Genomewide Approach Validates Thiopurine Methyltransferase Activity Is a Monogenic Pharmacogenomic Trait.

We performed a genomewide association study (GWAS) of primary erythrocyte thiopurine S-methyltransferase (TPMT) activity in children with leukemia (n = 1,026). Adjusting for age and ancestry, TPMT was the only gene that reached genomewide significance (top hit rs1142345 or 719A>G; P = 8.6 × 10-61 ). Additional genetic variants (in addition to the three single-nucleotide polymorphisms [SNPs], rs1800462, rs1800460, and rs1142345, defining TPMT clinical genotype) did not significantly improve classification accuracy for TPMT phenotype. Clinical mercaptopurine tolerability in 839 patients was related to TPMT clinical genotype (P = 2.4 × 10-11 ). Using 177 lymphoblastoid cell lines (LCLs), there were 251 SNPs ranked higher than the top TPMT SNP (rs1142345; P = 6.8 × 10-5 ), revealing a limitation of LCLs for pharmacogenomic discovery. In a GWAS, TPMT activity in patients behaves as a monogenic trait, further bolstering the utility of TPMT genetic testing in the clinic.

An Inherited Genetic Variant in CEP72 Promoter Predisposes to Vincristine-Induced Peripheral Neuropathy in Adults With Acute Lymphoblastic Leukemia.

Peripheral neuropathy is a major toxicity of vincristine, yet no strategies exist for identifying adult patients at high-risk. We used a case-control design of 48 adults receiving protocol therapy for acute lymphoblastic leukemia (ALL) who developed vincristine-induced neuropathy (NCI grade 2-4) during treatment, and 48 matched controls who did not develop grade 2-4 neuropathy. Peripheral neuropathy was prospectively graded by National Cancer Institute (NCI) criteria. CEP72 promoter genotype (rs924607) was determined using polymerase chain reaction (PCR)-based single nucleotide polymorphism (SNP) genotyping. Frequency of the CEP72 T/T genotype was higher in cases (31% vs. 10%, P = 0.0221) and the incidence of vincristine-induced neuropathy (grades 2-4) was significantly higher in patients homozygous for the CEP72 T/T genotype. 75% of the 20 patients homozygous for the CEP72 T allele developed grade 2-4 neuropathy, compared to 44% of patients with CEP72 CC or CT genotype (P = 0.0221). The CEP72 polymorphism can identify adults at increased risk of vincristine-induced peripheral neuropathy.

Comparison of genome sequencing and clinical genotyping for pharmacogenes.

We compared whole exome sequencing (WES, n = 176 patients) and whole genome sequencing (WGS, n = 68) and clinical genotyping (DMET array-based approach) for interrogating 13 genes with Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. We focused on 127 CPIC important variants: 103 single nucleotide variations (SNV), 21 insertion/deletions (Indel), HLA-B alleles, and two CYP2D6 structural variations. WES and WGS provided interrogation of nonoverlapping sets of 115 SNV/Indels with call rate >98%. Among 68 loci interrogated by both WES and DMET, 64 loci (94.1%, confidence interval [CI]: 85.6-98.4%) showed no discrepant genotyping calls. Among 66 loci interrogated by both WGS and DMET, 63 loci (95.5%, CI: 87.2-99.0%) showed no discrepant genotyping calls. In conclusion, even without optimization to interrogate pharmacogenetic variants, WES and WGS displayed potential to provide reliable interrogation of most pharmacogenes and further validation of genome sequencing in a clinical lab setting is warranted.

Clinical Pharmacogenetics Implementation Consortium guidelines for cytochrome P450 2D6 genotype and codeine therapy: 2014 update.

Codeine is bioactivated to morphine, a strong opioid agonist, by the hepatic cytochrome P450 2D6 (CYP2D6); hence, the efficacy and safety of codeine are governed by CYP2D6 activity. Polymorphisms are a major cause of CYP2D6 variability. We summarize evidence from the literature supporting this association and provide therapeutic recommendations for codeine based on CYP2D6 genotype. This document is an update to the 2012 Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for CYP2D6 genotype and codeine therapy.

A health-care system perspective on implementing genomic medicine: pediatric acute lymphoblastic leukemia as a paradigm.

The promise of genomic medicine has received great attention over the past decade, projecting how genomics will soon guide the prevention, diagnosis, and treatment of human diseases. However, this evolution has been slower than forecast, even where evidence is often strong (e.g., pharmacogenomics). Reasons include the requirement for institutional resources and the need for the will to push beyond barriers impeding health-care changes. Here, we illustrate how genomics has been deployed to advance the treatment of childhood leukemia.

Pharmacogenomics and individualized medicine: translating science into practice.

Research on genes and medications has advanced our understanding of the genetic basis of individual drug responses. The aim of pharmacogenomics is to develop strategies for individualizing therapy for patients, in order to optimize outcome through knowledge of the variability of the human genome and its influence on drug response. Pharmacogenomics research is translational in nature and ranges from discovery of genotype-phenotype relationships to clinical trials that can provide proof of clinical impact. Advances in pharmacogenomics offer significant potential for subsequent clinical application in individual patients; however, the translation of pharmacogenomics research findings into clinical practice has been slow. Key components to successful clinical implementation of pharmacogenomics will include consistent interpretation of pharmacogenomics test results, availability of clinical guidelines for prescribing on the basis of test results, and knowledge-based decision support systems.

Concordance of DMET plus genotyping results with those of orthogonal genotyping methods.

There are several hurdles to the clinical implementation of pharmacogenetics. One approach is to employ pre-prescription genotyping, involving interrogation of multiple pharmacogenetic variants using a high-throughput platform. We compared the performance of the Drug Metabolizing Enzymes and Transporters (DMET) Plus array (1,931 variants in 225 genes) with that of orthogonal genotyping methods in 220 pediatric patients. A total of 1,692 variants had call rates >98% and were in Hardy-Weinberg equilibrium. Of these, 259 were genotyped by at least one independent method, and a total of 19,942 single-nucleotide polymorphism (SNP)-patient sample pairs were evaluated. The concordance rate was 99.9%, with only 28 genotype discordances observed. For the genes deemed most likely to be clinically relevant (TPMT, CYP2D6, CYP2C19, CYP2C9, VKORC1, DPYD, UGT1A1, and SLCO1B1), a total of 3,799 SNP-patient sample pairs were evaluable and had a concordance rate of 99.96%. We conclude that the DMET Plus array performs well with primary patient samples, with the results in good concordance with those of several lower-throughput genotyping methods.

Clinical utility and implications of asparaginase antibodies in acute lymphoblastic leukemia.

Hypersensitivity to asparaginase is common, but the differential diagnosis can be challenging and the diagnostic utility of antibody tests is unclear. We studied allergic reactions and serum antibodies to E. coli asparaginase (Elspar) in 410 children treated on St. Jude Total XV protocol for acute lymphoblastic leukemia. Of 169 patients (41.2%) with clinical allergy, 147 (87.0%) were positive for anti-Elspar antibody. Of 241 patients without allergy, 89 (36.9%) had detectable antibody. Allergies (P=0.0002) and antibodies (P=6.6 × 10(-6)) were higher among patients treated on the low-risk arm than among those treated on the standard/high-risk arm. Among those positive for antibody, the antibody titers were higher in those who developed allergy than in those who did not (P<1 × 10(-15)). Antibody measures at week 7 of continuation therapy had a sensitivity of 87-88% and a specificity of 68-69% for predicting or confirming clinical reactions. The level of antibodies was inversely associated with serum asparaginase activity (P=7.0 × 10(-6)). High antibody levels were associated with a lower risk of osteonecrosis (odds ratio=0.83; 95% confidence interval, 0.78-0.89; P=0.007). Antibodies were related to clinical allergy and to low systemic exposure to asparaginase, leading to lower risk of some adverse effects of therapy.

Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for codeine therapy in the context of cytochrome P450 2D6 (CYP2D6) genotype.

Codeine is bioactivated to morphine, a strong opioid agonist, by the hepatic cytochrome P450 2D6 (CYP2D6); hence, the efficacy and safety of codeine as an analgesic are governed by CYP2D6 polymorphisms. Codeine has little therapeutic effect in patients who are CYP2D6 poor metabolizers, whereas the risk of morphine toxicity is higher in ultrarapid metabolizers. The purpose of this guideline (periodically updated at http://www.pharmgkb.org) is to provide information relating to the interpretation of CYP2D6 genotype test results to guide the dosing of codeine.

A genome-wide approach identifies that the aspartate metabolism pathway contributes to asparaginase sensitivity.

Asparaginase is an important component for treatment of childhood acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in the cytotoxicity of asparaginase, we used a genome-wide association approach using the HapMap lymphoblastoid cell lines (87 CEU trio members) and 54 primary ALL leukemic blast samples at diagnosis. Asparaginase sensitivity was assessed as the drug concentration necessary to inhibit 50% of growth (inhibitory concentration (IC)(50)). In CEU lines, we tested 2,390,203 single-nucleotide polymorphism (SNP) genotypes at the individual SNP (P<0.001) and gene level (P<0.05), and identified 329 SNPs representing 94 genes that were associated with asparaginase IC(50). The aspartate metabolism pathway was the most overrepresented among 199 pathways evaluated (P=8.1 × 10(-3)), with primary involvement of adenylosuccinate lyase and aspartyl-tRNA synthetase genes. We validated that SNPs in the aspartate metabolism pathway were also associated with asparaginase sensitivity in primary ALL leukemic blast samples (P=5.5 × 10(-5)). Our genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited genomic interindividual variation in a plausible candidate pathway can contribute to asparaginase sensitivity.

Combination of cladribine and cytarabine is effective for childhood acute myeloid leukemia: results of the St Jude AML97 trial.

Because cladribine can increase cytarabine triphosphate levels, we tested a cladribine-cytarabine combination in the St Jude AML97, trial in which this combination was administered before standard chemotherapy to 96 children with acute myeloid leukemia (AML) or myelodysplastic syndrome. Patients received a 5-day course of cladribine (9 mg/m(2) per dose) and cytarabine either as daily 2-h infusions (500 mg/m(2) per dose) (arm A) or a continuous infusion (500 mg/m(2) per day) (arm B). Ara-CTP levels and inhibition of DNA synthesis increased from day 1 to day 2, but were not different between the two arms. In addition, the median blast percentages at day 15 did not differ between arms A and B, but patients treated in arm A had shorter intervals between the initiation of the first and second courses of therapy. Thus, although there were trends toward better complete remission rates and overall survival for patients treated in arm B, the reduced efficacy of arm A may have been partially compensated by more intense timing of therapy for that group. For all patients, 5-year event-free survival and overall survival estimates were 44.1+/-5.4 and 50.0+/-5.5%. Our results suggest that cladribine in combination with continuous-infusion cytarabine is effective therapy for childhood AML.

Genetic polymorphism of inosine triphosphate pyrophosphatase is a determinant of mercaptopurine metabolism and toxicity during treatment for acute lymphoblastic leukemia.

The influence of genetic polymorphism in inosine triphosphate pyrophosphatase (ITPA) on thiopurine-induced adverse events has not been investigated in the context of combination chemotherapy for acute lymphoblastic leukemia (ALL). This study investigated the effects of a common ITPA variant allele (rs41320251) on mercaptopurine metabolism and toxicity during treatment of children with ALL. Significantly higher concentrations of methyl mercaptopurine nucleotides were found in patients with the nonfunctional ITPA allele. Moreover, there was a significantly higher probability of severe febrile neutropenia in patients with a variant ITPA allele among patients whose dose of mercaptopurine had been adjusted for TPMT genotype. In a cohort of patients whose mercaptopurine dose was not adjusted for TPMT phenotype, the TPMT genotype had a greater effect than the ITPA genotype. In conclusion, genetic polymorphism of ITPA is a significant determinant of mercaptopurine metabolism and of severe febrile neutropenia, after combination chemotherapy for ALL in which mercaptopurine doses are individualized on the basis of TPMT genotype.

Clinical and radiological characteristics of methotrexate-induced acute encephalopathy in pediatric patients with cancer.

BACKGROUND: Little information is available about the diagnosis and management of acute methotrexate (MTX)-induced encephalopathy. METHODS: We reviewed clinical and magnetic resonance imaging (MRI) [including diffusion-weighted imaging (DWI)] characteristics of this complication in pediatric cancer patients treated from 2000 to 2006. RESULTS: Six of 754 (0.8%) patients with leukemia or lymphoma and 2 of 44 (4.5%) with bone sarcoma experienced acute encephalopathy within 2 weeks (median, 7.5 days) after receiving high-dose i.v. and/or intrathecal MTX. The signs and symptoms varied at presentation and during episodes: hemiparesis (eight patients, alternating from side to side in four), dysphasia (six), confusion/emotionality (six), headache (three), choreoathetosis (two), and seizure (two). All patients recovered after 1-7 days (median, 5.5 days). DWI revealed restricted diffusion in anatomic brain regions associated with the symptoms; changes on T2-weighted and fluid-attenuated inversion recovery (FLAIR) imaging were consistently less marked. After recovery, DWI findings were normal but T2 and/or FLAIR imaging usually showed residual abnormalities. CONCLUSIONS: Acute MTX toxicity often manifests as fluctuating neurologic symptoms with alternating hemispheric involvement. Restricted diffusion on DWI is a reliable early sign of acute MTX encephalopathy and resolves as clinical status improves, despite the persistence of subtle abnormalities on MRI.

Experience with 2-chlorodeoxyadenosine in previously untreated children with newly diagnosed acute myeloid leukemia and myelodysplastic diseases.

PURPOSE: To develop more effective chemotherapy regimens for childhood acute myelogenous leukemia (AML). PATIENTS AND METHODS: Between June 1991 and December 1996, we administered the nucleoside analog 2-chlorodeoxyadenosine (2-CDA) to 73 children with primary AML and 20 children with secondary AML or myelodysplastic syndrome (MDS). Patients received one or two 5-day courses of 2-CDA (8.9 mg/m(2)/d) given by continuous infusion. All patients then received one to three courses of daunomycin, cytarabine, and etoposide (DAV) remission induction therapy. RESULTS: Seventy-two patients with primary AML were assessable for response. Their rate of complete remission (CR) was 24% after one course of 2-CDA, 40% after two courses of 2-CDA, and 78% after DAV therapy. Of the 57 patients who entered CR, 11 subsequently underwent allogeneic bone marrow transplantation (BMT), and 40 underwent autologous BMT. Twenty-nine patients remain in continuous CR after BMT. Two patients remain in CR after chemotherapy only. The 5-year event-free survival (EFS) estimate was 40% (SE = 0.080%). Patients with French-American-British (FAB) M5 AML had a higher rate of CR after treatment with 2-CDA (45% after one course and 70.6% after two courses) than did others (P =.002). In contrast, no patient with FAB M7 AML (n = 10) entered CR after treatment with 2-CDA. Similarly, no patient with primary MDS (n = 6) responded to 2-CDA. Seven patients with secondary AML or MDS (n = 14) had a partial response to one course of 2-CDA. CONCLUSION: This agent was well tolerated, and its toxicity was acceptable. Future trials should examine the effectiveness of 2-CDA given in combination with other agents effective against AML.

Lack of effect of ondansetron on the pharmacokinetics and analgesic effects of morphine and metabolites after single-dose morphine administration in healthy volunteers.

AIMS: The purpose of this investigation was to study the influence of ondansetron on the single-dose pharmacokinetics and the analgesic effects elicited by morphine and the 3- and 6-glucuronide metabolites of morphine in healthy volunteers. METHODS: This was a randomized, double-blind, placebo-controlled, two-way crossover study in which six male and six female subjects were administered a single 10 mg intravenous dose of morphine sulphate, followed 30 min later by a single 16 mg intravenous dose of ondansetron hydrochloride or placebo. Serum and urine concentrations of morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) samples were quantified over 48 h using high performance liquid chromatography with detection by mass spectrometry. Analgesia was assessed in the volunteers with a contact thermode device to provide a thermal pain stimulus. Four analgesic response variables were measured including thermal pain threshold, thermal pain tolerance, temporal summation of pain and mood state. RESULTS: The two treatments appeared to be equivalent based on the 90% confidence intervals (0.6, 1.67) of the least squares means ratio. All least squares means ratio confidence intervals for each parameter, for each analyte fell within the specified range, demonstrating a lack of an interaction. CONCLUSIONS: The results of this study suggest that administration of ondansetron (16 mg i.v.) does not alter the pharmacokinetics of morphine and its 3- or 6-glucuronide metabolites to a clinically significant extent, nor does it affect the overall analgesic response to morphine as measured by the contact thermode system.

Outpatient management vs in-hospital management of children with new-onset diabetes.

The long-term results of outpatient management of subjects with newly diagnosed insulin-dependent diabetes mellitus (IDDM) are unknown. The longest follow-up described to date is for only one year, a time when most children with IDDM still make endogenous insulin. In this study, 41 young subjects with IDDM, treated and educated in the outpatient setting (1980 to 1984), were compared to a group of 80 similar subjects who received their initial education as hospital inpatients. Long-term glycemic control, as measured by longitudinal glycohemoglobin (HbA1) values was not different in the two groups (p greater than .05). We conclude that outpatient education and management of newly diagnosed subjects with IDDM is as safe and effective as inpatient treatment.