Drug interactions may be important risk factors for methotrexate neurotoxicity, particularly in pediatric leukemia patients

Purpose: Methotrexate administration is associated with frequent adverse neurological events during treatment for childhood acute lymphoblastic leukemia. Here, we present evidence to support the role of common drug interactions and low vitamin B12 levels in potentiating methotrexate neurotoxicity. Methods: We review the published evidence and highlight key potential drug interactions as well as present clinical evidence of severe methotrexate neurotoxicity in conjunction with nitrous oxide anesthesia and measurements of vitamin B12 levels among pediatric leukemia patients during therapy. Results: We describe a very plausible mechanism for methotrexate neurotoxicity in pediatric leukemia patients involving reduction in methionine and consequential disruption of myelin production. We provide evidence that a number of commonly prescribed drugs in pediatric leukemia management interact with the same folate biosynthetic pathways and/or reduce functional vitamin B12 levels and hence are likely to increase the toxicity of methotrexate in these patients. We also present a brief case study supporting out hypothesis that nitrous oxide contributes to methotrexate neurotoxicity and a nutritional study, showing that patients. Conclusions: Use of nitrous oxide in pediatric leukemia patients at the same time as methotrexate use should be avoided especially as many suitable alternative anesthetic agents exist. Clinicians should consider monitoring levels of vitamin B12 in patients suspected of having methotrexate- induced neurotoxic effects

Reply: Methotrexate neurotoxicity due to drug interactions: an inadequate folinic acid effect

No abstract available

Application of four dyes in gene expression analyses by microarrays

BACKGROUND: DNA microarrays are widely used in gene expression analyses. To increase throughput and minimize costs without reducing gene expression data obtained, we investigated whether four mRNA samples can be analyzed simultaneously by applying four different fluorescent dyes. RESULTS: Following tests for cross-talk of fluorescence signals, Alexa 488, Alexa 594, Cyanine 3 and Cyanine 5 were selected for hybridizations. For self-hybridizations, a single RNA sample was labelled with all dyes and hybridized on commercial cDNA arrays or on in-house spotted oligonucleotide arrays. Correlation coefficients for all combinations of dyes were above 0.9 on the cDNA array. On the oligonucleotide array they were above 0.8, except combinations with Alexa 488, which were approximately 0.5. Standard deviation of expression differences for replicate spots were similar on the cDNA array for all dye combinations, but on the oligonucleotide array combinations with Alexa 488 showed a higher variation. CONCLUSION: In conclusion, the four dyes can be used simultaneously for gene expression experiments on the tested cDNA array, but only three dyes can be used on the tested oligonucleotide array. This was confirmed by hybridizations of control with test samples, as all combinations returned similar numbers of differentially expressed genes with comparable effects on gene expression

Comparing modeling strategies combining changes in multiple serum tumor biomarkers for early prediction of immunotherapy non-response in non-small cell lung cancer

BACKGROUND: Patients treated with immune checkpoint inhibitors (ICI) are at risk of adverse events (AEs) even though not all patients will benefit. Serum tumor markers (STMs) are known to reflect tumor activity and might therefore be useful to predict response, guide treatment decisions and thereby prevent AEs.OBJECTIVE: This study aims to compare a range of prediction methods to predict non-response using multiple sequentially measured STMs.METHODS: Nine prediction models were compared to predict treatment non-response at 6-months (n = 412) using bi-weekly CYFRA, CEA, CA-125, NSE, and SCC measurements determined in the first 6-weeks of therapy. All methods were applied to six different biomarker combinations including two to five STMs. Model performance was assessed based on sensitivity, while model training aimed at 95% specificity to ensure a low false-positive rate.RESULTS: In the validation cohort, boosting provided the highest sensitivity at a fixed specificity across most STM combinations (12.9% -59.4%). Boosting applied to CYFRA and CEA achieved the highest sensitivity on the validation data while maintaining a specificity &gt;95%.CONCLUSIONS: Non-response in NSCLC patients treated with ICIs can be predicted with a specificity &gt;95% by combining multiple sequentially measured STMs in a prediction model. Clinical use is subject to further external validation.</p

A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL

Distinctive genotypes in infants with T-cell acute lymphoblastic leukaemia

Infant T-cell acute lymphoblastic leukaemia (iT-ALL) is a very rare and poorly defined entity with a poor prognosis. We assembled a unique series of 13 infants with T-ALL, which allowed us to identify genotypic abnormalities and to investigate prenatal origins. Matched samples (diagnosis/remission) were analysed by single nucleotide polymorphism-array to identify genomic losses and gains. In three cases, we identified a recurrent somatic deletion on chromosome 3. These losses result in the complete deletion of MLF1 and have not previously been described in T-ALL. We observed two cases with an 11p13 deletion (LMO2-related), one of which also harboured a deletion of RB1. Another case presented a large 11q14·1-11q23·2 deletion that included ATM and only five patients (38%) showed deletions of CDKN2A/B. Four cases showed NOTCH1 mutations; in one case FBXW7 was the sole mutation and three cases showed alterations in PTEN. KMT2A rearrangements (KMT2A-r) were detected in three out of 13 cases. For three patients, mutations and copy number alterations (including deletion of PTEN) could be backtracked to birth using neonatal blood spot DNA, demonstrating an in utero origin. Overall, our data indicates that iT-ALL has a diverse but distinctive profile of genotypic abnormalities when compared to T-ALL in older children and adults

Phase II-like murine trial identifies synergy between dexamethasone and dasatinib in T-cell acute lymphoblastic leukemia

T-cell Acute Lymphoblastic Leukemia (T-ALL) is frequently characterized by glucocorticoid (GC) resistance, which is associated with inferior outcomes, thus highlighting the need for novel therapeutic approaches for GC resistant T-ALL. The pTCR/TCR signaling pathways play a critical role in cell fate decisions during physiological thymocyte development, with an interplay between TCR and glucocorticoid receptor (GR) signaling determining the T-lymphocyte selection process. We performed an shRNA screen in vitro and in vivo in T-ALL cell lines and patient derived xenograft (PDX) samples to identify vulnerabilities in the pTCR/TCR pathway and identified a critical role for the kinase LCK in cell proliferation. LCK knockdown or inhibition with dasatinib (DAS) caused cell cycle arrest. Combination of DAS with dexamethasone (DEX) resulted in significant drug synergy leading to cell death. The efficacy of this drug combination was underscored in a randomized phase II-like murine trial, recapitulating an early phase human clinical trial. T-ALL expansion in immunocompromised mice was significantly impaired using this drug combination, relative to mice receiving control vehicle or single drug treatment, highlighting the immediate clinical relevance of this drug combination for high risk T-ALL patients. Our results thus provide a strategy to improve the efficacy of current chemotherapy platforms and circumvent GC resistance

Molecular characterisation and clinical outcome of B-cell precursor acute lymphoblastic leukaemia with IG-MYC rearrangement

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) carries an immunoglobulin-MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukaemia and use of unproven individualised treatment schedules. Here we contrast the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered on a national BCP-ALL clinical trial/registry. Where present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analysis. Outcome was analysed to define 3-year event free survival (EFS) and overall survival (OS). IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either: established BCP-ALL specific abnormalities (high hyperdiploidy n=3, KMT2A-rearrangement n=6, iAMP21 n=1, BCR-ABL n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J rearranged cases, clearly distinct from Burkitt leukaemia/lymphoma. Children with IG-MYC-r within that subgroup had 3-year EFS of 47% and OS of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this patient group must be allowed access to contemporary, minimal residual disease adapted, prospective clinical trial protocols