进行大规模筛查和分子特征分析。
Large-scale screening and molecular characterization of EML4-ALK fusion variants in archival non-small-cell lung cancer tumor specimens using quantitative reverse transcription polymerase chain reaction assays.
机构信息
*University of California Davis Comprehensive Cancer Center, †Department of Internal Medicine, Division of Hematology & Oncology, University of California, Davis, School of Medicine, Sacramento, California; ‡Department of General-, Visceral- and Tumor Surgery, University of Cologne, Cologne, Germany; §Response Genetics, Inc., Los Angeles, California; ‖Department of Public Health Sciences, University of California, Davis, Davis, California; ¶Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California; and #formerly Response Genetics, Inc., Los Angeles, California.
出版信息
J Thorac Oncol. 2014 Jan;9(1):18-25. doi: 10.1097/JTO.0000000000000030.
INTRODUCTION
The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non-small-cell lung cancer (NSCLC) patients.
METHODS
We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients.
RESULTS
Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23-89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001).
CONCLUSIONS
This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.
简介
本研究旨在通过针对北美大型非小细胞肺癌（NSCLC）患者队列的变体特异性定量逆转录聚合酶链反应（RT-PCR）检测，确定并描述棘皮动物微管相关蛋白样 4 间变性淋巴瘤激酶融合（EML4-ALK+）癌症。
方法
我们开发了一组单重和多重 RT-PCR 检测，适用于快速准确地检测八种最常见的 EML4-ALK+变体和 ALK 基因在存档福尔马林固定、石蜡包埋 NSCLC 标本中的表达。在一部分患者中可进行 EGFR 和 KRAS 基因分型以及胸苷酸合成酶 RNA 水平的 RT-PCR 检测。
结果
在 2009 年 12 月至 2012 年 9 月期间，检测了 7344 例 NSCLC 标本。在 200 例标本（2.7%）中检测到 EML4-ALK+转录本，包括 109 例 V1（54.5%）、20 例 V2（10.0%）、68 例 V3（34.0%）和 3 例 V5a（1.5%）变体。中位年龄为 54.5 岁（范围，23-89），104 例患者（52.0%）为女性。绝大多数（n=188，94.0%）EML4-ALK+ NSCLC 肿瘤具有腺癌组织学。不同的 EML4-ALK+变体和单个肿瘤中 ALK 表达水平差异显著。仅检测到 1 例 EGFR 或 KRAS 突变同时存在。85 例 EML4-ALK+癌症的胸苷酸合成酶 RNA 中位水平明显低于 EML4-ALK-阴性肺腺癌（分别为 2.02 和 3.29，p<0.001）。
结论
本变体特异性定量 RT-PCR 检测试剂盒可检测存档福尔马林固定石蜡包埋 NSCLC 标本中的常见 EML4-ALK+变体和 ALK 基因表达水平。这些 RT-PCR 检测方法可能有助于辅助标准荧光原位杂交检测，以更好地了解间变性淋巴瘤激酶抑制剂的生物学变异性和反应模式。
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