Molecular landscape of acute promyelocytic leukemia at diagnosis and relapse.

Acute promyelocytic leukemia (APL) occurs in about 530% of adult de novo AML and is characterized by the presence of the chromosomal translocation t(15;17)(q24;q21). The resulting PML-RARA chimeric gene involves the retinoic acid receptor alpha-(RARA) gene on chromosome 17 and the PML gene, a putative transcription factor, on chromosome 15. The resulting PMLRARA chimeric protein is crucial to the pathogenesis of APL as it is thought to contribute two oncogenic hits in one: the block of differentiation and the aberrant selfrenewal of APL cells. Since the introduction of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) for the treatment of APL, the overall survival rate has improved dramatically. However, relapse/refractory patients showing resistance to ATRA and/or ATO are still recognized as a clinically significant problem. It is likely that concurrent genetic aberrations at initial diagnosis predispose patients to relapse or that additional genetic lesions acquired during the course of APL lead to treatment resistance. Genetic mutations resulting in amino acid substitution in the RARA ligand binding domain (LBD) and the PML-B2 domain of PML-RARA, respectively, have been reported as molecular mechanisms underlying resistance to ATRA and ATO. In the presence of LBD mutation, binding of ATRA to LBD is generally impaired, and ligand-dependent co-repressor dissociation and degradation of PMLRARA by the proteasome pathway, leading to cell differentiation, are inhibited. Mutations in the PML-B2 domain affect direct binding of ATO with PML-B2, and PML-RARA SUMOylation with ATO followed by multimerization and degradation is impaired. The aim of the study was to evaluate the mutational spectrum of APL both at initial diagnosis and relapse, and identify potential genetic defects leading to treatment resistance. We analyzed a cohort of 123 adult de novo APL cases including 14 cases who showed hematologic or molecular relapse. Patient characteristics are given in Table 1. All patients were proven to have t(15;17)(q24;q21)/PMLRARA by chromosome banding analysis (CBA) (n=113), fluorescence in situ hybridization (n=119), and/or RT-PCR (n=123). Sixty-nine patients were male and 54 patients were female, with a median age of 47 years (range: 16-84 years). All 123 samples were analyzed by next generation sequencing (NGS) using a 26-gene panel targeting ASXL1, CBL, CSF3R, DNMT3A, ETNK1, ETV6, EZH2, FLT3-TKD, GATA2, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1 and ZRSR2. Since mutations in the genes ARID1A and ARID1B were recently described in APL, NGS analysis of these genes was also included in this study. NGS was performed using the Fluidigm Access Array microfluidic chip system (Fluidigm, San Francisco, CA) in combination with MiSeq instrument (Illumina, San Diego, CA). Variants which cannot be assigned as mutation or SNP according to current knowledge were excluded from further studies (n=26)(for evaluation of variants see Online Supplementary Methods). FLT3-ITD was analyzed by gene scan. Relapse samples were additionally screened for mutations within the PML-RARA fusion transcript by direct Sanger sequencing as previously published. The patient cohort analyzed in the present study is unique and does not overlap with study cohorts described in previous evaluations. All patients gave their written informed consent for scientific evaluations. The study was approved by the Internal Review Board and adhered to the tenets of the Declaration of Helsinki. According to risk stratification of APL (Sanz index), 37/123 patients (30%) were stratified as high risk, 32/123 (26%) as intermediate risk, and 36/123 (30%) as low risk. Of the cases, 18/123 had no blood counts available for scoring, 67/123 cases (54%) had M3 subtype, and 55/123 (46%) had M3v subtype. In one case, cytomorphology was not available. Regarding treatment, 88/123 cases (72%) received ATRA in combination with chemotherapy, 12/123 cases (10%) received ATRA + ATO, and 10/123 cases (8%) received chemotherapy only. In 13 cases (10%), no detailed information about treatment was available. Using standard CBA, additional cytogenetic aberrations were observed in 44/113 patients (27%). The most frequent secondary chromosome aberration was trisomy 8 (14/44; 32%) (figure 1A). Using NGS, 82/123 patients (67%) had at least one mutation in addition to PML-RARA, and 22/82 (27%) had