Clinical evaluation of the effectiveness of fusion‐induced asymmetric transcription assay‐based reverse transcription droplet digital PCR for ALK detection in formalin‐fixed paraffin‐embedded samples from lung cancer

BACKGROUND: Accurate detection of anaplastic lymphoma kinase (ALK) rearrangement is the prerequisite for anti‐ALK therapy for the patient with non‐small cell lung cancer (NSCLC). Fusion‐induced asymmetric transcription assay (FIATA)‐based reverse transcription droplet digital PCR (RT‐ddPCR) was developed and performed for ALK status survey in NSCLC samples. METHODS: A total of 269 cases of formalin‐fixed paraffin‐embedded (FFPE) specimens from NSCLC, in which ALK status was confirmed by both fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), were analyzed by FIATA‐based RT‐ddPCR. RESULTS: In the ALK‐positive group, the 3′ ALK transcript copies range was 336.6–107 955.4, and the R3 [(the ratio of the 3′ ALK transcript copy numbers to the internal reference gene transcript copy numbers) × 100] was 17.23–672.77. In the ALK‐negative group, the 3′ ALK transcript copies range was 3.7–1370.6, and the R3 range was 0.10–15.57. The lowest R3 level in the ALK‐positive group was significantly higher than the highest R3 level in the ALK‐negative group. A positive correlation between the proportion of cancer cells in the tissue section and ALK RNA expression level (R3) was found (P < 0.05). There was no relationship between the percentage of FISH positive cells or FISH positive signal patterns and R3 level of the ALK gene. Compared with FISH and IHC, the clinical sensitivity and specificity of FIATA‐based RT‐ddPCR for ALK detection were 100%, respectively. CONCLUSIONS: An absolute quantitative FIATA‐based RT‐ddPCR was developed and validated for ALK fusion detection in NSCLC. This method can rapidly, accurately, and objectively classify ALK types and help with individual therapy.