Veena Vijayanathan

Persistent cognitive deficits, induced by intrathecal methotrexate, are associated with elevated CSF concentrations of excitotoxic glutamate analogs and can be reversed by an NMDA antagonist.

For patients with acute lymphoblastic leukemia or non-Hodgkin lymphoma, intrathecal (IT) methotrexate (MTX) significantly reduces the risk of relapse within the central nervous system, but is associated with neurotoxic sequelae. We established a rat model of MTX-induced cognitive deficits to further investigate the underlying pathophysiology and to develop protective therapeutic interventions. IT MTX 0.5 mg/kg was administered to 10-week old male Long Evans rats. Cerebrospinal fluid (CSF) was collected for measurement of folate, homocysteine, and excitotoxic glutamate analogs. Recognition and spatial memory were tested in the novel object recognition (NOR) task and the object placement (OP) task, respectively. Four doses of IT MTX in a two-week period induced cognitive deficits persisting at least three months after the final injection. CSF concentrations of the excitotoxic glutamate analogs homocysteic acid and homocysteine sulfinic acid were increased relative to baseline for the same three-month period. Dextromethorphan, a noncompetitive antagonist at the N-methyl-D-aspartate receptor, administered at a dose of 2 mg/kg intraperitoneally twice daily for a total of four doses, improved cognitive function among the MTX-treated rats, with no effect on control rats. Although this improvement was transient, each repeated treatment with dextromethorphan was followed by normalization of cognitive function. In conclusion, IT MTX induces persistent alterations in glutaminergic tone that may contribute to persistent cognitive deficits. Treatment with a glutamate receptor antagonist such as dextromethorphan may ameliorate the negative cognitive outcomes observed among patients with leukemia or lymphoma treated with repeated doses of prophylactic IT MTX.

Systemic methotrexate induces spatial memory deficits and depletes cerebrospinal fluid folate in rats

PURPOSE Although most children with acute lymphoblastic leukemia (ALL) are cured, a subset manifests persistent, focal cognitive deficits. Methotrexate (MTX), a key component of leukemia treatment, is suspected to contribute to treatment-induced cognitive dysfunction. We sought to establish a rodent model in order to further investigate the underlying pathophysiology. PROCEDURES Intraperitoneal MTX was given to Long-Evans rats on two schedules: acute (250 mg/kg once during adulthood), or chronic (1mg/kg twice weekly x4 doses, beginning at postnatal day 15, then weekly x6). Control rats were given saline injections on the same schedules. All male rats subsequently underwent behavioral testing designed to assess cognitive domains frequently impaired among children treated for ALL. Cerebrospinal fluid and serum folate concentrations were measured by HPLC. FINDINGS Both acute and chronic MTX administration produced spatial memory deficits, without significantly altering visual memory, general exploration, activity or motor coordination. MTX administration was also associated with a marked reduction in serum and CSF folate and a decrease in the ratio of CSF S-adenosylmethionine to S-adenosylhomocysteine. CONCLUSIONS Similar to children treated for ALL, rats given systemic MTX develop focal cognitive deficits along with expected alterations in folate physiology.

Intrathecal methotrexate induces focal cognitive deficits and increases cerebrospinal fluid homocysteine

Although most children with acute lymphoblastic leukemia (ALL) can be cured, a significant subset of survivors manifests focal deficits in cognitive function, even when the treatment regimen does not include cranial radiation. Intrathecal administration of the folate antagonist methotrexate (MTX) is necessary to prevent leukemic relapse within the central nervous system, but is suspected to contribute to treatment-induced cognitive dysfunction. To better elucidate the underlying pathophysiology, we sought to establish a rodent model of the cognitive and neurotoxic effects resulting from direct administration of MTX into the cerebrospinal fluid (CSF). MTX or artificial CSF was injected via transcutaneous puncture at the level of the cisterna magna. Subsequent behavioral tests were designed to assess cognitive domains frequently impaired among children treated for ALL. MTX administration produced both recognition and spatial memory deficits, without altering general activity or motor coordination. In addition, MTX significantly reduced folate levels in both CSF and serum and increased CSF homocysteine. Thus, we have established an animal model that mimics the clinical effects of prophylactic intrathecal MTX on cognitive function. Using this model we can further study the pathophysiology of MTX-induced cognitive dysfunction and test protective interventions.

Folate Homeostasis in Cerebrospinal Fluid During Therapy for Acute Lymphoblastic Leukemia

The neurotoxic effects of therapy for childhood acute lymphoblastic leukemia can result in leukoencephalopathy or measurable deficits in cognitive function. However, there are no validated biomarkers that allow the identification of those patients at greatest risk. With the objective of identifying such predictors, cerebrospinal fluid collected from 53 patients over 2.5 years of therapy for childhood acute lymphoblastic leukemia was retrospectively studied. Cerebrospinal fluid folate, concentrated relative to serum folate prior to therapy, dropped during the first month of therapy and remained below baseline throughout treatment. Cerebrospinal fluid homocysteine was inversely related to cognitive function prior to treatment. Oral methotrexate was associated with decreased cerebrospinal fluid folate and increased cerebrospinal fluid homocysteine, but these changes were not seen with oral aminopterin. Of 36 patients who had imaging after completion of therapy, 9 had periventricular or subcortical white matter abnormalities consistent with leukoencephalopathy. Peak cerebrospinal fluid tau concentrations during therapy were higher among patients who had leukoencephalopathy after completion of therapy than among those with normal imaging studies at the end of therapy. If confirmed prospectively, these markers may allow the identification of those patients at greatest risk of developing treatment-induced neurocognitive dysfunction, thus guiding preventive interventions.

Polymorphisms in Genes Related to Oxidative Stress Are Associated With Inferior Cognitive Function After Therapy for Childhood Acute Lymphoblastic Leukemia

PURPOSE Survivors of childhood acute lymphoblastic leukemia (ALL) exhibit increased rates of neurocognitive deficits. This study was conducted to test whether interpatient variability in neurocognitive outcomes can be explained by polymorphisms in candidate genes conferring susceptibility to neurocognitive decline. METHODS Neurocognitive testing was conducted in 350 pediatric leukemia survivors, treated on Dana-Farber Cancer Institute ALL Consortium Protocols 95-01 or 00-01. Genomic DNA was isolated from bone marrow collected at remission. Candidate polymorphisms were selected on the basis of prior literature, targeting genes related to drug metabolism, oxidative damage, altered neurotransmission, neuroinflammation, and folate physiology. Single nucleotide polymorphisms were detected using either a customized multiplexed Sequenom MassARRAY assay or polymerase chain reaction-based allelic discrimination assays. Multivariable logistic regression models were used to estimate the effects of genotype on neurocognitive outcomes, adjusted for the effects of demographic and treatment variables. False-discovery rate correction was made for multiple hypothesis testing, indicated as a Q value. RESULTS Inferior cognitive or behavioral outcomes were associated with polymorphisms in three genes related to oxidative stress and/or neuroinflammation: NOS3 (IQ, Q = 0.008; Vocabulary Q = 0.011; Matrix Reasoning Q = 0.008), SLCO2A1 (IQ Q = 0.043; Digit Span Q = 0.006; Block Design Q = 0.076), and COMT (Behavioral Assessment System for Children-2 Attention Q = 0.080; and Hyperactivity Q = 0.084). Survivors homozygous for NOS3 894T, with at least one SLCO2A1 variant G allele or with at least one GSTP1 variant allele, had lower mean estimated IQ scores than those without these genotypes. CONCLUSION These data are consistent with the hypothesis that oxidative damage contributes to chemotherapy-associated neurocognitive decline among children with leukemia.

Memantine protects rats treated with intrathecal methotrexate from developing spatial memory deficits.

Purpose: To test whether memantine can prevent methotrexate-induced cognitive deficits in a preclinical model. Experimental Design: After noting that methotrexate exposure induces prolonged elevations of the glutamate analog homocysteic acid (HCA) within cerebrospinal fluid, we tested whether intrathecal injection of HCA would produce memory deficits similar to those observed after intrathecal methotrexate. We then tested whether memantine, an antagonist of the N-methyl-d-aspartate (NMDA) subclass of glutamate receptors, could protect animals treated with clinically relevant doses of intrathecal methotrexate against developing memory deficits. Finally, we asked whether memantine affected this pathway beyond inhibiting the NMDA receptor by altering expression of the NMDA receptor or affecting concentrations of HCA or glutamate within the central nervous system. Results: Four intrathecal doses of methotrexate induced deficits in spatial memory, persisting at least one month following the final injection. Intrathecal HCA was sufficient to reproduce this deficit. Concurrent administration of memantine during the period of methotrexate exposure was protective, decreasing the incidence of methotrexate-induced spatial memory deficits from 56% to 20% (P < 0.05). Memantine neither altered expression of NMDA receptors within the hippocampus nor blunted the methotrexate-induced increases in glutamate or HCA. Conclusions: Excitotoxic glutamate analogs including HCA contribute to cognitive deficits observed after intrathecal methotrexate. Memantine, an NMDA receptor antagonist, reduces the incidence of cognitive deficits in rats treated with intrathecal methotrexate, and may therefore benefit patients with cancer receiving similar treatment. Clin Cancer Res; 19(16); 4446–54. ©2013 AACR.

A thymidylate synthase polymorphism is associated with increased risk for bone toxicity among children treated for acute lymphoblastic leukemia

Bone fractures and osteonecrosis frequently complicate therapy for childhood acute lymphoblastic leukemia (ALL). Bone toxicity has been associated with exposure to corticosteroids and methotrexate (MTX) and age greater than 10 years. We tested whether common genetic polymorphisms were associated with bone toxicity during treatment for ALL.

Long-term outcomes for children with acute lymphoblastic leukemia (ALL) treated on The Cancer Institute of New Jersey ALL trial (CINJALL).

Abstract The Cancer Institute of New Jersey Acute Lymphoblastic Leukemia trial (CINJALL) employed a post-induction regimen centered on intensive oral antimetabolite therapy, with no intravenous methotrexate (MTX). Fifty-eight patients enrolled between 2001 and 2005. A high rate of induction death (n = 3) or induction failure (n = 1) was observed. Among those who entered remission, five-year DFS is 80 ± 8.9% for those at standard risk of relapse and 76 ± 7.8% for high-risk patients, with median follow up over six years. The estimated cumulative incidence of testicular relapse among boys was elevated (13 ± 7.2%) compared to the rate observed on contemporary protocols. We conclude that post-induction therapy using intensive oral antimetabolites for children with acute lymphoblastic leukemia (ALL) can result in overall long-term DFS comparable to that observed among children treated with regimens including intravenous MTX. However, an increased risk of late extramedullary relapse among boys was observed, supporting the prevailing opinion that high-dose MTX improves outcome for children with ALL.

Charge and Size Dependent Effects of Cations on Triplex DNA Stabilization

Triplex DNA stabilization is affected by the microenvironment, including the presence of monovalent and divalent cations. We used melting temperature (Tm) measurements and circular dichroism spectroscopy to assess the stability and conformation of poly(dA)·2poly(dT). Monovalent cations (Li+, Na+, K+, Rb+, Cs+ and NH4+) promoted triplex DNA at concentrations ≥ 150 mM. The triplex DNA melting temperature varied from 49.8°C in the presence of 150 mM Li+ to 30.6°C in the presence of 150 mM K+. Among ammonium compounds, NH4+ was the most effective ion in stabilizing triplex DNA and its efficacy decreased with increasing substitution of the hydrogen atoms with bulky alkyl groups. Divalent cations were ∼1000-fold more efficacious than monovalent ions in stabilizing triplex DNA. Circular dichroism spectroscopic studies showed distinct conformational changes in triplex DNA stabilized by alkali metal and ammonium ions. These data demonstrate charge- and size-dependent effects of cations on triplex DNA stability.

Abstract 980: Intrathecal methotrexate induces focal cognitive deficits and alters CNS folate physiology in rats

Background: In order to reduce the persistent neurologic toxicity of therapy for childhood acute lymphoblastic leukemia (ALL), many treatment protocols avoid cranial radiation, relying instead on intrathecal chemotherapy to prevent central nervous system relapses. Nevertheless a significant subset of survivors treated without radiation manifest persistent focal deficits in cognitive function. The folate antagonist methotrexate (MTX), a key component of prophylactic intrathecal therapy, is suspected to contribute to treatment-induced cognitive dysfunction. Our goal was to establish a rodent model to isolate the cognitive and neurotoxic effects of MTX after direct administration into cerebrospinal fluid (CSF). Methods: Adult Long-Evans rats were given either intrathecal MTX 0.5mg/kg or artificial CSF via transcutaneous puncture at the level of the cisterna magna. Three days later, all rats subsequently underwent a battery of behavioral tests designed to assess cognitive domains frequently impaired among children treated for ALL, including spatial memory, visual memory, and motor coordination. Indicators of folate physiology (folate and homocysteine) in CSF and serum were measured using high performance liquid chromatography. Concentrations of interleukin-1β, IL-6, and tumor necrosis factor-α were measured in CSF to assess whether intrathecal MTX induces an inflammatory response. Results: Intrathecal MTX administration reliably produced both visual and spatial memory deficits, without altering general activity or motor coordination. MTX administration was associated with a significant reduction in both CSF and serum folate concentrations, along with an increase in CSF homocysteine. At 24 hours post-injection of MTX, CSF folate decreased by 75% and CSF homocysteine was increased 4-fold, relative to baseline. Intrathecal injection of both MTX and artificial CSF was associated with a significant rise in CSF IL-6 within 6 hours, which then returned to baseline by 24h. Conclusion: We have established an animal model in which intrathecal MTX induces cognitive dysfunction that mimics clinically observed toxicity, in association with a rapid fall in CSF folate and increase in CSF homocysteine. A non-specific, transient increase was seen in the proinflammatory cytokine IL-6 within CSF. This proinflammatory response does not appear sufficient to cause cognitive deficits, as it was observed among both groups of rats, while toxicity was demonstrated among only those who received MTX. This model will allow us both to further investigate the pathophysiology of treatment-induced cognitive dysfunction and to test protective interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 980.