The research encompassed biological material (peripheral blood) derived from 56 consecutive children diagnosed with acute lymphoblastic leukaemia who had undergone allogeneic haematopoietic stem cell transplantation at the Department of Paediatric Haematology, Oncology and Transplantology of Medical University in Lublin between 2002 and 2018. The patients’ characteristics are summarized in Table 1.
All patients were conditioned according to the European Bone Marrow Transplantation (EBMT) guidelines . Conditioning was myeloablative (MAC), and standard regimens were based on fractionated total body irradiation (FTBI) or busulfan. In reduced toxicity conditioning (RTC), treosulfan was used instead of busulfan. Cyclosporine was used as a graft versus host disease (GvHD) prophylaxis. Matched unrelated transplant recipients received anti-thymocyte globulin (ATG) to prevent GvHD. Mismatched related transplant recipients received ex-vivo T-cell depleted grafts. Engraftment was diagnosed when an absolute neutrophil count (ANC) of 500 or more was observed for 2 days.
Very early chimerism was evaluated from peripheral blood (PB) on days +7, +14, +21 and +28. Next, samples were collected, and chimerism was monitored according to the EBMT guidelines as a part of the routine follow-up post allo-HSCT [9, 10]. Depending on clinical indications, chimerism was monitored irrespective of the scheduled time points. A previously described STR PCR method that has been standardized in our laboratory based on Eurochimerism recommendations was used for chimerism assessment [11, 12]. The sensitivity of our method for detecting recipient cells was 1%, but patients with verified 1% autologous cells in 2 consecutive samples were considered mixed chimeras. Early mixed chimerism was determined as the presence of 1% or more recipient cells in peripheral blood.
The research material consisted of genomic DNA isolated from mononuclear peripheral blood cells. Peripheral blood was collected into anticoagulant (EDTA)-containing tubes. The isolation of the mononuclear cell (MNC) fraction was performed using Ficoll-Paque PLUS aqueous solution of 1.077 + 0.001 g/ml density (Amersham Biosciences, Inc., Piscataway, NJ, USA). DNA isolation was performed with the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). After extraction, DNA was eluted from the column with 60-70 ml of elution buffer. The quantity of DNA was determined with a Nanophotometer (Thermo Fisher Scientific, Waltham, MA, USA). According to the manufacturer’s recommendations, 1-2,5 ng of matrix DNA was used with the commercial AmpFISTR NGM Plus Kit (Thermo Fisher Scientific, Waltham, MA, USA). PCR was performed according to the manufacturer’s instructions, which were as follows: 95°C for 11 min, followed by 28 cycles at 94°C for 20 sec, 59°C for 3 min, with an additional elongation step for 10 min at 60°C (ProFlexTM PCR System, Thermo Fisher Scientific, Waltham, MA, USA) and a final hold at 4°C. The following loci were amplified: D10S1248, vWA, D16S539, D2S1338 labelled with 5-FAM, Amelogenina X/Y, D8S1179, D21S11, D18S51 labelled with VIC, D22S1045, D19S433, THO1, FGA labelled with NED and D2S441, D3S1358, D1S1656 and D12S391 labelled with PET. Separation and detection of the PCR products were performed on a Genetic Analyzer 3500 (Thermo Fisher Scientific, Waltham, MA, USA). Gene Mapper® ID-X software (Thermo Fisher Scientific, Waltham, MA, USA) was used to automatically determine the size of the amplified fragments.
Statistical analysis was performed using SPSS IBM Statistics (Version 24) and XLSTAT 2019 1.3. Non-parametric tests (Pearson’s Chi-square, chi-square test with simulating p values – test insensitive to small numbers, Kruskal-Wallis) were used for group comparison. OS and EFS were estimated using Kaplan-Meier method and Log-rank tests. Cumulative incidence of relapse was performed using STATA. Statistical significance was considered < 0.05.
The study was approved by the Ethics Committee of the Medical University of Lublin.