Quantitative detection of ALK rearrangements using droplet digital PCR (ddPCR) in liquid biopsies of patients with non-small cell lung cancer (NSCLC)

Background : The rearrangement of the anaplastic lymphoma kinase (ALK) is the oncogenic driver of 5% of advanced NSCLC and is identified on tumor by immunohistochemistry and FISH. Molecular pathologists are often hindered by the poor quality and quantity of available material to determine ALK -status and liquid biopsies represent an interesting alternative. Point mutations and indels are routinely detected in circulating-free DNA; however, data are scarce on detection of fusion transcripts in blood. Material and Methods : Sensitivity and specificity of specific- ALK rearrangements primer and probes using ddPCR were tested in tumors, cell lines, blood samples from healthy donors and patients with ALK -rearranged tumors. Results : On tissue, all FISH-positive and –negative samples were respectively positive and negative using ddPCR, indicating 100% specificity in tumors. Sensibility was 0.1% in cell lines. Interestingly, questionable cases by FISH were all positive by ddPCR. Blood samples of healthy donors were negative for ALK -rearrangement indicating a 100% specificity. Two non-smoking women with positive EML4 - ALK -positive stage IV adenocarcinoma confirmed by IHC and FISH in the tumor were found positive in the plasma with 3.45 and 13.36 copies/mL of plasma respectively. Conclusion : ddPCR is promising for the determination ALK -status in blood. It can be easily implemented in clinical practice. Detection of circulating free RNA may be extended to other fusion genes involved in lung carcinogenesis ( i.e. ROS1 -rearrangements) and to other tumor types ( i.e. sarcomas).