A group of 27 children aged 1.5 - 19 years (median 9.6 years) was referred to the Stem Cell Transplantation Centre of the University Children’s Hospital in Krakow and was included in this study. The patients were assessed twice—before HSCT (pre-HSCT group) and approximately 6 months after HSCT (post-HSCT group). Diseases that were the indication for HSCT are listed in Table 1. Patients with malignancies, except for juvenile myelomonocytic leukemia (JMML), were referred for HSCT in complete remission. Characteristics of the transplantation procedures are detailed in Table 2.
In more than half of the patients (16 patients, n=27) a conditioning regimen was based on Busulfan/Treosulfan. Total body irradiation (TBI) was used in 7 of patients, 4 patients received regimen based on Cyclofsphamide. Most patients (85%) in whom graft-versus-host disease (GvHD) prophylaxis was used received methotrexate combined with cyclosporine. Mucositis was diagnosed in 82% cases (22 patients), grade III and IV mucositis in 26% (7 patients). The key clinical data of the HSCT recipients are presented in Table 3. Mucositis requiring parenteral nutrition was found in almost half (48%) of the patients. Systemic glucocorticoids were used in 19 children in the post-HSCT group to treat complications of HSCT. In 11 of patients aGvHD was seen, including intestinal involvement in one. According to the aGvHD grader (agvhd.com), grade II and III aGvHD was found in 22% cases (6 patients). In two cases multiple locations of aGvHD occurred (II/C - skin+liver, III/C - skin+GI+liver). The patients with aGvHD were treated with additional immunosuppressive agents, including tacrolimus, mycophenolate mofetil, and etanercept. Six months after HSCT, four children still received tapered doses of immunosuppressive agents other than glucocorticoids. The control group consisted of 11 boys and 15 girls aged 4.3 to 16.0 years (median 12.2 years). The control children were recruited among family donors, siblings of patients treated with HSCT, and unrelated healthy children. They all had negative medical history, no signs or symptoms of acute or chronic diseases, and no abnormalities in laboratory tests (CBC, serum ALT, and creatinine levels).
Height and body weight measurements were performed by an anthropometrist. Body mass index (BMI), BMI percentile (BMIPerc) and BMI SDS (BMISDS) were calculated using online WHO BMI calculators . The results were compared to regional reference values, and the reference values were published by the WHO [18, 19, 17]. The BF mass and BF% were measured using bioimpedance and calculated according to the method described by Kushner RF and Schoeller DA .
Fasting blood samples were collected in the morning. Patients treated with HSCT were assessed immediately before conditioning and after a median of 6.3 months after HSCT. In the control group samples were obtained once, after enrollment to the study. Blood samples were collected in EDTA and aprotinin tubes, (Becton-Dickinson; UK), and tubes with no anticoagulants. The tubes were delivered to the laboratory immediately and centrifuged for 15 minutes at 3000 rpm using a horizontal rotor. Serum and plasma samples were stored at -80oC until the time of measurement. Subsequently, mononuclear cells were separated for microarray followed by total RNA extraction.
Plasma concentrations of the peptides were measured using EIA kits: ghrelin, CCK, GLP-1 (Phoenix Pharmaceuticals, Inc., USA), and FGF-21 (Millipore Corporation, USA). The sensitivity of the methods are provided by kit suppliers and are as follows: ghrelin – 0.08 ng/ml (14% intra- assay and 5% interassay variability),CCK – 0.06 ng/ml (5% intra- assay and 9% interassay variability), GLP-1 – 0.18 ng/ml (14% intra- assay and 5% interassay variability), FGF-21 – 0.016 ng/ml (5.8% intra- assay and 9% interassay variability).
Microarray analysis used a GeneChip Human Gene 1.0 ST Arrays (Affimetrix, Santa Clara, USA) according to the manufacturer’s protocol. GLP-1 expression data were not available in the Affimetix database, and thus we checked the results of GLP-1 receptor gene expression. Gene loci and Affimetrix codes of the tested peptides are presented in Table 4.
All the primary microarray data were submitted to GEO public repository and are accessible using GEO Series accession number GSE69421 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69421). In our study a part of submitted microarray data was used.
Continuous clinical and biochemical variables are presented as the mean values and standard error or as the median values and quartiles, as appropriate. Categorical variables are presented as frequencies and percentages. The Shapiro-Wilk test was used to assess the normal distribution of the continuous variables. To examine the differences between two or more independent groups, ANOVA/Student's t-test (for variables with normal distribution) or Kruskal-Wallis/Mann-Whitney tests (for variables with non-normal distribution) were used. To assess the correlations between two continuous variables, Spearman rank correlation coefficient was calculated. The two-sided p-values < 0.05 were considered statistically significant. Gene expression data were RMA-normalized and presented as the mean and standard deviation. ANOVA was used to examine the differences in gene expression between two independent groups. The Benjamini-Hochberg (B-H)-corrected p-values < 0.05 were considered statistically significant. The statistical analyses were performed using the R 3.4.3 software.
The Permanent Ethical Committee for Clinical Studies of the Jagiellonian University Medical College approved the study protocol. All parents, adolescent patients, and adult patients signed a written informed consent before blood sample collection. Study was conducted in accordance with the Declaration of Helsinki.