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Letters to the Editor
Figure 1. Flowchart of sequencing and filtering methods to identify and prioritize variants. Post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) samples were matched against their donor peripheral blood (PB) and previous post allo-HSCT samples to identify the acquisition of mutations along post allo- HSCT period until donor cell myeloid neoplasm (DCMN) development. Those somatic variants detected in coding region and donor or acceptor splicing zone, mis- sense, nonsense and frameshift variants with minor allele frequency (MAF) <0.01 were further studied. Copy number variation (CNV) analysis was performed in all samples. To perform the donor analysis a cancer-associated gene list was compiling, the list comprised of a total of 2197 genes and variants detected in cod- ing or splice-site, with variant allele frequency (VAF) between 0.45-0.55, MAF <0.01 were further studied. BWA: Burrows-Wheeler Aligner; COSMIC: Catalogue Of Somatic Mutations In Cancer; HGMD: Human Gene Mutation Database). 1See Online Supplementary Tables S2 and S3. 2See Figure 2, Online Supplementary Table S1 and Online Supplementary Figures S1-S9.
donor stem cells were also detected in all the correspon- ding follow-up samples after allo-SCT. Based on the vari- ant allele frequency (VAF), all variants seem to have germline origin except for those in MOS (donor 5) and that in SETBP1 (donor 6), which appear be of somatic ori- gin. Unfortunately, germline samples were not available to confirm this observation.
Interestingly, although acquired or inherited genetic alterations, which might be related to a predisposition to cancer, were observed in all donors, none of them had developed overt neoplasia at the moment of DCMN diag- nosis in the recipients.
Considering the results in patients and donors described above, we propose a plausible model of leukemogenesis for each case, with progressive emergence of mutations in donor cells related to the development of AML or MDS after allo-HSCT (Figure 2). In cases 2, 4, 6 and 7, in which the median time to DCMN diagnosis was 21.5 months, the acquisition of mutations and the evolution of the leukemic clone occurs early after allo-HSCT, a period char- acterized by a marked immunosuppression state in the patients. Moreover, these four patients had received con- ditioning regimens based on chemotherapy combined with total body irradiation (TBI) and/or timoglobulin, which would also contribute to the development of MN.
The present study shows that MN evolve by an iterative
process of genetic diversification derived from clonal selec- tion and expansion that begin before the clinical onset, in accordance with the current multistep pathogenesis model of leukemogenesis.8 After the initiating mutation, malig- nant clones evolve and accelerate disease progression through the acquisition of new mutations. Although no clear pattern of clonal mutation acquisition has been observed, initiating mutations appear to affect epigenetic regulators of transcription (DNMT3A, TET2) or genes involved in intracellular signal transduction (SNX13, RHPN2, IRS1, CSF3R).
Different factors can influence the development of a neoplasm in donor cells. Interestingly, cytogenetic abnor- malities involving chromosome 7 were the most frequent in our DCMN cohort (6 of 7 patients). The frequency of - 7 is particularly high among therapy or radiation-induced MDS or AML,9 as well as TP53 and epigenetic modifying gene somatic mutations,10 as in 4 out of 7 patients. In the present cohort, 6 of 7 patients received an alkylating agent or antimetabolites within the conditioning regimen. Furthermore, four of them also received ionizing radiation therapy, and the time of leukemogenic transformation in these patients was shorter than in those who only received chemotherapy. Due to the high prevalence of chromo- some 7 anomalies in this entity (a characteristic previously observed by others authors2), it seems that the residual
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