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NPM-ALK quantification by RQ-PCR and dPCR in ALCL
groups, the differences in results might be related to the use of standard curves that were not centrally distributed and to the fact that the cut-off at 10 NCN NPM-ALK is close to the detection limit of the assay. The latter point is an unchangeable limitation to inter-laboratory compara- bility for quantification of NPM-ALK transcripts and a major difference from MRD quantification in leukemia.
In order to overcome some of the technical problems inherent to RQ-PCR we developed a dPCR method for the quantification of NPM-ALK transcripts and compared the
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results obtained with this method to those obtained with RQ-PCR in a large cohort of patients. Using dPCR with a cut-off at 30 NCN NPM-ALK for quantitative measure- ments of MDD in blood and BM in the presented study allowed measurements near to the detection limit without needing standard curve calibration. The dPCR assay might be more suitable for quantitative measurements of NPM- ALK in a multinational setting, because it overcomes sev- eral limitations of the RQ-PCR assay. First, it is independ- ent of a calibration curve, thereby excluding the impact of
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Figure 4. Outcome according to NPM-ALK copy numbers measured by digital polymerase chain reaction in bone marrow. (A) Cumulative incidence of relapse, (B) event-free survival and (C) overall sur- vival at 3 years according to a cut-off of 30 normal- ized copy numbers of NPM/ALK/104 copy numbers of ABL1. BM: bone marrow, NCN: normalized copy numbers.
haematologica | 2020; 105(8)
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