Prognostic Impact of Minimal Disseminated Disease By Digital PCR for Pediatric ALK-Positive Anaplastic Large Cell Lymphoma

[Introduction] Anaplastic large cell lymphoma (ALCL) is a rare type of peripheral T-cell lymphoma. According to anaplastic lymphoma kinase (ALK) expression, the World Health Organization classifies ALCL into two subtypes: ALK-positive and ALK-negative. In children, more than 80% of ALK-positive ALCLs are associated with the t(2;5)(p23;q35) translocation, which results in expression of the NPM-ALK fusion gene. The detection of NPM-ALK transcripts at diagnosis by quantitative real-time PCR (RQ-PCR) in peripheral blood (PB) or bone marrow (BM), defined as minimal disseminated disease (MDD)-positive, is associated with poor prognosis. However, RQ-PCR requires standard curve calibration, and thus, the assay results cannot be compared between multiple laboratories. Recently, the use of digital PCR (dPCR) has been reported for the reproducible quantification of fusion gene transcription, including NPM-ALK transcripts, in ALK-positive ALCL. This study aimed to confirm the prognostic impact of MDD analyzed by dPCR in children with ALK-positive ALCL. Although approximately 90% of patients with ALK-positive ALCL have NPM-ALK fusions, other variant translocations are rarely observed. Thus, we assessed NPM-ALK and universal ALK dPCR based on 3'ALK transcript amplification, to detect NPM-ALK fusion and other ALK translocation variants, according to a previous report.

[Methods] Patients aged < 20 years with ALCL who were enrolled in the ALCL99 trial in Japan between January 2000 and April 2012 were eligible for our study. ALK-positive ALCL was confirmed by a central pathological review of all patients. NPM-ALK and 3'ALK transcripts at diagnosis were analyzed and quantified using RQ-PCR and dPCR in 50 BM and 52 PB samples from 61 patients. NPM-ALK and 3'ALK copy numbers in RQ-PCR and dPCR were calculated per 10,000 copies of the ABL reference gene (normalized copy number; NCN).

[Results] NPM-ALK NCNs detected by dPCR and RQ-PCR were highly concordant in 101 samples (r =0.953). NPM-ALK MDD was analyzed in 60 patients. In the RQ-PCR assay of NPM-ALK, the 3-year progression-free survival (PFS) rate of 24 patients with more than 10 NCN was significantly poorer than that of 36 patients with 10 or less NCN (58% vs. 100%, P < 0.001). In the NPM-ALK dPCR assay, we defined more than 30 NCN by dPCR as MDD-positive for NPM-ALK transcripts, and the 3-year PFS rates of 19 MDD-positive and 41 MDD-negative patients were 58% and 95%, respectively (P < 0.001, figure 1). For the analysis of 3'ALK transcripts in dPCR, applying a cut-off of 50 NCN, the 3-year PFS rates of 16 MDD-positive and 45 MDD-negative patients were 50% and 93%, respectively (P < 0.001, figure 2).

[Conclusion] NPM-ALK MDD assessment using dPCR, as well as MDD assessment using RQ-PCR, is useful as a prognostic factor at diagnosis in pediatric ALK-positive ALCL. MDD assessment by dPCR does not require standard curve calibration, and its reproducibility might be higher than that of RQ-PCR. Our findings suggest that 3'ALK MDD assessments will be beneficial for patients with ALK-positive ALCL, because 3'ALK MDD can be assessed without confirming ALK fusion partners by karyotyping of the lymphoma tissue. Stratification of patients with ALK-positive ALCL based on MDD using dPCR can be beneficial for the establishment of new standard treatments.

Fukano:Pfizer: Research Funding. Mori:Pfizer: Research Funding; Chugai: Honoraria; Takeda: Honoraria. Horibe:Novartis Japan: Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Speakers Bureau; Amgen Inc: Speakers Bureau; Kyowa Kirin Co.,Ltd.: Consultancy; Pfizer Japan Inc.: Consultancy.