Differences in Vitamin D Levels at Diagnosis and after Induction Phase Chemotherapy in Children with Acute Lymphoblastic Leukemia in Tertiary Hospital in West Java Indonesia

Objective: We examined differences of vitamin D levels at initial diagnosis and after induction phase chemotherapy in children with Acute Lymphoblastic Leukemia (ALL). Methods: A cross-sectional study was conducted on newly diagnosed children with ALL from October 2021 until October 2022 at Dr. Hasan Sadikin Hospital−Bandung, West Java, Indonesia. Vitamin D levels were measured twice using blood samples: at initial diagnosis and after the remission induction phase of chemotherapy. A paired t-test was applied and the value of p<0.05 is considered as statistically significant. Result: There were 53 subjects that fulfilled research criteria. Thirty−six subjects met the inclusion criteria while 17 were excluded. Majority of ALL patients were male (52.8%) and aged 1−10 years (83.3%). Mean vitamin D level of ALL children at initial diagnosis was 16.68±9,56 ng/mL. After the induction phase of chemotherapy, mean vitamin D level was 11.84±7,8 ng/mL. Paired t-test was performed to obtain p value = 0.000. Conclusion: The prevalence of hypovitaminosis D in ALL children is 88.8%. Vitamin D levels after chemotherapy are lower than at initial diagnosis.

However, serum vitamin D levels in acute leukemias such as ALL have received less attention. Hasanein H. Ghali et al (2020) [4] studied about 66 childrens who newly diagnosed acute leukemia and 50 childrens as control group. Serum vitamin D levels were below normal in children with acute leukemia; at the time of diagnosis, the mean serum level was 13 ng/dl, while the control group had 21 ng/dl. There was a statistically significant difference between the patient and control groups (p-value 0.001) [4]. Arhsi Naz at al (2012) studied the majority of patients with acute leukemia had vitamin D insufficiency, and vitamin D levels were further reduced after remission induction when compared to the untreated group, a difference that was statistically significant when compared to each group [8].
Previous study in Padang Indonesia showed the average vitamin D level in patients with acute leukemia was 24.017.21 ng/ml and vitamin D status was 50.9% deficient [9]. The studied about effect chemotherapy in ALL patients were limited in Indonesia and this research aims to analyze the differences in vitamin D levels in children with ALL before and after chemotherapy.

Materials and Methods
We performed a cross−sectional study of children newly diagnosed with ALL undergoing induction phase chemotherapy from October 2021 to October 2022 at Dr. Hasan Sadikin Hospital, Bandung. The inclusion criteria were patients aged ≤18 years who had just been diagnosed with ALL based on the results of Bone Marrow Puncture or immunophenotyping. Parents gave written consent for participating in the research (filling in informed consent). Exclusion criteria were ALL patients who consumed vitamin D supplementation and did not complete the induction phase of chemotherapy.
Patients newly diagnosed with ALL and fulfilling the inclusion criteria were included as research samples. Each parent/guardian of the subject diagnosed with ALL was given a detailed explanation of the research before signing the informed consent form. Subjects' identities were recorded in the case record form, anamnesis was carried out, and medical record data were recorded. The data includes name, age, gender, type of leukemia, address, and telephone number. Blood is taken for the first vitamin D level check before starting chemotherapy and stored in the laboratory. After completing the induction phase of chemotherapy (after BMP evaluation), a second sample of vitamin D levels will be taken. An examination of vitamin D levels was carried out after all samples were collected. Examination of 25(OH)D levels was carried out using Competitive Enzyme-linked immunosorbent assay (ELISA). Descriptive analysis was performed on demographic data, type of ALL, and vitamin D levels. Statistical analysis was performed to compare the differences in vitamin D levels at diagnosis and after induction phase chemotherapy. Numerical data that are normally distributed will be analyzed using t-test analysis, while the Wilcoxon test will be applied for data that is not normally distributed to compare before and after chemotherapy. The significancy of the results was determined based on the p-value <0.05. This study has obtained ethical approval from the Ethics Committee of Hasan Sadikin Hospital Bandung, No. LB. 02.01/X.6.5/252/2021.

Results
The Comparative research on vitamin D levels at diagnosis and after induction phase chemotherapy was carried out in newly diagnosed ALL children. In this research, 53 samples met the research criteria. Of the 53 samples, 36 met the inclusion criteria, and 17 were excluded because seven samples dropped out and ten died. Seven samples dropped out due to the difficulty of access to the hospital and financial problems. Ten samples died due to; intracranial bleeding in 2 patients, 4 with septic shock, and 4 due to hospital-acquired pneumonia. The data collected included demographic data; age, gender, clinical signs and symptoms, hemoglobin, leukocyte, platelet,  (2018) researching gender and the incidence of cancer in children, differences in genomic characteristics on the X chromosome between males and females during prenatal development and childhood determine the biological mechanisms underlying sex differences and the risk of childhood cancer [15].
The majority age at diagnosis was ≤10 years (83.3%). This research divides age into two groups. The basis for grouping is based on age stratification on the distribution of ALL types, namely standard risk or high risk. Research by Ameer K et al. found that ALL most often occurs at 5−9 years old [12].
Laboratory results showed that when the patient was diagnosed with leukemia, most patients (41.7%) had leukocyte levels <10,000/uL. Hemoglobin level was 7−11 g/dL respectively 61.1%. Most patients had a platelet level of 20,000−99,000/uL at 52.8%. Previous research at Hasan Sadikin Hospital found that severe anemia with hemoglobin levels <7 g/dL occurred in 44.8% of cases, leukocytosis occurred in 46.9% of cases, and severe thrombocytopenia occurred in 55% of cases [10] Other studies showed that at the time of diagnosis, the median leukocytes were 7120/uL (450−600,000/uL), the median hemoglobin was 7.5 g/dL (2,4−15.3 d/dL), the median platelets were 47,400/uL (4,000−544,000 /uL). Leukocytosis occurred in 36.6% of cases, leukopenia occurred in 36.1% of cases, and anemia was found in 82.9% of cases [18].
The average vitamin D level of ALL patients at diagnosis was 16.68±9,56 ng/mL and after the and vitamin D levels, which were examined two times, at the time of diagnosis and after the induction phase of chemotherapy. The characteristics of the subjects in this study included gender, age, clinical signs and symptoms, leukocyte count, hemoglobin level, platelet count, and ALL types, which can be seen in Table 1. Table 1 shows that most ALL patients were male (52.8%) and aged 1−10 years (83.3%). The most frequent clinical signs and symptoms were pallor (94.4%), fever (86.1%), hepatomegaly (72.2%), lymphadenopathy (69.4%), and splenomegaly (63.9%) consecutively. Laboratory examination results at the time of diagnosis showed that the majority of patients had leukocyte levels <10,000/uL (41.7%), hemoglobin levels 7−11 g/dL (61.1%), and platelet levels 20,000−99,000/uL (52.8%). Bone marrow puncture results showed the same high-risk ALL and standard risk (50%). Table 2 shows that the average vitamin D level in ALL at diagnosis was 16.68±9.56 ng/mL and after induction chemotherapy was 11.84±7.18 ng/mL.
The hypovitaminosis D status of standard-risk and high-risk ALL patients at diagnosis can be seen in Table 3 (a-e). The prevalence of vitamin D hypovitaminosis in this study was 88.8%. The incidence of hypovitaminosis D after induction phase chemotherapy is higher in high-risk ALL (100%) than in standard risk (94.4%). Tabel 3 (a-e) showed the average vitamin D level at diagnosis was 16.68±9,56 ng/ml and after induction phase chemotherapy was 11.84±7,18 ng/ml. Paired t-test was performed to obtain p value = 0.000.

Discussion
The research showed that LLA was more common in boys (52.8%). Previous research at Hasan Sadikin Hospital in Bandung in 2016 obtained the same results, namely, the majority of ALL occurred in boys, as did other studies [10][11][12]. A systematic review and meta-analysis conducted in Indonesia showed that ALL was more common in boys, 2.45 per 100,000 children, than in girls, 2.05 per 100,000 children [13]. Most leukemia cases occur in boy of all ages, the cause of which is still not clearly explained. A study by Jackson N et al (2009) [14] in Malaysia showed that most leukemia occurs in boys, presumably related to the type of blood group and the ABO gene on chromosome     induction phase of chemotherapy was 11.84±7,18 ng/ mL. The incidence of hypovitaminosis D in this study was 88.8%, and there was an increase in the number of cases of hypovitaminosis D in both standard-risk and high-risk ALL after induction phase chemotherapy. A study by Bhattacharya et al (2020) concluded that the prevalence of vitamin D deficiency in pediatric patients with ALL in India was 84.95%, and vitamin D deficiency was associated with outcomes in ALL [19].
Research by Hassanein G et al (2020) stated that vitamin D levels in pediatric acute leukemia patients were below normal, an average of 13 ng/mL, and in the control group was 21 ng/mL. If supplementation is not given, vitamin D levels will decrease after the induction phase of chemotherapy [4]. Research in West Sumatra, Indonesia [9] showed that the average vitamin D level in acute leukemia patients was 24.01 ± 7.91 ng/ml. Vitamin D status was at the level of insufficiency in 50.9% of acute leukemia patients [9]. Several risk factors contribute to Vitamin D deficiency in cancer. Cancer children are more susceptible to a lack of ultraviolet rays than healthy children. Nutritional problems can add to an existing deficiency status. Decreased vitamin D levels may be a cofactor in chemotherapy-induced mucocutaneous toxicity, and changes in the sense of taste (dysgeusia) and vitamin D supplementation may improve the condition. Other factors that affect vitamin D levels are genetic and iatrogenic [20].
Vitamin D has anti-cancer effects through cellular mechanisms, namely suppressing cell proliferation, increasing apoptosis, reducing the incidence of metastasis and invasion, reducing angiogenesis, suppressing inflammatory processes, and enhancing the immune response [20]. Decreased levels of vitamin D in children with cancer are caused by the effects of the disease or the therapy given, one of which is chemotherapy [21]. Vitamin D receptors essentially regulate cytochrome P4503A4 (CYP3A4), cytochrome P4503A5 (CYP3A5), and p-glycoprotein (MDR1), which are involved in the metabolism of vincristine, prednisone, dexamethasone, etoposide, and daunorubicin. This chemotherapy drug is given to ALL patients, and the effects of chemotherapy can cause a decrease in RVD [22]. A systematic review of vitamin D supplementation in children with cancer recommends a standardized diet high in vitamin D and calcium and regular monitoring of 25(OH)D levels. Vitamin D and calcium supplementation are recommended in children with low vitamin D levels to maintain vitamin D levels >20 ng/mL [23] Several limitations of our study deserve comments. We did not assess the nutritional status and there was no control group. The advantage of this research is prospective research so that the data needed in this research can be obtained.
In conclusion, the prevalence of hypovitaminosis D in ALL patients is 88.8%. There are differences in vitamin D levels in leukemia patients at the time of diagnosis and after the induction phase of chemotherapy. The average vitamin D level of children with ALL at the diagnosis was 16.68 ±9.56 ng/mL which is higher than the vitamin D level after the induction phase of chemotherapy (11.84 ±7,18 ng/mL).

Approval
It is part of an approved student thesis.

Conflict of Interest
None declared.

Ethical Declaration
Hereby, Wan Rita Mardhiya, Nur Suryawan, Budi Setiabudiawan consciously assure that for the manuscript Differences in Vitamin D Levels at Diagnosis and After Induction Phase Chemotherapy in Children with Acute Lymphoblastic Leukemia in Tertiary Hospital in West Java Indonesia the following is fulfilled: 1) This material is the authors' own original work, which has not been previously published elsewhere.
2) The paper is not currently being considered for publication elsewhere.
3) The paper reflects the authors' own research and analysis in a truthful and complete manner.
4) The paper properly credits the meaningful contributions of co-authors and co-researchers.
5) The results are appropriately placed in the context of prior and existing research. 6) All sources used are properly disclosed (correct citation). Literally copying of text must be indicated as such by using quotation marks and giving proper reference. 7) All authors have been personally and actively involved in substantial work leading to the paper, and will take public responsibility for its content.
The violation of the Ethical Statement rules may result in severe consequences.

Authors Contribution
Wan Rita Mardhiya, Nur Suryawan and Budi Setiabudiawan designed and reviewed the results, develop research method and data analysis, wrote and revised the manuscript. All authors read and approved the final version of the manuscript.

Data Availability
Study Registration: This study has obtained ethical approval from the Ethics Committee of Hasan Sadikin