Evaluation of Transplanted Stem Cell Dynamic Variables in the Bone Marrow of Children with Malignancies

Objective: According to risk classifications, the transplant of healthy hematopoietic stem cells in the bone marrow is a priority treatment option for hematopoietic diseases. The effectiveness of hematopoietic stem cell transplantation after bone marrow transplantation is largely dependent on the capacity of the bone marrow microenvironment to accept transplanted cells. Detailed analysis of hematopoietic stem cell kinetics after transplantation is very important, as initial transplantation of hematopoietic stem cells affects prognosis. In this study, hematopoietic stem and progenitor cell percentage values were evaluated together with other transplantation-related parameters to determine the capacity of stem cell dynamic variables in the bone marrow. Methods: Dynamic variables in hematopoietic stem cell fate in bone marrow transplantation include many parameters; hematopoietic stem cell transplantation rates in the apheresis product of 13 patients and cell counts per kilogram and engraftment times, CD34 + cell rates


Introduction
Hematopoietic stem cell transplantation (HSCT) is a medical procedure that infuses stem cells after a short course of chemotherapy, radiotherapy, or both namely a conditioning regimen [1]. Bone marrow transplantation has become a curative therapy for an increasing number of malignant and non-malignant diseases [2]. HSCT was first performed by E. Donnell Thomas in 1957 as a new form of cancer therapy [3]. Although initial attempts were largely unsuccessful, the procedure has improved dramatically over the past decades [4]. Today, more than 50,000 HSCT procedures are performed annually for a variety of malignant and benign diseases worldwide [5]. In an HSCT procedure, a recipient's unhealthy natural bone marrow cells and immune system are replaced with grafted healthy stem cells and immune cells (grafts) after a brief chemotherapy and/or radiotherapy administration. The procedure can eliminate residual cancer by taking advantage of the graft-tumor effect. The primary goal of most transplants is to treat an underlying malignancy or hematological disorder [1]. Detailed analysis of hematopoietic stem cell (HSC) kinetics after transplantation is very important, as initial transplantation of HSCs affects prognosis. Although the number of autologous and allogeneic stem cell transplantations is increasing, relatively little information is available about recovery after transplantation. The adult bone marrow (BM) niche is a complex space in which the dynamic homeostatic and hematopoietic system is converged between different cellular and non-cellular factors. Signaling mechanisms triggered by cell-cell, cellextracellular matrix, cell-cytokine interactions, and local microenvironment parameters play a role in the self-renewal and differentiation of stem cells and the control of the migration of hematopoietic stem and progenitor cells (HSPCs) [6]. It is also an important question to what extent the amounts given during bone marrow transplantation (BMT) of HSCTs, which have significant potential in many ways controlled by the dynamics of the BM niche, affect the transplantation. In their study, Carroll, et al., stated that the in vitro leukemic niche they created for understanding the molecular events responsible for the functional failure of HSCT may be useful for therapeutic evaluation [7]. In addition, medicinal products based on autologous HSCTs created using lentiviral and gammaretroviral vectors are now approved for clinical use [8]. In this study, to determine the optimal dose of HSCT for rapid and stable engraftment after HSCT, the relationship between engraftment times, which is one of the most important variables of the hematopoietic healing process, and HSCT cells infused during transplantation was analyzed.

Material and method
Peripheral blood progenitor cells (PBPC) were mobilized with granulocyte colony-stimulating factor (G-CSF) alone (10 μg/kg/day SC, Neupogen (R); Amgen, Thousand Oaks, California, USA) and a Spectra Optia Apheresis System-Terumo BCT (Colorado, Colorado, USA) and mononuclear cells including CD34 + cell contents were collected. Immunophenotypic analyses were performed on samples from apheresis products by flow cytometry. Local protocols and techniques routinely used at our center were used for instrument calibration, sample preparation, immunostaining, and data collection [9,10]. CD34 + cells were studied with the BD Stem Cell Enumeration kit ( Engraftment is an important indicator for the evaluation of graft function in the early posttransplantation period, and the following criteria were evaluated and followed up: a) Neutrophil engraftment: The first day when the fragmented neutrophil count is >500/mm³ for 3 consecutive days. b) Platelet engraftment: The first day when the platelet count is >20.000/mm 3 for 3 consecutive days without platelet support for 7 days. c) Erythrocyte engraftment: The first day when the reticulocyte count is >60.000/mm³ for 3 consecutive days.
Measurements were made on the Sysmex XN-1000 SA-01 hemogram device to determine neutrophil, platelet, and erythrocyte engraftment times.

Result
In this study, 13 pediatric patients aged between 2 and 15 years underwent 18 HSCT procedures for acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), lymphoma, and neuroblastoma diseases (able 1). After relapse, 2 nd HSCT was performed in 1 of the patients and 3 rd HSCTs were performed in 2 of the patients. Due to the recurrence of malignancies in 3 of the patients, 2 nd and 3 rd HSCTs were performed after relapse. To analyze the relationships between variables affecting HSCT and HSCT cell values, each transplant of 3 patients with more than one transplantation was considered a different transplantation procedure.
The cumulative incidence of chronic GvHD was determined as 38.8% in our 18 HSCT procedures that make up our case series. In our case series, the mean platelet engraftment time was 19 days ( Figure 1). As observed in Table 1, it has been shown that there is a statistically significant and positive correlation between the percentage and number of HSCT (M=0.29, SE=0.16; M=2.57 × 10 6 , SE=1.83 × 10 6 ) and the platelet engraftment time (M=19.25, SE=3.62).   According to the results of the Pearson correlation analysis (Nonparametric Correlations), a strong positive and significant relationship was found between the HSPC number per kg (M=2.57 × 10 6 , SE=1.83 × 10 6 ) and the HSPC percentage values (M=0.29, SE=0.16) of the patients (r=.733, p<.05).
According to the results of the analysis, there was a moderately significant and positive relationship (r=.631, p<.05) between HSPC percentage and platelet engraftment time (M=19.25, SE=3.62), while erythrocyte engraftment time (M=23.06, SE=1.53) and the existence of a strong inverse relationship (r=-.730, p<.01).
In this study, the mean erythrocyte engraftment time was determined to be 23 days in a series of 16 cases (Figure 2). The median CD34 + cell dose of all patients in allogeneic transplants in our study was 5.8 × 10 6 /kg (range 1.5 to 27.2). The median platelet engraftment time of the patients was 15.5 (range 11 to 71) days, and the median erythrocyte engraftment time was 21.5 (range 16 to 35) days.
A strong positive and significant correlation was found between the number of HSPC per kilogram infused into patients (M=2.57 × 10 6 , SE=1,83 × 10 6 ) and platelet engraftment times (r=.780, p<.05). No correlation was noted between neutrophil engraftment time, WBC, and numbers and percentages of HSPC infused per kilogram.

Discussion and conclusion
This study confirms that G-CSF-mobilized PBPC provides rapid short-term and longterm platelet engraftment in pediatric patients undergoing autologous and allogeneic transplantation if the HSPC dose is infused on a mean of ≥ 2.57 × 10 6 /kg. Since this dose of HSPC appears to have clinical and potential economic implications, it may be considered the optimal dose for apheresis. It is useful to determine the average value of the optimum HSPC dose by increasing the number of patients to answer the question of how much the amounts of HSPCs controlled by the dynamics of the BM niche affect the transplantation.
Ringden O, et al., the mean time to platelet engraftment was reported to be 23 days in the PBSC transplant patient group [11]. In the study, this period is 19 days. The study is important in terms of revealing the possible relationship between the shortening of platelet engraftment time and the number and percentage of HSPC in transplant patients.
It is also important that a statistically significant inverse relationship was determined between HSPC values and mean erythrocyte engraftment times. It would be beneficial to examine the relationship between these factors in more detail and in a high number of patient populations. Kulkarni U, et al., showed in their study that the median time to platelet engraftment was 17 days (range, 10 to 44) in patient groups whose median CD34 cell dose was 4.87 × 10 6 /kg [12]. In our study, the median CD34 + cell dose was 5.8 × 10 6 /kg (range 1.5 to 27.2) while the median platelet engraftment time was 15.5 (range 11 to 71). A study shows, the pulling forward of the median platelet engraftment time may also be associated with an increase in the dose of median CD34 + cells and HSPC. It may positively affect platelet engraftment, especially in the allogeneic transplant process.