Infection-related complications during treatment for childhood acute lymphoblastic leukemia.

Background: Comprehensive studies on neutropenia and infection-related complications in patients with acute lymphoblastic leukemia (ALL) are lacking. Patients and methods: We evaluated infection-related complications that were grade ≥3 on National Cancer Institute's Common Terminology Criteria for Adverse Events (version 3.0) and their risk factors in 409 children with newly diagnosed ALL throughout the treatment period. Results: Of the 2420 infection episodes, febrile neutropenia and clinically or microbiologically documented infection were seen in 1107 and 1313 episodes, respectively. Among documented infection episodes, upper respiratory tract was the most common site (n = 389), followed by ear (n = 151), bloodstream (n = 147), and gastrointestinal tract (n = 145) infections. These episodes were more common during intensified therapy phases such as remission induction and reinduction, but respiratory and ear infections, presumably viral in origin, also occurred during continuation phases. The 3-year cumulative incidence of infection-related death was low (1.0±0.9%, n = 4), including 2 from Bacillus cereus bacteremia. There was no fungal infection-related mortality. Age 1-9.9 years at diagnosis was associated with febrile neutropenia (P = 0.002) during induction and febrile neutropenia and documented infection (both P < 0.001) during later continuation. White race was associated with documented infection (P = 0.034) during induction. Compared with low-risk patients, standard- and high-risk patients received more intensive therapy during early continuation and had higher incidences of febrile neutropenia (P < 0.001) and documented infections (P = 0.043). Furthermore, poor neutrophil surge after dexamethasone pulses during continuation, which can reflect the poor bone marrow reserve, was associated with infections (P < 0.001). Conclusions: The incidence of infection-related death was low. However, young age, white race, intensive chemotherapy, and lack of neutrophil surge after dexamethasone treatment were associated with infection-related complications. Close monitoring for prompt administration of antibiotics and modification of chemotherapy should be considered in these patients.

Genetics of ancestry-specific risk for relapse in acute lymphoblastic leukemia.

The causes of individual relapses in children with acute lymphoblastic leukemia (ALL) remain incompletely understood. We evaluated the contribution of germline genetic factors to relapse in 2225 children treated on Children's Oncology Group trial AALL0232. We identified 302 germline single-nucleotide polymorphisms (SNPs) associated with relapse after adjusting for treatment and ancestry and 715 additional SNPs associated with relapse in an ancestry-specific manner. We tested for replication of these relapse-associated SNPs in external data sets of antileukemic drug pharmacokinetics and pharmacodynamics and an independent clinical cohort. 224 SNPs were associated with rapid drug clearance or drug resistance, and 32 were replicated in the independent cohort. The adverse risk associated with black and Hispanic ancestries was attenuated by addition of the 4 SNPs most strongly associated with relapse in these populations (for blacks: model without SNPs hazard ratio (HR)=2.32, P=2.27 × 10-4, model with SNPs HR=1.07, P=0.79; for Hispanics: model without SNPs HR=1.7, P=8.23 × 10-5, model with SNPs HR=1.31, P=0.065). Relapse SNPs associated with asparaginase resistance or allergy were overrepresented among SNPs associated with relapse in the more asparaginase intensive treatment arm (20/54 in Capizzi-methorexate arm vs 8/54 in high-dose methotrexate arm, P=0.015). Inherited genetic variation contributes to race-specific and treatment-specific relapse risk.

Genome-Wide Study Links PNPLA3 Variant With Elevated Hepatic Transaminase After Acute Lymphoblastic Leukemia Therapy.

Remission induction therapy for acute lymphoblastic leukemia (ALL) includes medications that may cause hepatotoxicity, including asparaginase. We used a genome-wide association study to identify loci associated with elevated alanine transaminase (ALT) levels after induction therapy in children with ALL enrolled on St. Jude Children's Research Hospital (SJCRH) protocols. Germline DNA was genotyped using arrays and exome sequencing. Adjusting for age, body mass index, ancestry, asparaginase preparation, and dosage, the PNPLA3 rs738409 (C>G) I148M variant, previously associated with fatty liver disease risk, had the strongest genetic association with ALT (P = 2.5 × 10-8 ). The PNPLA3 rs738409 variant explained 3.8% of the variability in ALT, and partly explained race-related differences in ALT. The PNPLA3 rs738409 association was replicated in an independent cohort of 2,285 patients treated on Children's Oncology Group protocol AALL0232 (P = 0.024). This is an example of a pharmacogenetic variant overlapping with a disease risk variant.

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.

Clinical impact of minimal residual disease in children with different subtypes of acute lymphoblastic leukemia treated with Response-Adapted therapy.

To determine the clinical significance of minimal residual disease (MRD) in patients with prognostically relevant subtypes of childhood acute lymphoblastic leukemia (ALL), we analyzed data from 488 patients treated in St Jude Total Therapy Study XV with treatment intensity based mainly on MRD levels measured during remission induction. MRD levels on day 19 predicted treatment outcome for patients with hyperdiploid >50 ALL, National Cancer Institute (NCI) standard-risk B-ALL or T-cell ALL, while MRD levels on day 46 were prognostic for patients with NCI standard-risk or high-risk B-ALL. Patients with t(12;21)/(ETV6-RUNX1) or hyperdiploidy >50 ALL had the best prognosis; those with a negative MRD on day 19 had a particularly low risk of relapse: 1.9% and 3.8%, respectively. Patients with NCI high-risk B-ALL or T-cell ALL had an inferior outcome; even with undetectable MRD on day 46, cumulative risk of relapse was 12.7% and 15.5%, respectively. Among patients with NCI standard-risk B-ALL, the outcome was intermediate overall but was poor if MRD was ⩾1% on day 19 or MRD was detectable at any level on day 46. Our results indicate that the clinical impact of MRD on treatment outcome in childhood ALL varies considerably according to leukemia subtype and time of measurement.

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.

Inherited variation in OATP1B1 is associated with treatment outcome in acute myeloid leukemia.

Using broad interrogation of clinically relevant drug absorption, distribution, metabolism, and excretion (ADME) genes on the DMET platform, we identified a genetic variant in SLCO1B1 (rs2291075; c.597C>T), encoding the transporter OATP1B1, associated with event-free (P = 0.006, hazard ratio = 1.74) and overall survival (P = 0.012, hazard ratio = 1.85) in children with de novo acute myeloid leukemia (AML). Lack of SLCO1B1 expression in leukemic blasts suggested the association might be due to an inherited rather than a somatic effect. rs2291075 was in strong linkage with known functional variants rs2306283 (c.388A>G) and rs4149056 (c.521T>C). Functional studies in vitro determined that four AML-directed chemotherapeutics (cytarabine, daunorubicin, etoposide, and mitoxantrone) are substrates for OATP1B1 and the mouse ortholog Oatp1b2. In vivo pharmacokinetic studies using Oatp1b2-deficient mice further confirmed our results. Collectively, these findings demonstrate an important role for OATP1B1 in the systemic pharmacokinetics of multiple drugs used in the treatment of AML and suggest that inherited variability in host transporter function influences the effectiveness of therapy.

A revised definition for cure of childhood acute lymphoblastic leukemia.

With improved contemporary therapy, we reassess long-term outcome in patients completing treatment for childhood acute lymphoblastic leukemia (ALL) to determine when cure can be declared with a high degree of confidence. In six successive clinical trials between 1984 and 2007, 1291 (84.5%) patients completed all therapies in continuous complete remission. The post-therapy cumulative risk of relapse or development of a second neoplasm and the event-free survival rate and overall survival were analyzed according to the presenting features and the three treatment periods defined by relative outcome. Over the three treatment periods, there has been progressive increase in the rate of event-free survival (65.2% vs 74.8% vs 85.1% (P<0.001)) and overall survival (76.5% vs 81.1% vs 91.7% (P<0.001)) at 10 years. The most important predictor of outcome after completion of therapy was the type of treatment. In the most recent treatment period, which omitted the use of prophylactic cranial irradiation, the post-treatment cumulative risk of relapse was 6.4%, death in remission 1.5% and development of a second neoplasm 2.3% at 10 years, with all relapses except one occurring within 4 years of therapy. None of the 106 patients with the t(9;22)/BCR-ABL1, t(1;19)/TCF3-PBX1 or t(4;11)/MLL-AFF1 had relapsed after 2 years from completion of therapy. These findings demonstrate that with contemporary effective therapy that excludes cranial irradiation, approximately 6% of children with ALL may relapse after completion of treatment, and those who remain in remission at 4 years post treatment may be considered cured (that is, less than 1% chance of relapse).

The role of inherited TPMT and COMT genetic variation in cisplatin-induced ototoxicity in children with cancer.

Ototoxicity is a debilitating side effect of platinating agents with substantial interpatient variability. We sought to evaluate the association of thiopurine S-methyltransferase (TPMT) and catechol O-methyltransferase (COMT) genetic variations with cisplatin-related hearing damage in the context of frontline pediatric cancer treatment protocols. In 213 children from the St. Jude Medulloblastoma-96 and -03 protocols, hearing loss was related to younger age (P = 0.013) and craniospinal irradiation (P = 0.001), but did not differ by TPMT or COMT variants. Results were similar in an independent cohort of 41 children from solid-tumor frontline protocols. Functional hearing loss or hair cell damage was not different in TPMT knockout vs. wild-type mice following cisplatin treatment, and neither TPMT nor COMT variant was associated with cisplatin cytotoxicity in lymphoblastoid cell lines. In conclusion, our results indicated that TPMT or COMT genetic variation was not related to cisplatin ototoxicity in children with cancer and did not influence cisplatin-induced hearing damage in laboratory models.

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.

Aurora kinases in childhood acute leukemia: the promise of aurora B as therapeutic target.

We investigated the effects of targeting the mitotic regulators aurora kinase A and B in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Aurora protein expression levels in pediatric ALL and AML patient samples were determined by western blot and reverse phase protein array. Both kinases were overexpressed in ALL and AML patients (P<0.0002), especially in E2A-PBX1-translocated ALL cases (P<0.002), compared with normal bone-marrow mononuclear cells. Aurora kinase expression was silenced in leukemic cell lines using short hairpin RNAs and locked nucleic acid-based mRNA antagonists. Aurora B knockdown resulted in proliferation arrest and apoptosis, whereas aurora A knockdown caused no or only minor growth delay. Most tested cell lines were highly sensitive to the AURKB-selective inhibitor barasertib-hydroxyquinazoline-pyrazol-anilide (AZD1152-HQPA) in the nanomolar range, as tested with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. But most importantly, primary ALL cells with a high aurora B protein expression, especially E2A-PBX1-positive cases, were sensitive as well. In adult AML early clinical trials, clear responses are observed with barasertib. Here we show that inhibition of aurora B, more than aurora A, has an antiproliferative and pro-apoptotic effect on acute leukemia cells, indicating that particularly targeting aurora B may offer a new strategy to treat pediatric ALL and AML.

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.

ETV6-RUNX1-positive childhood acute lymphoblastic leukemia: improved outcome with contemporary therapy.

ETV6-RUNX1 fusion is the most common genetic aberration in childhood acute lymphoblastic leukemia (ALL). To evaluate whether outcomes for this drug-sensitive leukemia are improved by contemporary risk-directed therapy, we studied clinical features, response and adverse events of 168 children with newly diagnosed ETV6-RUNX1-positive ALL on St Jude Total Therapy studies XIIIA (N=36), XIIIB (N=38) and XV (N=94). Results were compared with 494 ETV6-RUNX1-negative B-precursor ALL patients. ETV6-RUNX1 was associated with age 1-9 years, pre-treatment classification as low risk and lower levels of minimal residual disease (MRD) on day 19 of therapy (P<0.001). Event-free survival (EFS) or overall survival (OS) did not differ between patients with or without ETV6-RUNX1 in Total XIIIA or XIIIB. By contrast, in Total XV, patients with ETV6-RUNX1 had significantly better EFS (P=0.04; 5-year estimate, 96.8±2.4% versus 88.3±2.5%) and OS (P=0.04; 98.9±1.4% versus 93.7±1.8%) than those without ETV6-RUNX1. Within the ETV6-RUNX1 group, the only significant prognostic factor associated with higher OS was the treatment protocol Total XV (versus XIIIA or XIIIB) (P=0.01). Thus, the MRD-guided treatment schema including intensive asparaginase and high-dose methotrexate in the Total XV study produced significantly better outcomes than previous regimens and demonstrated that nearly all children with ETV6-RUNX1 ALL can be cured.

Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing.

Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TPMT alleles) experience severe myelosuppression, 30-60% of individuals who are heterozygotes (~3-14% of the population) show moderate toxicity, and homozygous wild-type individuals (~86-97% of the population) show lower active thioguanine nucleolides and less myelosuppression. We provide dosing recommendations (updates at http://www.pharmgkb.org) for azathioprine, mercaptopurine (MP), and thioguanine based on TPMT genotype.