*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.
J Thorac Oncol. 2014 Jan;9(1):18-25. doi: 10.1097/JTO.0000000000000030.
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.
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.
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).
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.
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。
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。
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）。
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。
