This study included one hundred and thirty Egyptian children who were newly diagnosed to have acute lymphoblastic leukemia (ALL). The study was approved by medical research ethical committee of our university hospitals during the period from January 2014 to December 2018. The study was done in the departments of pediatrics, clinical pathology, neurology, rheumatology, and radiodiagnosis. Assessments of the patients were performed twice: at inclusion into the current study (the first assessment) and after 3 years (the second assessment), but only one assessment (at the start of the study) was done for genotyping for all the participants in this study. Data of the patients who did not complete this study (20 patients were lost during follow-up and 10 patients died) were eliminated. So, this study included 100 patients (58 girls and 42 boys), “the patients’ group.” Patients’ mean age/years (± SD) were 11 years (± 4). Patients with hormonal therapy or central nervous system irradiation were excluded. Other criteria for exclusion were allogeneic bone marrow transplantation or stem cell transplantation; patients with history of any serious gastrointestinal problems, malabsorption, and liver diseases; and those with concomitant medical diseases that may affect bone metabolism (hyperuricemia, syndrome of inappropriate secretion of antidiuretic hormone, type B lactic acidosis, non-islet cell tumor hypoglycemia, and hyperglycemia are other potential metabolic abnormalities occurring in patients with hematological malignancies) were also excluded from the study. None of our patients had history of neurological disorder prior to the onset of ALL.
One hundred apparently healthy Egyptian subjects, age and sex matched with the patients, were included as controls. Their mean age/years (± standard deviation) were 10 years (± 3.5); they were 57 girls and 43 boys. Informed consent was obtained from all subjects’ parents or responsible relative before enrollment. Systematic assessment at inclusion into the study was done to identify the patients who had pre-existing low level of lumbar spine bone mineral density (BMDLS), levels of osteocalcin (OC), and alkaline phosphatase (ALP) {specific markers of bone formation} were also measured [15].
Clinical examination
All the patients were subjected to complete history taking, thorough general and neurological examination. Body mass index (BMI) percentiles were calculated and kids who measure at the 85th to 94th percentiles are considered overweight. A child whose BMI is between the 5th percentile and 85th percentile is in the healthy weight range. A child with a BMI below the 5th percentile is considered underweight [16]. The diagnosis of ALL was made by cytomorphological and immunological examination of blood and bone marrow smears of our local institution. For the diagnosis of ALL, 25% blasts or more in the bone marrow was mandatory. Immunological markers were judged positive if expressed in 20% or more of the malignant cells. All cases were classified as precursor B-ALL. Data at inclusion of the patients into the current study were collected, including the address, sex, date of birth, age at disease onset, peripheral white blood cell (WBC) count, immunophenotyping, neurological examination, states of lymph nodes, liver, spleen, testes, bone marrow aspiration, % blast cells at diagnosis, and bone marrow (BM) status at day 14 of induction, and according to these initial data, the eligibility criteria were put and the type of CCG protocol therapy was assigned (M1 marrow with blast count less than 5%, M2 with blast count 5–25%, and M3 marrow with blast count more than 25%). Risk stratification was based on clinical data, morphological and immunological studies, day 14 bone marrow response, as well as conventional cytogenetic. Standard risk ALL group involved patients with age 1–9.99 years, WBC count < 50 × 109/l, and precursor B immunophenotype. High-risk standard arm group involved patients with age ≥ 10 years and/or WBC ≥ 50 × 109/l. High-risk augmented arm group included patients with neurological disease and/or BM blast day14 > 5% (slow early responders). Standard risk ALL patients were treated according to the CCG 1991 protocol6 using a single delayed intensification (DI) arm. The CCG 1991 protocol and high-risk patients received post-induction intensification therapy [17].
Sample collection
After an overnight fast, blood samples were taken from all children and were divided into three tubes:
1. The 1st K3-EDTA tube for osteocalcin determination: blood was centrifuged immediately and EDTA-plasma was stored for 3 months at − 20 °C. Osteocalcin concentrations were determined by the electrochemiluminescence immunoassay using cobas e601 analyzer, normal range 6.6–35.7 ng/ml.
2. The 2nd plain tube for total calcium, alkaline phosphatase, vitamin D (cutoff for normal value is ≥ 20 ng/ml), and parathyroid hormone: the total calcium and alkaline phosphatase were measured photometrically on cobas c 311/501 analyzer. Vitamin D and parathyroid hormone were assayed by the electrochemiluminescence immunoassay on cobas e601 immunoassay analyzer.
Molecular analysis
The 3rd EDTA tube for molecular assay of COLIA1 genotypes was according to manufacturer’s instructions. The COLIA1 genotype measurement was done by digestion using restriction enzyme (Bal1) of DNA amplified by the polymerase chain reaction (PCR-RFLP) for all subjects at the start of the study.
DNA extraction
DNA was extracted from leukocytes of peripheral blood samples using the QIAamp® UltraSens virus® extraction kit (Qiagen) USA according to manufacturer protocol. The extracted DNA was stored at − 20 °C until analysis.
PCR amplification
DNA was amplified using Maxime PCR PreMix Kit composed of Ready- to-Go PCR Beads which were designed and manufactured by iNtRON Biotechnology. PCR reaction with 25 μl final volume was prepared by adding 12.5 μl Master mix, 1.0 μl forward primer, 1.0 μl reverse primer, 10 μl extracted DNA, and 0.5 μl sterile high-quality water in PCR wells. PCR was done by the following conditions: initial denaturation at 94 ° C for 3 min, 35 cycles (94 °C for 50 s for denaturation, 62 °C for10 s for annealing, and 72 °C for 15 s for extension), and for final extension step 72 °C for 5 min. The PCR products were stored at − 80 °C until used.
Collagen type I alpha-1gene primers: the primer sequences were as follows:
Forward primer: (5′ GTCCAGCCCTCATCCTGGCC-3′).
Reverse primer: (5′TAACTTCTGGACTATTTGCGGACTTTTTGG-3′).
Detection of amplified PCR product by agarose gel electrophoresis: 5 μl of each sample was slowly loaded into the sample well and 5 μl PCR markers were also loaded into one of the wells. The power supply was programmed as 150 V and 100 mA for 20 min (Consort E 844). Then the gel was placed on the filter area of the ultraviolet transilluminator (Biometra). The amplified PCR product gave rise to 264 bp fragment.
Restriction digests reaction
The amplified DNA was digested by using Bal 1 (Biolabs, USA, part No. R0534S, 250 units) 5,000 units/ml. Ten microliters of amplified product were added to 16 μl sterile distilled water, 2 μl of restriction enzyme, and 2 μl of reaction buffer then incubated at 37 °C.
Detection of the band of polymorphism
The digested segments were subjected to electrophoresis on 8% non-denaturing polyacrylamide gel; the gel was stained using ethidium bromide (1 mg/ml) for 30 min at room temperature followed by visualization by the ultraviolet transilluminator. The amplified product 264 bp fragment with Bal 1 restriction enzyme gave rise to the following:
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Undigested 264 bp fragment indicated the presence of G allele.
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Appearance of 246 bp fragment indicated the presence of T allele and identified as G-T substitution.
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-The homozygous variant (G/G) results in one fragment at 264 bp, homozygous variant (T/T) results in one fragment at 246 bp, while the heterozygous variant (G/T) results in two fragments at 264 bp and 246 bp.
Lumbar spine bone mineral density (BMDLS) was measured by dual-energy X-ray absorptiometry (DXA). Osteoporosis should be defined as a BMDLS Z score lower than − 2.0 and osteopenia as a condition in which the Z score lies between − 1.0 and − 2.0 ISCD [18].
Radiological evaluation
Symptomatic fractures were confirmed by the plain X-ray imaging. Patients presented with neurological complication were subjected to computer tomography with or without magnetic resonance imaging.
Electrophysiological studies
A patient who had clinical manifestations suggesting some neuromuscular disorders (neuropathy, radiculopathy, plexopathy or muscular affection) were subjected to motor and sensory nerve conduction studies. Electromyography was elicited according to the recommended protocols for diagnosis of different possible disorders. All tests were done by the same examiners using a Nicolet Viking Quest cart electrodiagnostic system. Extremity temperature was maintained at or above 30 °C at time of examination. Any patient presented with seizures was subjected to electroencephalography (EEG) [19].
Statistical analysis
Presentation of quantitative data was done in the form of a mean (± standard deviation) or median (range) in parametric and nonparametric data respectively. Qualitative data are represented in number and percentages. t test was used in comparing two groups or Mann–Whitney U test according to the type of data. ANOVA or Kruskal-Wallis test was used (according to the type of data; parametric or nonparametric respectively) for comparing more than two groups. Chi-squared test was done for comparing numerical data. Multivariate analysis as well as logistic regression analysis was used. All statistical analysis was performed using statistical program SPSS version 10 for Windows (SPSS, Chicago, IL, USA).
