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C. Damm-Welk et al.
by the same persons, according to standard operating pro- cedures for RQ-based MDD measurement and analysis. However, there are still significant inter-laboratory differ- ences and quantification of minimal disease in patients with ALCL can currently not be compared between labo- ratories.12,13,17,33 This is exemplified by the comparison of the Japanese study group’s data with our data. Both groups used the same therapy and the same RQ-PCR assay for quantification. Twenty per cent of patients showed >10 NCN NPM-ALK in our cohorts and 37% of patients had high copy numbers in the Japanese cohort even though the relapse rate in the Japanese cohort was somewhat lower. Accordingly, the relapse risk of patients with >10 NCN NPM-ALK was higher in our cohorts (65%) than in the Japanese cohort (40%). In order to use quantification of copy numbers for patient stratification or to follow the course of individual patients in multinational studies, the RQ-PCR method needs very strict protocol harmonization and quality control. The experiences from quantification of BCR-ABL1 transcripts can partly guide this development.34 The introduction of calibrators, specif- ic conversion factors to the calibrators for each laboratory and calibrated reference material led to a high standardi- zation of BCR-ABL1 measurements.19,35-37 In Philadelphia- positive acute leukemia the optimization and standardiza- tion process for RQ-PCR-based measurement of m-BCR-
ABL1 transcripts underscores the importance of standard- ization of all steps for quantitative PCR, including data interpretation and quality controls.38 In the standardiza- tion process of MRD assessment of m-BCR-ABL1 fusion gene transcripts organized by the Euro MRD consortium, the usage of a common primer and probe set as well as a centrally distributed plasmid standard curve had the great- est impact on overcoming inter-laboratory variability.38
Since the same primer/probe sets and the same RQ-PCR protocol were applied by the Japanese and BFM study
Table 2. Normalized copy numbers of NPM-ALK measured by digital poly- merase chain reaction analysis in a 10-fold serial dilution of a NPM-ALK anaplastic large cell lymphoma cell line in 106 mononuclear cells.
Dilution
10-1
10-2 10-3 10-4 10-5
ALK+ cells in 106 MNC
100,000
10,000 1,000 100 10
Copies of NPM-ALK/104 ABL1
9222
974 93 8 0.4
000
MNC: mononuclear cells.
Figure 3. Comparison of NPM- ALK copy numbers measured by quantitative real-time poly- merase chain reaction and digi- tal polymerase chain reaction. Normalized copy numbers of NPM/ALK/104 copy numbers of ABL1 measured in 132 blood and bone marrow samples. quantPCR: quantitative real-time polymerase chain reaction; dPCR: digital polymerase chain reaction; NCN: normalized copy number.
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haematologica | 2020; 105(8)