Quantification of minimal disseminated disease by quantitative PCR and digital PCR for NPM-ALK as prognostic factor in children with anaplastic large cell lymphoma

Detection of minimal disseminated disease is a validated prognostic factor in ALK-positive anaplastic large cell lymphoma. We previously reported that quantification of minimal disease by quantitative real-time PCR in bone marrow applying a cut-off of 10 copies NPM-ALK/104 copies of the reference transcript ABL1 identifies a very high-risk group of patients. In the present study, we aimed to confirm the prognostic value of quantitative minimal disseminated disease evaluation by real-time PCR and to validate digital PCR as alternative method. Among 91 patients analyzed by real-time PCR in bone marrow, the cumulative incidence of relapses of 18 patients with more than 10 normalized copy-numbers of NPM-ALK was 61+/-12% compared to 21+/-5% for the remaining 76 patients (p=.0002). Results in blood correlated with bone marrow (r=0.74) in 70 patients with both materials. Copy numbers tended to be higher in blood compared to bone marrow. Transcripts were quantified in addition by digital PCR in 75 bone marrow and 57 blood samples. Copy number estimation by using digital PCR and real-time PCR correlated in 132 samples (r=.85). Applying a cut-off of 30 copies NPM-ALK/104 copies ABL1 for quantification by digital PCR, almost identical patient groups were separated compared to real-time PCR. In summary, the prognostic impact of quantification of minimal disseminated disease in bone marrow could be confirmed for patients with anaplastic large cell lymphoma. Quantification of minimal disease by digital PCR provides a promising tool to facilitate harmonization of minimal disease measurement between laboratories and for clinical studies.