The impact of homocysteine level on methotrexate induced neurotoxicity in children treated with St. Jude total XV acute lymphoblastic leukemia protocol

1Department Pediatrics Oncology, National Cancer Institute, Cairo, Egypt 2Department of Pediatrics, National Research Centre, Guiza, Egypt 3Department of Pharmacy, National Cancer Institute Cairo University, Cairo, Egypt 4Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt Received October 15, 2015; Revised January 10, 2016; Accepted January 23, 2016; Published Online February 03, 2016 Original Article


Introduction
Methotrexate (MTX) is a folic acid antagonist used since many years in the treatment of hematological malignancies. Its high dose (HD MTX) was prompted for the hematological, central nervous system (CNS) and testicular prophylaxis. [1][2][3][4][5][6] MTX is an inhibitor of dihydrofolate reductase (DHFR) resulting in a cellular depletion of tetrahydrofolates (THF) that are normally essential for the conversion of Homocysteine (Hcy) to methionine. This results into hyperhomocysteinemia 7-10 that is associated with neurotoxicity 11 and has been reported for years in the medical literature to present in acute, sub-acute or chronic syndromes 12, 13 . It may be transient and reversible but severe neurological disorders leading to coma or even death may occur as well. [14][15][16][17][18][19][20][21] In this study, assessment of the changes in Hcy concentration in plasma was done for newly diagnosed acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL) pediatric patients treated with HDMTX. The purpose of the study is to determine whether the increase in plasma Hcy concentration is related to MTX-induced neurotoxicity.

Methods and Materials
Pediatric patients newly diagnosed as ALL or non-Hodgkin's LL, treated at the National Cancer Institute, Cairo University, Egypt were the subject of this prospective study. They were treated according to St. Jude total XV protocol to receive > 1 g/m 2 MTX according to hematological and CNS initial status and disease stratification. 22 They were selected between 1 st of October 2009 and end of January 2010 to assess the correlation between the Hcy level and MTX induced neurotoxicity. The local institute ethical committee approved the study and an informed consent was obtained from the patients guardians prior to their inclusion in the study.

Consolidation treatment
After the patient has achieved remission induction following a 6-week chemotherapeutic treatment including triple intrathecal therapy 22 , consolidation consisted of HDMTX given every 2 weeks. The HD MTX (5 g/m 2 or 2.5 g/m 2 over 24 hours whether standard/high-SR/HR-or low-risk -LR-arms respectively), was given the 1 st day in association with the same triple intrathecal therapy including aracytine (Arac), hydrocortisone (HC) and MTX (Table 1). Folinic acid rescue was given intravenously at a dose of 15 mg/m 2 for standard/high-risk patients or 10 mg/m 2 for low-risk patient. It started 42 hours later and was given every 6 hours with modifications according to a specific schedule. 22 Oral 6-mercaptopurine (6MP), 50 mg / m 2 / day was associated for 8 weeks to all risk groups.

Homocysteine assay
Hcy measures were timed at diagnosis and relative to the HDMTX treatment ( Figure 1). Blood (3 to 4 mL) was collected in EDTA tubes at diagnosis, immediately before 1 st and 2 nd high dose MTX and 42 hours after each of them but before leucovorin treatment. The samples were stored at -20°C till time of analysis. Homocysteine was estimated using the Hcy enzyme immunoassay Any increase in Hcy level above 15 µmol/L following at least one of the two HDMTX, the final Hcy value was considered positive. However, normal levels (5 -15 µmol/L) were considered negative when Hcy level after both 1 st and 2 nd HDMTX were negative.

Clinical neurotoxicity
Neurotoxicity was evaluated from the results of clinical neurological examination. This was carried out initially at diagnosis and every other week coinciding with HDMTX during the consolidation phase. The target was to elicit any abnormal cognitive, motor or sensory findings. Moreover to detect any increase in intracranial tension in the form of headache, vomiting, blurring of vision or signs of meningeal irritation and finally to find out any cranial nerve affection. These were assessed using a form based on common terminology criteria for adverse events version 4.0 CTCAE, national institutes of health, national cancer institute. 25 Neurotoxicity was evaluated to be positive and given a score I when was present within one day of methotrexate infusion and included one of the following: severe intractable vomiting > thrice not gastro intestinal tract (GIT) induced, headache, low activity, speech impairment, memory impairment, disturbed level of consciousness, convulsions, syncopal attack (not cardiovascular induced) and coma. In its absence the clinical score was zero.

Radiological neurotoxicity
Radiological neurotoxicity was evaluated from the results of brain magnetic resonance imaging (MRI) findings. A base line brain MRI was initially carried out at diagnosis and subsequently at the end of consolidation phase 2 weeks after the last HD MTX and intrathecal therapy. All cases were examined by using 1.5 Tesla superconducting MR imager (magnotom ESPREE 1.5 T, Erlangen, Siemens, Germany).
After intravenous administration of Gadolonium -DTPA 0.3 mg/kg contrasted enhanced T1W1 in axial, sagittal and coronal places were obtained.
Radiological methotrexate toxicity was to be considered positive and given a score I at the presence of one or more of the following: demyelination, necrotizing lesions, mineralizing microangiopathy, vascular complications, cerebral infarcts, leukencephalopathy, white matter lesions or atrophy. In its absence the score was zero.
If both clinical and radiological examination were of score zero, the MTX neurotoxicity net result was considered negative and given a grade of I. If at least one of both was score I, MTX neurotoxicity net result was considered positive and given a grade of II. If both were of score I, the MTX neurotoxicity net result was considered positive and given a grade of III.

Statistical methods
Data was analyzed using SPSS win statistical package version 20 (SPSS Inc., Chicago, IL). Numerical data were expressed as mean and standard deviation. Comparisons between means where carried out using one-way analysis of variance (ANOVA) test followed by Dunnett test for multiple comparisons.
Qualitative data were expressed as frequency and percentage. Chi-square test (Fisher's exact test) was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was done using either student t-test or Mann-Whitney test (non-parametric t-test) as appropriate. Values of pre and post assessments were analyzed by paired t-test or wilcoxon signed rank test. P-value ≤ 0.05 was considered significant.

Results
Out of 40 cases, 29 cases were newly diagnosed ALL or LL. Of them, 20 were males and 9 were females. Their age ranged from 2 to 17 with a mean of 8 ± 4.4 years. Eleven patients were excluded from analysis as they did not complete the study. The patient's characteristics are shown in Table 2 and 3.

Plasma Hcy concentrations at different check points
Regarding the final Hcy value, 18 cases (62%) were with positive (above 15 µmol/L) values corresponding to increase Hcy levels after at least one of the two HDMTX cycles.

Methotrexate-induced neurotoxicity
Neurotoxicity was assessed qualitatively as the aim was to correlate it with the Hcy level. After HDMTX administration, 17 (58%) patients had features suggestive of neurotoxicity with newly developed changes in either MRI findings or clinical neurological examination or both of them. Clinical neurological manifestations were shown in 14 patients where it was the solitary finding in 12 patients and associated to MRI changes in the remaining 2 patients. On the other hand MRI changes were shown in 5 patients where it was associated to clinical neurological findings in 2 patients and as a solitary finding in the remaining 3 patients Table 3.

Correlation between Hcy levels and various factors
A. Final positive Hcy: A highly significant correlation was found between final positive Hcy value (>15 µmol/L) whenever detected at least once in relation to HDMTX and the development of neurotoxicity (p = 0.05). The same was found between final positive Hcy value and the HD MTX dose whether 5 g/m 2 or 2.5 g/m 2 (P = 0.023). However, Hcy was positive if age ≤ 10 years and CNS I or II, but not statistically significant Table 4.
A highly significant relation was found between final Hcy level and initial Hcy level as in 11 patients, the initial Hcy level was 9 µmol/L +/-1.82 SD. This corresponded to negative final Hcy level, while in 18 patients; the initial level was 14 µmol/L +/-4.09 SD. This corresponded to positive final Hcy level p = 0.001.
B. Hcy at different checkpoints: A significant relation between Hcy level post 1 st HDMTX and Hcy level post 2 nd HDMTX as (p = 0.006) Table 5.

Correlation between neurotoxicity and various factors
No significant relation was found between MTX neurotoxicity and various factors including age, MTX dose, patients CNS status and initial Hcy levels. Table 6  Moreover, there was no correlation between MTX neurotoxicity and initial Hcy levels, as 14 patients showed a mean of 11 µmol/L +/-3.69 SD corresponding to negative neurotoxicity oppositely to13 µmol/L +/-4.54 SD corresponding to positive neurotoxicity in 15 patients.

Discussion
In our study, we found a statistically significant correlation between higher plasma Hcy levels and higher dose of MTX 5 g versus 2.5 g and patients with neurotoxicity either clinically or radiologically or both. Kishi et al. 2003, recorded a significant higher plasma Hcy levels among patients with seizures following HD MTX. 26 The same as for an increased risk for encephalopathy after the administration of HD MTX and coinciding with higher MTX level at hour 42 and a higher Hcy concentration in the 1st HD MTX. 27 Hyperhomocysteinemia is classified as mild (15-30 µmol/L), moderate (31-100 µmol/L) or severe (>100 µmol/L). 28 However, the correlation between neurotoxicity and higher level of Hcy was not proved after the administration of MTX 1 or 3 gm/m 2 . 29 In the current study, Hcy levels post 1 st HDMTX were higher than their levels post 2 nd HDMTX and both were higher than their corresponding levels prior to HDMTX. These levels prior to HDMTX were however lesser than those at diagnosis. This goes with the higher baseline Hcy level before therapy among our patients that might have probably reflected their disease burden and occasional folate deficiency. 8 However, such baseline differences between risk groups were shown by the end of a 6-week remission induction therapy in other studies. 26 In the study of Kubota et al. 29 a significant rise of the mean Hcy levels at 24 hours was observed as compared to those before HDMTX treatment, then at 48 and 72 hours there were slight decreases in Hcy levels but these values were still significantly higher than the initial levels. 29 In the current study more than half of the cases developed neurotoxicity attributed to MTX. It included mainly solitary clinical manifestations representing 4 times the solitary radiological manifestations and 6 times combined clinical and radiological ones. These results might be due to over expression of clinical manifestations especially if confounded with GI manifestations of MTX toxicity. In other studies, neurotoxicity was observed after the administration of HDMTX (2g/m 2 or 5g/m 2 ) to children with newly diagnosed ALL.

Conclusion
In conclusion, the results of the present study showed that plasma Hcy concentration was significantly elevated after HDMTX administration and this elevation is related to the observed neurotoxicity. Whether the elevation in Hcy concentration can prove an informative biomarker for neurotoxicity requires additional testing with alternative regimens of MTX.