Monitoring minimal residual disease (MRD) provides important information during treatment of hematologic malignancies. Chimerism analysis also provides key information after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recent advances in next-generation sequencing (NGS) have enabled identification of various mutations and quantification of mutant allele burden. In this study, we developed a new analytic algorithm to monitor chimerism applicable to NGS multi-gene panel in use to identify mutations of myelodysplastic syndrome (MDS). We enrolled patients who were diagnosed with MDS and received allo-HSCT and their corresponding donors. Monitoring MRD by NGS assay was performed using 53 DNA samples by calculating mutant allele burden after treatment. For monitoring chimerism by NGS, we selected 121 single nucleotide polymorphisms (SNPs) after careful stepwise evaluation and calculated average donor allele burden. Data obtained from NGS were compared with bone marrow findings, chromosome analysis and short tandem repeat (STR)-based chimerism. SNP-based NGS chimerism analysis was accurate and even superior to conventional STR method by overcoming the various technical limitations of STR. In addition, simultaneous monitoring of mutation and chimerism using NGS could implement comprehensive pre- and post-HSCT monitoring of various clinical conditions such as complete donor chimerism, persistent mixed chimerism, early relapse, and even donor cell-derived diseases.

Patients with myeloid neoplasms who relapsed after allogenic hematopoietic stem cell transplant (HSCT) have poor prognosis. Monitoring of chimerism and specific molecular markers as a surrogate measure of relapse is not always helpful; therefore, improved systems to detect early relapse are needed. We hypothesized that the use of next generation sequencing (NGS) could be a suitable approach for personalized follow-up post-HSCT. To validate our hypothesis, we analyzed by NGS, a retrospective set of peripheral blood (PB) DNA samples previously evaluated by high-sensitive quantitative PCR analysis using insertion/deletion polymorphisms (indel-qPCR) chimerism engraftment. Post-HCST allelic burdens assessed by NGS and chimerism status showed a similar time-course pattern. At time of clinical relapse in 8/12 patients, we detected positive NGS-based minimal residual disease (NGS-MRD). Importantly, in 6/8 patients, we were able to detect NGS-MRD at time points collected prior to clinical relapse. We also confirmed the disappearance of post-HCST allelic burden in non-relapsed patients, indicating true clinical specificity. This study highlights the clinical utility of NGS-based post-HCST monitoring in myeloid neoplasia as a complementary specific analysis to high-sensitive engraftment testing. Overall, NGS-MRD testing in PB is widely applicable for the evaluation of patients following HSCT and highly valuable to personalized early treatment intervention when mixed chimerism is detected.

Severe aplastic anemia (SAA) is a bone marrow failure syndrome that can be treated with hematopoietic cell transplantation (HCT) or immunosuppressive (IS) therapy. A retrospective cohort of 56 children with SAA undergoing transplantation with fludarabine-cyclophosphamide-ATG-based conditioning (FluCyATG) was analyzed. The endpoints were overall survival (OS), event-free survival (EFS), cumulative incidence (CI) of graft versus host disease (GVHD) and CI of viral replication. Engraftment was achieved in 53/56 patients, and four patients died (two due to fungal infection, and two of neuroinfection). The median time to neutrophil engraftment was 14 days and to platelet engraftment was 16 days, and median donor chimerism was above 98%. The overall incidence of acute GVHD was 41.5%, and that of grade III-IV acute GVHD was 14.3%. Chronic GVHD was diagnosed in 14.2% of children. The probability of 2-year GVHD-free survival was 76.1%. In the univariate analysis, a higher dose of cyclophosphamide and previous IS therapy were significant risk factors for worse overall survival. Episodes of viral replication occurred in 33/56 (58.9%) patients, but did not influence OS. The main advantages of FluCyATG include early engraftment with a very high level of donor chimerism, high overall survival and a low risk of viral replication after HCT.

Activated phosphoinositide 3-kinase delta syndrome 1 (APDS-1) is a recently described inborn error of immunity caused by monoallelic gain-of-function mutations in the PIK3CD gene. We reviewed for the first time medical records and laboratory data of eight Italian APDS-1 patients. Recurrent sinopulmonary infections were the most common clinical feature at onset of disease. Seven patients presented lymphoproliferative disease, at onset or during follow-up, one of which resembled hemophagocytic lymphohistiocytosis (HLH). Genetic analysis of the PIK3CD gene revealed three novel mutations: functional testing confirmed their activating nature. In the remaining patients, the previously reported variants p.E1021K (n = 4) and p.E525A (n = 1) were identified. Six patients were started on immunoglobulin replacement treatment (IgRT). One patient successfully underwent hematopoietic stem cell transplantation (HSCT), with good chimerism and no GVHD at 21 months post-HSCT. APDS-1 is a combined immune deficiency with a wide variety of clinical manifestations and a complex immunological presentation. Besides IgRT, specific therapies targeting the PI3Kδ pathway will most likely become a valid aid for the amelioration of patients' clinical management and their quality of life.

The effects of total body irradiation (TBI) to the capacity of skeletal muscle hypertrophy were quantified using the compensatory muscle hypertrophy model. We additionally assessed the responses of stem and/or progenitor cells in the muscles. A single TBI of 9.0, 5.0 and 2.5 Gy was delivered to C57BL/6 mice. Bone marrow stromal cells were obtained from GFP-Tg mice, and were injected into the tail vein of the recipient mice (1 × 106 cells/mouse), for bone marrow transplantation (BMT). Five weeks after TBI, the mean GFP-chimerism in the blood was 96 ± 0.8% in the 9 Gy, 83 ± 3.9% in the 5 Gy, and 8.4 ± 3.4% in the 2.5 Gy groups. This implied that the impact of 2.5 Gy is quite low and unavailable as the BMT treatment. Six weeks after the TBI/BMT procedure, muscle hypertrophy was induced in the right plantaris muscle by surgical ablation (SA) of the synergist muscles (gastrocnemius and soleus), and the contralateral left side was preserved as a control. The muscle hypertrophy capacity significantly decreased by 95% in the 9 Gy, 48% in the 5 Gy, and 36% in the 2.5 Gy groups. Furthermore, stem/progenitor cells in the muscle were enzymatically isolated and fractionated into non-sorted bulk cells, CD45-/34-/29+ (Sk-DN), and CD45-/34+ (Sk-34) cells, and myogenic capacity was confirmed by the presence of Pax7+ and MyoD+ cells in culture. Myogenic capacity also declined significantly in the Bulk and Sk-DN cell groups in all three TBI conditions, possibly implying that skeletal muscles are more susceptible to TBI than bone marrow. However, interstitial Sk-34 cells were insusceptible to TBI, retaining their myogenic/proliferative capacity.