With the aim of describing the long-term follow-up and to define the prognostic role of the clinical/pathological/molecular characteristics at diagnosis for childhood, adolescent and young adults affected by anaplastic large cell lymphoma (ALCL), we analyzed 420 patients aged up to 22 years homogeneously treated within the international ALCL99 trial. The 10-year progression free survival (PFS) was 70% and overall survival was 90%, rare late relapses occurred but no secondary malignancies were reported. Among clinical/pathological characteristics, only patients presenting a small cell/lymphohistiocytic (SC/LH) pattern were independently associated with risk of failure (hazard ratio = 2.49). Analysis of minimal disseminated disease (MDD), available for 162 patients, showed that both SC/LH pattern (hazard ratio = 2.4) and MDD positivity (hazard ratio = 2.15) were significantly associated with risk of failure in multivariate analysis. Considering MDD and SC/LH results, patients were separated into three biological/pathological (bp) risk groups: a high-risk group (bpHR) including MDD-positive patients with SC/LH pattern; a low-risk group (bpLR) including MDD-negative patients without SC/LH pattern; and an intermediate-risk group (bpIR) including remaining patients. The 10-year PFS was 40%, 75% and 86% for bpHR, bpIR and bpLR, respectively (p < 0.0001). These results should be considered in the design of future ALCL trials to tailor individual treatments.

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.

Characterization and diagnostic application of genomic NPM-ALK fusion sequences in anaplastic large-cell lymphoma Characterization and diagnostic application of genomic NPM-ALK fusion sequences in anaplastic large-cell lymphoma

Minimal disseminated and residual disease (MDD/MRD) analyzed by qualitative PCR for NPM-ALK fusion transcripts are validated prognostic factors in pediatric ALK-positive anaplastic large cell lymphoma (ALCL). Although potentially promising, MDD quantification by quantitative real-time PCR in international trials is technically challenging. Quantification of early MRD might further improve risk stratification. We aimed to assess droplet digital PCR for quantification of minimal disease in an inter-laboratory setting in a large cohort of 208 uniformly treated ALCL patients. Inter-laboratory quality control showed high concordance. Using a previously described cut-off of 30 copies NPM-ALK/10⁴ copies ABL1 (NCN) in bone marrow and peripheral blood, MDD quantification allowed identification of very high-risk patients (5-year PFS% 34 ± 5 for patients with ≥30 NCN compared to 74 ± 6 and 76 ± 5 for patients with negative or <30 NCN, respectively, p < 0.0001). While MRD positivity was confirmed as a prognostic marker for the detection of very high-risk patients in this large study, quantification of MRD fusion transcripts did not improve stratification. PFS% was 80 ± 5 and 73 ± 6 for MDD- and MRD-negative patients, respectively, versus 35 ± 10 and 16 ± 8 for MRD-positive patients with <30 and ≥30 NCN, p < 0.0001. Our results suggest that MDD quantification by dPCR enables improved patient stratification in international clinical studies and patient selection for early clinical trials already at diagnosis.

Minimal residual disease (MRD) detection is established routine practice for treatment stratification in leukemia and used for treatment optimization in adult lymphomas. Minimal disease studies in childhood non-Hodgkin lymphomas are challenged by stratified treatment in different subtypes, high cure rates, low patient numbers, limited initial tumor material, and early progression. Current clinical applications differ between the subtypes. A prognostic value of minimal disseminated disease (MDD) could not yet be clearly established for lymphoblastic lymphoma using flow cytometry and PCR-based methods for T-cell receptor (TCR) or immunoglobulin (IG) rearrangements. MYC–IGH fusion sequences or IG rearrangements enable minimal disease detection in Burkitt lymphoma and -leukemia. An additional prognostic value of MDD in Burkitt lymphoma and early MRD in Burkitt leukemia is implicated by single studies with risk-adapted therapy. MDD and MRD determined by PCR for ALK-fusion transcripts are independent prognostic parameters for patients with ALK-positive anaplastic large cell lymphoma (ALCL). They are introduced in routine clinical practice and used for patient stratification in clinical studies. Early MRD might serve as an endpoint for clinical trials and for guiding individual therapy. Validation of MDD and MRD as prognostic parameters is required for all subtypes but ALCL. Next-generation sequencing-based methods may provide new options and applications for minimal disease evaluation in childhood lymphomas.

Summary: Frequency, distribution and prognostic meaning of ALK‐partner genes other than NPM1 in ALK‐positive anaplastic large‐cell lymphoma (ALCL) are unknown. Forty‐nine of 316 ALCL diagnosed in the NHL‐BFM study group showed no nuclear ALK expression suggestive of a variant ALK‐partner; 41 were analysed by genomic capture high‐throughput sequencing or specific RT‐PCRs. NPM1::ALK was detected in 13 cases. Among the 28 patients with a non‐NPM1::ALK‐fusion partner, ATIC (n = 8; 29%) and TPM3 (n = 9; 32%) were the most common. Five of eight patients with ATIC::ALK‐positive ALCL relapsed, none of nine with TPM3::ALK. Variant ALK‐partners are rare and potentially associated with different prognoses.