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C. Damm-Welk et al.
Detection of blasts in bone marrow (BM) and blood by cytomorphological analysis is a rare event in ALCL.6,7 The chimeric fusion gene transcript NPM-ALK has been used to investigate the prognostic value of submicroscopic mini- mal disseminated disease (MDD) in BM and blood at diag- nosis in independent cohorts of patients.10-13 Polymerase chain reaction (PCR) analysis allows the reliable detection of one circulating tumor cell among 100,000 normal cells.10 The detection of MDD by qualitative PCR in BM or blood (55% of patients) conferred a relapse risk of 50% in several studies.10-12,14 Measurement of minimal residual disease (MRD) using the qualitative PCR assay enabled identifica- tion of a very high-risk group of patients.14
We previously reported the possibility of identifing patients bearing a very high risk of relapse already at diag- nosis by quantification of fusion gene transcripts using an NPM-ALK-specific quantitative real-time (RQ)-PCR assay. Applying a cut off of 10 copies NPM-ALK per 104copies of the reference transcript ABL1 (normalized copy numbers, NCN), 16 patients (22%) with more than 10 NCN NPM- ALK in the BM at diagnosis had a 5-year probability of event-free survival of 23±11% compared to 78±6% for the 58 patients with NCN below the cut-off. MDD levels measured in blood provided comparable results.12
The Japanese NHL study group recently reported the outcomes of 60 ALCL-patients according to MDD in blood or BM using the same cut-off of 10 NCN NPM-ALK.13 The patients received comparable therapies. Compared to the Berlin-Frankfurt-Münster (BFM) group, however, more patients showed MDD levels above 10 NCN. The progres- sion-free survival of 37 patients with >10 NCN NPM-ALK in blood or BM was 58±12% compared to 85±6% for the 22 patients with ≤10 NCN.13
The differences regarding the prognostic value of MDD assessment by RQ-PCR in these two studies illustrate the need for standardization before the implementation of quantification of NPM-ALK transcripts for initial risk assessment or MRD evaluation in clinical studies. Currently, quantitative values from different laboratories cannot be directly compared to each other, whereas MRD quantification within one laboratory has been reported to enable the course of the disease to be monitored.15-17
To achieve comparability of MRD quantification for NPM-ALK obtained by RQ-PCR in different reference lab- oratories, extensive protocol harmonization is necessary, as was done for the quantification of BCR-ABL1 fusion gene transcripts in acute lymphoblastic leukemia and chronic myelogenous leukemia.18,19 Since quantification is performed at the lowest end of the necessary standard curve in NPM-ALK-specific RQ-PCR, a quantitative PCR approach with improved reproducibility without the need for a standard curve would be advantageous. Digital PCR (dPCR) may represent a quantitative PCR method that could be used as a replacement for RQ-PCR for NPM-ALK copy number estimation in ALK-positive ALCL. dPCR is a quantitative PCR method based on the distribution of the target RNA or DNA molecules in many partitions.20 The amount of partitions with a positive PCR results allows the concentration of a given target to be determined without the need for standard curve calibration.21
The aim of this work was to validate the prognostic meaning of quantitative MDD measurements of NPM- ALK fusion gene transcripts by RQ-PCR in an independent cohort of uniformly treated ALK-positive ALCL patients of the BFM group. In addition, in an effort to facilitate quali-
ty-controlled quantification between different laboratories, a dPCR assay for quantification of NPM-ALK transcripts was developed and validated.
Methods
Patients
Patients with ALCL from Austria, Germany and Switzerland enrolled in the ALCL99 trial or the NHL-BFM registry 2012 between January 2006 and December 2016 were eligible after con- firmation of NPM-ALK positivity of the ALCL. Both studies were approved by the institutional ethics committee of the primary investigators. Informed consent from the patients/caregivers to the studies included consent for future research on MDD.
Controls and cell lines
Blood from 20 healthy donors and eight ALK-negative ALCL patients included in ALCL99 or the NHL-BFM registry served as negative controls after written informed consent.
The cell lines HL-60 (acute myeloid leukemia), SU-DHL-1 and Karpas-299 (NPM-ALK-positive ALCL) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany).
Complementary DNA synthesis and quantitative real- time polymerase chain reaction
Complementary DNA (cDNA) synthesis and RQ-PCR were performed as described previously.12 In total four replicates were analyzed (two with undiluted cDNA and two with 1+1 diluted cDNA, as an additional control for RQ-PCR inhibition). Samples which were positive for NPM-ALK in one to three of four repli- cates only or had NCN below one copy were considered as low positive not quantifiable. Negativity for NPM-ALK in all four repli- cates was considered as negative.
Digital polymerase chain reaction assay
Primer and probe sequences for the NPM-ALK- and ABL1-spe- cific dPCR assay were identical to those used for the RQ-PCR assay.12 Probes used for dPCR were ordered with 5’FAMTM as the reporter dye and the double quencher dyes ZENTM and 3’Iowa- Black®FQ (IDT, Leuven, Belgium).
Ten microliters of dPCRTM supermix for probes (no dUTP; BIO- RAD, Munich, Germany), 0.6 mL forward primers, 0.6 mL reverse primers (10 mM, final concentration 300 pM) and 1 mL probe (final concentration 250 pM) were used in a reaction volume of 20 mL. Droplets were generated with the QX-200 droplet generator (BIO- RAD, Munich, Germany). The PCR was performed at 95°C for 10 min for enzyme activation, 44 cycles at 94°C for 30 s, followed by 1 min at 54.1°C for annealing and extension, and enzyme inacti- vation at 98°C for 10 min. Droplets were measured with the QX200 droplet reader and were analyzed with Quanta Soft pro analysis software (BIO-RAD, Munich, Germany). Four replicates per sample were measured. Only replicates with ≥10,000 accepted droplets were included in the analysis. The threshold for discrim- ination between positive and negative droplets was set manually with an adequate distance from the background. cDNA from the ALK-positive cell line SU-DHL-1 (positive control), HL-60 (nega- tive control) and no template controls were included in each meas- urement. Copy numbers were normalized to 10,000 copies of the reference gene ABL1 (NCN). Samples with <1,000 copies of ABL1 were excluded. Samples with detectable fusion gene transcripts in one to three, but not in all four replicates were defined as low pos- itive, not quantifiable. Samples were defined as negative if no pos- itive droplets were observed.
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haematologica | 2020; 105(8)