Abstract
Introduction
Therapy related myeloid neoplasms account for 10 to 20% of all malignant myeloid malignancies. Several cytotoxic agents have been implicated in the development of therapy related myelodysplastic syndromes (t-MDS) including alkylating agents, topoisomerase inhibitor, antimetabolite and radiotherapy. Acquired mutations and cytogenetic abnormalities between therapy and diagnosis of t-MDS are more frequent and confer poor prognosis. Using Azacitidine (AZA) therapy, t-MDS had a similar response rate but significantly shorter overall survival (OS) (Bally et al. Leuk Res 2013). Comprehensive mutation profiling has become essential to reveal molecular aberrations and follow the underlying clonal evolution. Here we focused on the clonal evolution pattern in post radiotherapy AML patients.
Material and methods
DNA was isolated from bone marrow (BM) using the QIAamp DNA blood mini Kit (Qiagen) according to the manufacturer's protocol in 7 t-MDS patients at diagnosis and during AZA courses. A total of 16 samples were sequenced with Ion Torrent Personal Genome Machine (PGM) on 316 v2 Chips. We used the Ion AmpliSeq™ AML Cancer Research Panel comprising 237 amplicons to analyze 19 genes implicated in AML (entire coding regions: CEPBA, DNMT3A, GATA2, TET2, TP53 ; hotspot regions: ASXL1, BRAF, CBL, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1, WT1). Sanger sequencing analysis of ASXL1 DupG (c.1934dupG) was performed in parallel. Sequence alignment and mutation calling were performed using ion reporter software (v 5.4). Candidate mutations with a variant allele frequency (VAF) > 2% were selected.
Results
Seven t-MDS patients previously treated by radiotherapy for breast (n=6) and thyroid (n=1) cancers were included in this study. Median age was 73 years (range, 56-82). Four and 3 patients were high and very high risk respectively according to IPSS-R. Median AZA cycles was 9 (range, 3-13). Response rate was 57% and median OS was 13 months (range, 8-22). A total of 17 pathogenic or potentially pathogenic variants was found, with a median of one variant per patient (range 0-3), and a VAF ranging from 4.5% to 78.6% (mean = 30%). Sequencing and cytogenetic data are summarized in Table 1. We observed 3 different situations. First, we found TP53 mutation in patients #1 and #3. Under AZA course, we observed a disappearance of TP53 mutation correlated with complete remission. In patient #1, relapse was associated with recurrence of TP53 mutation. As already known, patient #1 and #3 had complex karyotype in association with TP53 mutation. This observation confirmed efficiency of AZA in TP53 mutated patient and could be a good strategy to prepare to allo stem cell transplantation. Second, we observed in patients #5 and #6 a disappearance of PTPN11 and DNMT3A mutations whereas we showed an increase of VAF in ASXL1 mutation patient #2. This observation suggested that AZA had selective effect depending to mutational status at baseline. Nevertheless, this selective effect was not associated with response. Third, in patients #2, #4, #5, #6 and #7, we observed emergence of new mutated clones during AZA courses: TET2 (2), WT1 (1), IDH1 (1), NRAS (2), ASXL1 (1), RUNX1 (1), GATA2 (1) and DNMT3A (2). This observation could explain cytopenias persistence or progression in these patients and suggested clonal selection of AZA-resistant clones.
Conclusion
These data confirmed molecular complexity of t-MDS. We observed AZA positive effect on TP53 mutation correlated with AZA response. We suggested clonal selection during AZA courses. Molecular profile before and during AZA treatment could be of interest to define which combination or targeted therapies could be used. Five others post alkylating agent t-MDS are ongoing and will be presented at the meeting.
No relevant conflicts of interest to declare.
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