Donor‐derived gene mutations in sex chromosome loss after stem cell transplantation

Sex chromosome loss (SCL) in blood cells is often observed in an age-dependent manner. The loss of chromosome Y (LOY) in older men was associated with various diseases and shorter survival. In sex-mismatched allogeneic stem cell transplantation (SCT), sex chromosome fluorescence in situ hybridization (FISH) is the established method for chimerism analysis. However, the 45,X karyotype (i.e., loss of an X chromosome in women or LOY in men) can occur after SCT and can be derived from the donor or the recipient. If the SCL occur in the recipient cells, it may be associated with relapse. Given the poor prognosis of post-transplant recurrence, the origin of SCL is clinically essential. Clonal hematopoiesis has been reported in older individuals and SCT recipients. However, most studies documented single-nucleotide substitutions and indels, and the interplay between gene mutations and chromosomal aberrations has rarely been analyzed. Frequent mutations were reported in patients with a high proportion of LOY, but it remains primarily unknown in the post-transplant setting. The aim of this retrospective study was to clarify the origin of SCL cells and investigate the relationship between SCL and gene mutations. This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Tokyo Metropolitan Komagome Hospital (approval number 2220). Written informed consent for mutational analyses was obtained from each patient. In this study, we report four cases of SCL without relapse among 414 recipients who underwent sex-mismatched SCT at our hospital from 2008 to 2019. No sex chromosome aberrations were found before SCT. The frequency of FISH testing was four times for the first year and left to the discretion of their physicians thereafter. The FISH analyzed 500 cells per assay, and only patients with repetitively detected SCL were included in this study. The patient characteristics are described in Table I. There were three women and one man, and the age at SCT was 22, 37, 42, and 68 years old. After SCT, complete donor-chimera was confirmed by sexchromosome FISH in all patients. Then, the SCL cells were identified using the FISH analyses (Fig S1A,B). The interval from SCT to SCL detection was 3 2, 6 4, 11 7, and 15 2 months, and the frequency of SCL cells during the first detection was 0 6% in three and 3 2% in one. In all cases, SCL cells persisted throughout the clinical course, and the frequency during the last follow-up was 1 4%, 2 8%, 4 0%, and 45 0%. Three patients developed mild thrombocytopenia during SCL detection, but the blood cell counts gradually improved. However, pancytopenia developed in case 1 (Fig 1A). The follow-up period after SCL detection was 32 2, 34 3, 41 8, and 133 9 months. All four survived without relapsing, and no patient was diagnosed with a hematological malignancy after SCT (Fig S1C–F). In case 2, the reversetranscriptase polymerase chain reaction for TCF3-PBX1 remained negative repeatedly. DNA was extracted from peripheral blood when SCL was detected in each patient and 1 or 2 years after the first sampling. Next generation sequencing (NGS) targeting 68 myeloid malignancy-associated genes was performed with AmpliSeq for Illumina Myeloid Panel and On-demand Panel using a MiniSeq system (Illumina, San Diego, CA, USA). Gene variants were detected by analysis pipeline, as previously described, with Mutect2. Mutations were detected in three out of four patients. The detected mutations included KMT2A S3314Pfs (variant allele frequency [VAF] 23 1%) in case 1, DNMT3A W860R (VAF 4 2%) in case 2, and TP53 C238Y (VAF 5 2%) in case 3. The transition of VAF in each mutation and the frequency of SCL cells in cases 1–3 are shown in Fig 1B. These results showed that the mutations and SCL were persistent. Mutation analyses were also performed using pre-SCT recipient specimens, but the relevant mutations were not detected. Next, single-nucleotide polymorphisms (SNPs) were compared for chimerism evaluation. In the analyses, autosomal variants with a prevalence greater than 5% among East Asians and those of X chromosome with a prevalence greater than 1% were extracted as SNPs. The VAF patterns of the SNPs were compared before and after SCT, and visualized using R. These analyses revealed that the samples, including the SCL cells, did not contain recipient-derived cells (Fig 1C). In our study, the frequent concurrence of gene mutations in SCL patients suggested the close relationship between gene mutations and chromosomal aberrations. It is reported that 63% of cancer patients with mosaic chromosomal alterations in blood cells also had gene mutations. The exact mechanism is unclear, but considering the high incidence of leukemia among cancer patients with gene mutations and chromosomal alterations, a detailed medical follow-up is required. Gibson et al. reported that donor-engrafted clonal hematopoiesis was associated with cytopenia after SCT. Likewise, mild cytopenia was seen during the first SCL detection. Correspondence