Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

Abstract

Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

ALK‐rearrangement in non‐small‐cell lung cancer (NSCLC)

“…First of all, while FISH and IHC detect relatively indirect signs of the presence of ALK translocation, RT‐PCR usually reveals the exact variant of the rearrangement and therefore provides definitive evidence of ALK fusion. Furthermore, RT‐PCR has high sensitivity and specificity, with a rapid turnaround time and ease of analysis, and can detect a small number (1%) of ALK ‐driven NSCLC cells in the presence of normal tissues . In addition, RT‐PCR analysis utilizes the same technical platform as other kinds of molecular NSCLC diagnosis, for example EGFR testing.…”

“…Furthermore, RT-PCR has high sensitivity and specificity, with a rapid turnaround time and ease of analysis, and can detect a small number (1%) of ALK-driven NSCLC cells in the presence of normal tissues. [64][65][66] In addition, RT-PCR analysis utilizes the same technical platform as other kinds of molecular NSCLC diagnosis, for example EGFR testing. Finally, a number of commercial RT-PCR kits for detection of ALK rearrangements have been developed recently, including: the ALK RGQ RT-PCR Kit (Qiagen, Valencia, CA, USA); the EML4-ALK Fusion Gene Detection Kit (Amoy Diagnostics, Xiamen, China); and EML4 ALK Gene Fusion, PCR (Quest Diagnostics, Secaucus, NJ, USA).…”

Potential anti-vitiligo properties of cynarine extracted from Vernonia anthelmintica (L.) Willd

“…The cDNA from each sample was used as a template in the RT-qPCR to detect the mRNA level of the 1,5-diCQA target genes. qPCR was conducted as previously described, with certain modifications ( 24 ). qPCR was performed in a 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in a final volume of 25 µ l [forward and reverse primers, 0.25 mol/l each; Power SYBR ® -Green PCR Master mix (cat.…”

<p>Comparison of Next-Generation Sequencing and Ventana Immunohistochemistry in Detecting ALK Rearrangements and Predicting the Efficacy of First-Line Crizotinib in Patients with Advanced Non-Small Cell Lung Cancer</p>

“…23,26,27 Numerous studies have compared the reliability of ALK detection using different methods, including IHC, FISH, and RT-PCR. 23,26,[28][29][30][31][32] However, only a few studies investigated the reliability of NGS as compared with other traditional detection methods. [18][19][20][21][22][23] In this study, we compared NGS -a method that has become routinely used in clinical oncology due to its multiplex-ability-with IHC -an established method that is routinely used for the molecular diagnosis of ALK in the clinical setting.…”