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.

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作者：

K Gruber，H Horn，Kalla, J?rg，P Fritz，A Rosenwald，Kohlh?ufl, Martin，G Friedel，M Schwab，G Ott，C Kalla

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摘要：

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 ofALK. 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 detectALKdysregulation was shown by comparison with FISH and immunohistochemistry. qRT-PCR analysis detected unbalancedALKexpression indicative of a gene rearrangement in 24 (4.6%) and full-lengthALKtranscript 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 identifiedALKderegulation in two cases with insufficient FISH. Six tumors expressing full-lengthALKtranscripts 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-lengthALKexpression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding. Our qRT-PCR assay reliably identifies and distinguishesALKrearrangements 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 ofALKactivation. The expression of full-lengthALKtranscripts may be relevant for ALK inhibitor therapy in NSCLC.

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关键词：

Non-small-cell lung cancer Anaplastic lymphoma receptor tyrosine kinase Translocation and overexpression Quantitative expression analysis Routine diagnosis

DOI：

10.1097/JTO.0000000000000068

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年份：

2014