ALK positive anaplastic large cell lymphoma: Molecular diagnosis and minimal residual disease monitoring

Abstract

Background: ALK positive anaplastic large cell lymphoma (ALCL) is characterized by expression of the anaplastic lymphoma kinase (ALK), frequently associated with the t(2;5)(p23;q35), fusing the ALK and nucleophosmin (NPM) genes. In the remaining cases immunohistochemically (IHC) proven ALK+ALCL, the ALK gene was shown to have various translocation partners. Methods: In our study molecular diagnostics included detection of the most frequent fusion geneNPM/ALK by PCR.NPM/ALK negative tumorswere analyzed by ALK specific Rapid Amplification of 5'cDNA Ends (5'RACE).Molecular findings were correlated with IHC localization of ALK protein, I-FISH andwith expression of the 3'end of ALKmRNAusing quantitative PCR (Q-RT-PCR).Wemonitoredminimal residual disease (MRD) usingQ-RT-PCR of the 3'end of ALK mRNA. 5'RACE is amethod for detection of unknown translocation partners of a fusion gene. We prepared Q-RT-PCR assay for the quantitative assessment of quantification of 3'end ALKmRNA, and housekeeping gene. Results: We analysed ALK+ALCL from 76 patients (1-80 years, median 17 years). Chromosomal breakpoints affecting the ALK locus were detected by I-FISH in all investigated tumors. The NPM/ALK was detected in 57/76 patients. 5'RACE identified ATIC/ALK in 7 patients, CLTC/ALK in 2 patients. Other fusion genes (TPM4/ALK, TPM3/ALK, ALO17/ALK, MYH9/ALK) were each found separately in single individual patients. In all specimens, overexpression of 3'end ALK mRNA suitable for the MRD detection was found (median 69462 copies). We evaluated MRD by the monitoring 3'end of ALK mRNA levels in 430 residual samples (149x bone marrow, 269x peripheral blood) from 31 ALK+ ALCL patients with median follow-up time 798 days. Levels of 3'end ALK mRNA showed a good correlation with clinical course of the disease. Conclusions: Molecular diagnostics allows for detecting translocation partners of ALK gene. Q-RT-PCR based analysis of 3'end ALK mRNA is a promising and rapid approach for diagnostics and MRD monitoring of patients with ALK+ ALCL.