Minimal residual disease monitoring using a 3'ALK universal probe assay in ALK-positive anaplastic large cell lymphoma: ddPCR, an attractive alternative method to real-time quantitative PCR

In ALK-positive anaplastic large cell lymphomas, positive qualitative PCR for NPM1-ALK in peripheral blood and/or bone marrow at diagnosis and during treatment are associated with a higher risk of treatment failure. Real-time quantitative PCR allows identification of very high risk patients. However, this latter technique initially designed for patients with lymphomas carrying the most frequent NPM1-ALK translocation necessitates calibration curves, limiting inter-laboratory reproducibility. We designed an ALK universal quantitative PCR based on 3'ALK transcript amplification to allow the detection of all ALK fusion transcripts. We validate the absolute concordance of 3'ALK quantitative PCR results with the routine NPM1-ALK qualitative and quantitative PCR on 46 samples. The universality of ALK fusion transcript detection was also validated on TPM3-, ALO17- and ATIC-ALK-positive samples, and EML4-ALK-positive cell line. Then, we show that digital droplet PCR using 3'ALK universal probe gives highly concordant results with 3'ALK universal quantitative PCR. A major benefit of digital droplet PCR is a reduced experimental set-up compared with quantitative PCR, without generation of standard curves, leading to a reliable protocol for multilaboratory validation, in multicenter clinical trials essential for this rare pathology. Our ALK universal method could be used for the screening of ALK fusion transcripts in liquid biopsy of other ALK positive tumors, including non-small cell lung carcinomas.