IDENTIFICATION OF ANAPLASTIC LYMPHOMA KINASE-EXPRESSING LUNG ADENOCARCINOMA BY NEWLY-DEVELOPED ULTRASHORT RT-PCR ASSAY
Anaplastic lymphoma kinase (ALK) fusion is highly correlated with ALK inhibitor therapy response rate in patients with non-small cell lung cancer (NSCLC). Reverse-transcription PCR (RT-PCR) is a highly sensitive technique but it suffers from a lack of diagnostic value in formalin-fixed paraffin embedded (FFPE) tissue specimens and detection of all known and unknown ALK fusion variant patterns.
Isolated RNA samples were subjected to complementary DNA synthesis, followed by expression analysis using real-time PCR based on published assays developed by our research group. Detection of 18S ribosomal RNA gene were utilized for evaluation of FFPE tissue quality. Complementary DNA samples from cell lines were included as positive and negative controls. Sections of tissues from 47 Thai lung adenocarcinoma patients were subjected to determination of ALK expression, an indirect indicator of ALK fusion, by immunohistochemistry (IHC) and our RT-PCR.
We successfully developed a new quantitative RT-PCR method for amplifying ultrashort (approximately 45 bases) target sequences in 3’ region of ALK and 18S ribosomal RNA gene. We were able to confirm the presence of the ALK fusion in the NCI-H2228 human NSCLC cell line and the absence of the ALK fusion in the non-cancerous HEK293 cells. Our newly-developed assay revealed ALK expression in 3 of 47 FFPE lung adenocarcinoma tissues and achieved a high concordance rate with IHC results. It is noteworthy that extremely degraded RNA from improper tissue collection, handling, processing, or storage may affect the performance of the assay.
We demonstrate the first ultrashort RT-PCR assay in detecting ALK expression which potentially overcome the limitations of currently available RT-PCR assays. Careful attention of preanalytical factors is critically important in molecular analysis.