J Thorac Oncol：检测肺癌ALK基因重组的新方法——定量RT-PCR
来源：生物谷 2014-02-25 20:28
2014年2月25日讯 /生物谷BIOON/--近日，研究人员开发出一种用于检测非小细胞肺癌中ALK重排的新技术，其比当前可用的技术更敏感，更容易执行。这项技术可以帮助提高对非小细胞肺癌ALK的检测，检测ALK对于确定晚期NSCLC患者中谁最有可能从靶向治疗ALK抑制剂受益很重要。
目前用于检测非小细胞肺癌ALK重排的三个常规方法各有缺点，特别是对于那些需要用到福尔马林固定，石蜡包埋组织标本的方法。荧光原位杂交（FISH）是唯一批准测试ALK的技术，但它由于成本高，测试所需时间长，并且需要专门的设备和专门知识，因此并不总是可行的。
免疫组化（IHC）结果因为弱的和可变的免疫反应性，解读实验结果变得具有挑战性。逆转录-聚合酶链反应（RT-PCR）是高度敏感的，但需要高品质的RNA，而这往往是很难获得的。
量化（q）的RT-PCR克服了这些问题，通过利用RT-PCR方法的敏感性和两个特征：RNA分离方法优化扭转甲醛改性，小的RT-PCR扩增子允许使用零散核酸来使ALK cDNA高效扩增。
新的定量RT-PCR检测测ALK转录部分5 '和3'蛋白的表达，它检测到不平衡的ALK表达以此指示基因重排，在研究中523例非小细胞肺癌标本中检测到24例（4.6％）基因重排和六例肿瘤（1.1％）全长ALK的转录表达。
这项研究的结果发表在Journal of Thoracic Oncology杂志上。该定量RT-PCR技术能可靠的检测ALK重排，也可检测基因全长转录表达的肿瘤，这是FISH无法检测的。这项技术似乎是高敏感性、易于执行、高通量、适于肺癌ALK活化常规诊断的新方法。（生物谷Bioon.com）
doi:10.1097/JTO.0000000000000068
Detection of Rearrangements and Transcriptional Up-Regulation of ALK in FFPE Lung Cancer Specimens Using a Novel, Sensitive, Quantitative Reverse Transcription Polymerase Chain Reaction Assay.
Kim Gruber, Heike Horn, J?rg Kalla, Peter Fritz, Andreas Rosenwald, Martin Kohlhufl, Godehard Friedel, Matthias Schwab, German Ott, Claudia Kalla.
INTRODUCTION:
The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK.
METHODS:
We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry.
RESULTS:
qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding.
CONCLUSIONS:
Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.
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