Clinical evaluation of the effectiveness of fusion-induced asymmetric transcription assay-based reverse transcription droplet digitalPCRforALKdetection in formalin-fixed paraffin-embedded samples from lung cancer

Background Accurate detection of anaplastic lymphoma kinase (ALK) rearrangement is the prerequisite for anti-ALKtherapy 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 forALKstatus survey in NSCLC samples. Methods A total of 269 cases of formalin-fixed paraffin-embedded (FFPE) specimens from NSCLC, in whichALKstatus was confirmed by both fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), were analyzed by FIATA-based RT-ddPCR. Results In theALK-positive group, the 3 ' ALKtranscript copies range was 336.6-107 955.4, and the R3 [(the ratio of the 3 ' ALKtranscript copy numbers to the internal reference gene transcript copy numbers) x 100] was 17.23-672.77. In theALK-negative group, the 3 ' ALKtranscript copies range was 3.7-1370.6, and the R3 range was 0.10-15.57. The lowest R3 level in theALK-positive group was significantly higher than the highest R3 level in theALK-negative group. A positive correlation between the proportion of cancer cells in the tissue section andALKRNA 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 theALKgene. Compared with FISH and IHC, the clinical sensitivity and specificity of FIATA-based RT-ddPCR forALKdetection were 100%, respectively. Conclusions An absolute quantitative FIATA-based RT-ddPCR was developed and validated forALKfusion detection in NSCLC. This method can rapidly, accurately, and objectively classifyALKtypes and help with individual therapy.