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C. Damm-Welk et al.
standard curve differences for inter-laboratory compar- isons. Second, partitioning of target molecules leads to a more precise detection especially of rare events so that the assay is able to detect low copy numbers more accurately than RQ-PCR.39,40 To exclude that templates are not ampli- fiable during the dPCR reaction it is still unavoidable to verify the quality of a given cDNA by parallel estimation of a reference gene. Furthermore, for measurement of clin- ical samples, appropriate positive and negative controls must be included in order to control for the overall per- formance of a given dPCR experiment.
The applicability of dPCR for MRD measurement has already been shown for several hematologic malignancies. The accuracy of dPCR and high concordance with RQ- PCR was demonstrated for DNA-based MRD measure- ments of the BCL2/IGH rearrangement in blood and BM from patients with low stage follicular lymphoma41 and immunoglobulin/T-cell receptor rearrangements in patients with acute lymphoblastic leukemia.42 dPCR was shown to be reliable for quantifying BCR-ABL1 transcripts for MRD monitoring in chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia.43-46 Altogether, dPCR is a valuable tool for highly reproducible quantification of minimal disease at both DNA and RNA levels in patients’ samples without requiring standard curves. In addition, for MDD and MRD in ALCL, it might have the advantage over RQ-PCR that the quantification is more accurate at lower copy numbers. Stringent proto- col standardization and quality control are needed for this technique, as well.47,48
In summary, our data validate that quantification of NPM-ALK transcripts by RQ-PCR using a cut-off of 10 NCN identifies very high-risk patients if performed in one laboratory. Quantification of MRD is indicated to follow the course of disease and response to treatment modules in MDD-positive or relapsed ALCL patients. In a rare dis- ease such as ALCL, with planned and ongoing internation- al trials, both methods for transcript quantification require
Table 3. Concordance of patients’ stratification according to quantity of NPM-ALK transcripts determined using digital polymerase chain reac- tion and quantitative real-time polymerase chain reaction in (A) bone marrow and (B) blood.
(A)
RQ-PCR
(B)
RQ-PCR
≤30 NCN
≤30 NCN
dPCR
dPCR
>30 NCN 0
12
>10 NCN
≤10 NCN
>10 NCN
63
0
44
1
>30 NCN 0
12
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14. Damm-Welk C, Mussolin L, Zimmermann M, et al. Early assessment of minimal resid-
≤10 NCN
The cut-off for the digital polymerase chain reaction was 30 normalized copy num- bers (NCN), while that for quantitative real-time polymerase chain reaction was 10 NCN. dPCR: digital polymerase chain reaction; RQ-PCR: quantitative real time poly- merase chain reaction.
inter-laboratory comparability of measurements. Since harmonization is difficult and expensive with RQ-PCR, we developed and validated a dPCR assay enabling reli- able quantification of NPM-ALK transcripts at very low copy numbers without the need for standard calibration curves.
Acknowledgments
The authors would like to thank Claudia Keller for excellent technical assistance and Ulrike Meyer for excellent assistance with data management. The NHL-BFM Registry 2012 is sup- ported by the Deutsche Kinderkrebsstiftung. CDW, NK, JS and WW were additionally supported by Forschungshilfe Peiper. The pediatric lymphoma research of IO and WK is supported by the KinderKrebsInitiative Buchholz, Holm-Seppensen.
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