Each single-nucleotide polymorphism (SNP) is plotted with respect to its chromosomal location (x-axis) and the corresponding meta P value for association with vincristine neuropathy (left y-axis). The blue line indicates the recombination rate (right y-axis) at the corresponding region of chromosome 5 (x-axis). SNPs are colored to indicate extent of linkage disequilibrium with the indexed SNP (pairwise r2 values from HapMap CEU). The National Center for Biotechnology Information Build 37 corresponding to the 1000 Genomes Project March 2012 reference was used.
The cumulative incidence of vincristine-induced neuropathy (grades 2-4) differed by CEP72 promoter SNP (rs924607) genotype in the St Jude cohort (A) (P < .0001), the Children’s Oncology Group (COG) group A cohort (B) (P = .0087), and the combined St Jude and COG group A cohort (C) (P < .0001). Likewise, the cumulative incidence of severe neuropathy (grades 3-4) was also significantly higher in patients homozygous for the CEP72 risk allele (TT) (D). P values were computed based on weighted logistic regression adjusting for ancestry and accounting for multiple episodes of neuropathy. The number of patients evaluable and at risk of neuropathy is provided for each CEP72 genotype (diplotype). The x-axis represents time since the start of continuation therapy for ALL.
eAppendix 1. Supplemental Methods
eAppendix 2. Supplemental Results
eTable 1. National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) Version 1.0 Used to Assess Peripheral Neuropathy in the St. Jude Total XIIIB Cohort
eTable 2. Modified (“Balis”) Pediatric Scale of Peripheral Neuropathies Used to Assess Peripheral Neuropathy in the COG AALL0433 Cohort
eTable 3. Patient Characteristics and Relation to Occurrence of Vincristine-Induced Peripheral Neuropathy During Continuation Phase of Treatment in the St Jude Total XIIIB Cohort
eTable 4. Patient Characteristics and Relation to Occurrence of Vincristine-Induced Peripheral Neuropathy During Continuation Phase of Treatment in the COG AALL0433 Cohort
eTable 5. Top SNPs Associated With Vincristine-Induced Peripheral Neuropathy
eTable 6. Allele Frequencies of rs924607 in the Different Race Groups
eTable 7. Hazard Ratio Estimates by Cox Regression Model (With Time to Failure Defined as Time to the First Episode of Grade 2 or Higher Vincristine-Induced Neuropathy) in Combined Cohort of Total XIIIB and COG AALL0433 Group A (1.5 mg/m2 vincristine), Along With Number of Patients With Neuropathy and Number of Neuropathy Episodes in Each Factor Level
eTable 8. Accelerated Failure Time (AFT) Regression Modeling of Time to the First Episode of grade 2 or Higher of Vincristine Related Neuropathy by the rs924607 Genotypes (TT vs. CT or CC) Adjusting for Cumulative Dose and Genetically Determined Ancestry
eTable 9. Association of CEP72 SNP With Severity (Grade) of Vincristine-Induced Neuropathy
eTable 10. Analysis of Free Energy Components of the NKX-6.3 Homeodomain Bound With Wild Type (C) and Mutated (T) DNA Molecules
eFigure 1. CONSORT Flow Diagram of St Jude Total XIIIB Cohort
eFigure 2. CONSORT Flow Diagram of COG AALL0433 Cohort
eFigure 3. Significance of SNPs Associated With Vincristine-Induced Peripheral Neuropathy
eFigure 4. Genotyping Using the Allelic Discrimination Assay
eFigure 5. Cumulative Incidence of Vincristine-Induced Neuropathy in the COG Cohort Group B
eFigure 6.CEP72 Promoter SNP Genotype (Rs924607) and CEP72 Expression
eFigure 7. CEP72 and Vincristine Sensitivity in iPSC Neurons and Human Leukemia Cells
Diouf B, Crews KR, Lew G, Pei D, Cheng C, Bao J, Zheng JJ, Yang W, Fan Y, Wheeler HE, Wing C, Delaney SM, Komatsu M, Paugh SW, McCorkle JR, Lu X, Winick NJ, Carroll WL, Loh ML, Hunger SP, Devidas M, Pui C, Dolan ME, Relling MV, Evans WE. Association of an Inherited Genetic Variant With Vincristine-Related Peripheral Neuropathy in Children With Acute Lymphoblastic Leukemia. JAMA. 2015;313(8):815-823. doi:10.1001/jama.2015.0894
With cure rates of childhood acute lymphoblastic leukemia (ALL) exceeding 85%, there is a need to mitigate treatment toxicities that can compromise quality of life, including peripheral neuropathy from vincristine treatment.
To identify genetic germline variants associated with the occurrence or severity of vincristine-induced peripheral neuropathy in children with ALL.
Design, Setting, and Participants
Genome-wide association study of patients in 1 of 2 prospective clinical trials for childhood ALL that included treatment with 36 to 39 doses of vincristine. Genome-wide single-nucleotide polymorphism (SNP) analysis and vincristine-induced peripheral neuropathy were assessed in 321 patients from whom DNA was available: 222 patients (median age, 6.0 years; range, 0.1-18.8 years) enrolled in 1994-1998 in the St Jude Children’s Research Hospital protocol Total XIIIB with toxic effects follow-up through January 2001, and 99 patients (median age, 11.4 years; range, 3.0-23.8 years) enrolled in 2007-2010 in the Children’s Oncology Group (COG) protocol AALL0433 with toxic effects follow-up through May 2011. Human leukemia cells and induced pluripotent stem cell neurons were used to assess the effects of lower CEP72 expression on vincristine sensitivity.
Treatment with vincristine at a dose of 1.5 or 2.0 mg/m2.
Main Outcomes and Measures
Vincristine-induced peripheral neuropathy was assessed at clinic visits using National Cancer Institute criteria and prospectively graded as mild (grade 1), moderate (grade 2), serious/disabling (grade 3), or life threatening (grade 4).
Grade 2 to 4 vincristine-induced neuropathy during continuation therapy occurred in 28.8% of patients (64/222) in the St Jude cohort and in 22.2% (22/99) in the COG cohort. A SNP in the promoter region of the CEP72 gene, which encodes a centrosomal protein involved in microtubule formation, had a significant association with vincristine neuropathy (meta-analysis P = 6.3×10−9). This SNP had a minor allele frequency of 37% (235/642), with 50 of 321 patients (16%; 95% CI, 11.6%-19.5%) homozygous for the risk allele (TT at rs924607). Among patients with the high-risk CEP72 genotype (TT at rs924607), 28 of 50 (56%; 95% CI, 41.2%-70.0%) developed at least 1 episode of grade 2 to 4 neuropathy, a higher rate than in patients with the CEP72 CC or CT genotypes (58/271 patients [21.4%; 95% CI, 16.9%-26.7%]; P = 2.4×10−6). The severity of neuropathy was greater in patients homozygous for the TT genotype compared with patients with the CC or CT genotype (2.4-fold by Poisson regression [P<.0001] and 2.7-fold based on mean grade of neuropathy: 1.23 [95% CI, 0.74-1.72] vs 0.45 [95% CI, 0.3-0.6]; P = .004 by t test). Reducing CEP72 expression in human neurons and leukemia cells increased their sensitivity to vincristine.
Conclusions and Relevance
In this preliminary study of children with ALL, an inherited polymorphism in the promoter region of CEP72 was associated with increased risk and severity of vincristine-related peripheral neuropathy. If replicated in additional populations, this finding may provide a basis for safer dosing of this widely prescribed anticancer agent.
Cancer remains the leading cause of death by disease in US children despite major advances in the last 20 years. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, and as cure rates have surpassed 85%,1,2 it becomes increasingly important to mitigate the acute and chronic toxicities of treatment that adversely affect quality of life and longevity. Vincristine is one of the most widely used and effective anticancer agents for treating leukemias, lymphomas, brain tumors, and solid tumors in both adults and children1,2 and is administered multiple times as part of treatment protocols for every child with ALL and for children with neuroblastoma, Wilms tumor, rhabdomyosarcoma, Ewing sarcoma, and retinoblastoma.3
Vincristine exerts its cytotoxic effects by interfering with microtubule formation and mitotic spindle dynamics, leading to mitotic arrest and cell death.4- 6 The dose-limiting toxic effect of vincristine is peripheral neuropathy,7,8 characterized by neuropathic pain and sensory and motor dysfunction (eg, impaired manual dexterity, balance and deep tendon reflexes, and altered gait). A substantial percentage of patients develop neuropathy that causes considerable morbidity and often disrupts curative treatment.9 Currently, there are no reliable means of identifying patients at high risk of vincristine-induced neuropathy nor strategies to mitigate this drug toxicity. Although prior studies have documented racial differences in the incidence of vincristine neuropathy, candidate gene studies have failed to identify consistent genetic variants associated with an increased risk of vincristine-induced neuropathy.9- 12
To determine whether there are genetic polymorphisms associated with vincristine-induced neuropathy, we performed a genome-wide association study (GWAS) in 2 independent cohorts of children receiving protocol-defined treatment for ALL.
Two independent patient cohorts were treated either by St Jude Children’s Research Hospital or the Children’s Oncology Group (COG) according to contemporary institutional review board–approved ALL protocols that prospectively assessed and graded vincristine neuropathy according to National Cancer Institute (NCI) criteria. We analyzed all consenting patients who had germline DNA available for genome-wide single-nucleotide polymorphism (SNP) analyses in May 2011. The first cohort included children with newly diagnosed ALL enrolled and treated in the St Jude Total XIIIB protocol13- 15 (available from the authors). The second cohort included patients with relapsed ALL enrolled in the COG AALL0433 protocol (https://clinicaltrials.gov/show/NCT00381680). Written informed consent was obtained from patients or their guardians and assent from patients, as appropriate.
The St Jude Total XIIIB protocol was divided into 3 phases: 8 weeks of remission induction therapy, 2 weeks of consolidation therapy, and 120 weeks of continuation therapy (including a reinduction phase at weeks 16-21). Vincristine was administered as a component of combination chemotherapy during the induction and continuation phases of therapy at a dose of 1.5 mg/m2 with a maximum dose of 2 mg. Altogether, St Jude patients were scheduled to receive a total of 36 vincristine doses. Consecutive patients were enrolled between August 1994 and July 1998 and were followed up for toxic effects through 2001.
The COG AALL0433 protocol was a cooperative group-wide phase 3 randomized study of intensive therapy for patients with recurrent childhood precursor B-cell ALL, including late bone marrow or combined relapse (≥36 months since diagnosis), or early (<18 months since diagnosis) isolated extramedullary relapse in the central nervous system or testes. The COG AALL0433 treatment was divided into induction, intensification (with a reinduction phase at weeks 28-32 or 29-33), and continuation phases. Vincristine was administered as a component of combination chemotherapy during the induction, intensification, reinduction, and continuation phases of therapy; a total of 39 doses of vincristine were scheduled to be given per this protocol. Patients were randomly assigned to receive a vincristine dose of 1.5 mg/m2 with a maximum dose of 2 mg (group A) or 2 mg/m2 with a maximum dose of 2.5 mg (group B). Group B treatment and the vincristine dose randomization were permanently suspended by COG in 2010 because of a high incidence of peripheral neuropathy, most prominently in patients aged 13 years or older. Patients were enrolled between March 2007 and September 2010 at the time of the suspension of group B treatment and were followed up for toxic effects through May 2011.
As part of required clinical trial adverse event monitoring, children in both cohorts were prospectively assessed for the presence of peripheral neuropathy via physical examination by the treating oncologist at each clinic visit during treatment.16,17 Children in the St Jude cohort were assessed for toxicity weekly throughout all phases of therapy. Children in the COG cohort were assessed for toxicity weekly throughout the first year of therapy and monthly thereafter. For these analyses, children in the St Jude Total XIIIB study were graded according to NCI Common Terminology Criteria for Adverse Events (CTCAE) version 1.0 and those in the COG cohort according to a modified Balis scale for neuropathy, which is a modification of the NCI CTCAE version 2.0 (eTables 1 and 2 in the Supplement). Both scales classify neuropathy events as mild (grade 1), moderate (grade 2), serious/disabling (grade 3), or life threatening (grade 4).18 Those with grades 2, 3, or 4 motor and/or sensory neuropathy were considered neuropathy cases in our analyses. There were no neuropathy-related deaths (grade 5). Follow-up for the St Jude cohort continued through January 2001; follow-up for the COG cohort continued through May 2011.
Germline DNA (500 ng) was extracted from normal peripheral blood leukocytes, digested with restriction enzymes, amplified, labeled, and hybridized to the Affymetrix GeneChip Human Mapping 500K array (532 552 SNPs) or the SNP 6.0 array (906 600 SNPs) (Affymetrix). SNPs were excluded if genotyping call rates were less than 95% or if the minor allele frequency was less than 1% (eAppendix 1 in the Supplement). SNP imputation was carried out using MaCH version 184.108.40.206 The total number of SNPs that were imputed was 21 764 463 (eAppendix 1 in the Supplement).
Race was genetically determined following a method previously described.20 Briefly, the genetic ancestry estimates were determined by applying STRUCTURE (version 2.2.3) using HapMap project data as references (eAppendix 1 in the Supplement). SNP genotype data for HapMap cells were downloaded from release 22 on the International HapMap Project website (http://hapmap.ncbi.nlm.nih.gov/).
The human precursor B leukemia cell line Nalm6, the human T-lineage leukemia cell line CEM, and the neuroblastoma cell line SH-SY5Y were cultured in RPMI 1640 medium containing 2 mM of glutamine and 10% (vol/vol) fetal bovine serum at 37°C (98.6°F) with 5% CO2. iCell neurons derived from human induced pluripotent stem cells were obtained from Cellular Dynamics International and maintained per manufacturer’s protocol. Primary ALL cells were isolated by applying a Ficoll-Hypaque gradient to bone marrow aspirates obtained at diagnosis (with a median of 97% blast cells). CEP72 expression (GenBank BC001750) was impaired by using short hairpin RNA. In vitro drug sensitivity of primary ALL cells and of Nalm6 and CEM cell lines was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) assay as previously reported.21 The drug concentration lethal to 50% of the leukemia cells (LC50) was used as the measure of sensitivity to vincristine. Vincristine-induced neurotoxicity in iCell neurons was determined by individual cell measurements of total neurite outgrowth, number of processes, and number of branches in 1000 cells for each replicate experiment. Luciferase assays to measure CEP72 expression were performed as described in eAppendix 1 in the Supplement. Gene expression of HapMap cells measured by the Affymetrix GeneChip Human Exon 1.0 ST Array was downloaded from http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7761.
Analyses were conducted using R (http://www.r-project.org/) and SAS (SAS Institute Inc) software. Weighted logistic regression model fitting for recurrent events was used to test the associations between SNP genotypes and the phenotype of peripheral neuropathy (ie, occurrence of grade ≥2 vincristine-induced neuropathy during continuation therapy), including cumulative vincristine dose and genetically determined ancestry as covariates, as previously described (eAppendix 1 in the Supplement).15 After filtering out SNPs with a call rate of less than 95% (SNP call rate is the percentage of patients who have a confident genotype call for each SNP according to Affymetrix quality control criteria) or minor allele frequency of less than 1%, the remaining SNPs (484 623 typed SNPs and 1 091 393 imputed SNPs) were assessed in a GWAS for their association with vincristine-induced neuropathy in the St Jude and COG cohorts separately using weighted logistics regression models, then combined by pooling the P values from the 2 cohorts via the Fisher combined probability test. All P values were computed using 2-sided tests; statistical significance was determined at the genome-wide significance level of P<5×10−8.
Post-GWAS analyses focusing on the CEP72 SNP rs924607 were performed as follows: the relationship between the time to first episode and genotype was analyzed using cumulative incidence function estimates22 and the Gray test,23 Cox regression, and accelerated failure time regression22 with cumulative dose and genetic ancestry as covariates. The relationship between the cumulative vincristine dose at the first toxic episode and genotype was analyzed by Cox regression with genetically determined ancestry as a covariate. The relationship between severity (grade) of toxicity at the first episode and genotype was analyzed by Poisson regression model with cumulative dose and genetic ancestry as covariates. The t test was used to examine the differences in luciferase reporter activity. A linear regression model was used to test the association between vincristine sensitivity (LC50) and the SNP genotype in primary ALL cells. In post-GWAS analyses and laboratory experiments, all tests were 2-sided and significance was determined at the P<.05 level.
Genome-wide SNP analysis and vincristine-induced neuropathy were assessed in 321 patients enrolled in 2 ALL treatment protocols. Of 247 consecutive patients enrolled in the St Jude Total XIIIB protocol, 222 patients with newly diagnosed ALL (median age at diagnosis, 6.0 years; range, 0.1-18.8 years) had germline DNA available for analysis and were included in this study (eFigure 1 in the Supplement). Of the 137 patients who were enrolled in the COG AALL0433 protocol, 99 patients with relapsed ALL (median age at diagnosis, 11.4 years; range, 3.0-23.8 years) had germline DNA available for analysis and were included in this study (eFigure 2 in the Supplement).
The demographic and clinical characteristics of these 321 patients are summarized in Table 1 and for each cohort in eTables 3 and 4 in the Supplement.
Grade 2 to 4 vincristine-induced peripheral neuropathy during continuation therapy occurred in 28.8% (64/222) and 22.2% (22/99) of patients in the St Jude and COG protocols, respectively. Genetically determined ancestry and vincristine cumulative dose were the demographic or clinical characteristics significantly related to the occurrence of vincristine-induced neuropathy in each of the cohorts, as summarized in eTables 3 and 4 in the Supplement. Within the COG trial, the rate of vincristine-induced neuropathy was higher in the cohort of patients randomized to the higher dose of vincristine (2.0 mg/m2 with a maximum dose of 2.5 mg) compared with those randomized to the conventional dose (1.5 mg/m2 with a maximum dose of 2 mg) (19 of 48 patients [39.6%] vs 3 of 51 patients [5.9%]; P < .0001), leading to early closure of the higher-dose treatment group of the COG protocol.
Assessing SNPs that had a P<.05 in both cohorts and concordant effects in both populations yielded 5051 typed SNPs and 10 195 imputed SNPs. The Manhattan plot (eFigure 3 in the Supplement) depicts P values of SNPs from the 2 patient cohorts assessed via a meta-analysis. The SNPs with the lowest P values for their association with vincristine-induced neuropathy in the 2 cohorts are summarized in Table 2 and in eTable 5 in the Supplement. In a multivariable analysis that included genetically defined ancestry and cumulative dose of vincristine, only rs924607 remained genome-wide significantly (P = 4.7×10−8) related to the development of vincristine-induced neuropathy (Table 2 and eTable 5). rs924607 is in the promoter region of the CEP72 gene in chromosome 5 (Figure 1).
Twenty (62%) of 32 patients with the TT genotype in the St Jude cohort and 8 (44%) of 18 patients with the TT genotype in the COG cohort developed at least 1 episode of vincristine-induced neuropathy. In total, 28 of 50 patients (56%) with the TT genotype developed at least 1 episode of grade 2 to 4 vincristine-induced neuropathy (Table 3) compared with 58 (21.4%) of 271 patients with the CC or CT genotype (P = 2.4×10−6). The allelic odds ratios for the development of neuropathy for rs924607 were 2.43 (95% CI, 1.70-3.49) and 4.1 (95% CI, 1.86-9.01) in the St Jude and COG cohorts, respectively. As summarized in eTable 6 in the Supplement, the frequency of the CEP72 risk allele (T) differed by ancestry, with a lower frequency in patients with African ancestry and in the HapMap cells from individuals of African ancestry (Yoruba in Ibadan, Nigeria; African ancestry in southwestern United States; Luhva in Webuve, Kenya; Maasai in Kinyama, Kenya) compared with cells from individuals of European ancestry.
Because rs924607 is an imputed SNP, we validated the genotype in a subset of 30 patients randomly chosen to represent the 3 diplotypes for CEP72. This documented complete concordance between the imputed genotypes and those obtained by direct genotyping (eFigure 4 in the Supplement).
The cumulative incidence of all neuropathy episodes (grade 2-4) differed significantly by CEP72 rs924607 genotype (diplotype) in both patient cohorts treated with 1.5 mg/m2 of vincristine, with P values in the St Jude, COG, and combined cohorts of P < .0001, P = .0087, and P < .0001, respectively, by Gray test (Figure 2, A-C). Likewise, the cumulative incidence of severe neuropathy episodes (grades 3-4) was also significantly higher in patients homozygous for the CEP72 risk allele (P < .0001) (Figure 2D). Based on binomial proportions, 21 of 38 (55.3%; 95% CI, 39.7%-69.9%) patients with the CEP72 TT genotype experienced grade 2 to 4 neuropathy during the monitoring period compared with 46 of 235 (19.6%; 95% CI, 14.8%-25.3%) patients with the CC/CT genotype (P<.001). The cumulative incidence of the first neuropathy episode (grade 2-4) was 60.8% (95% CI, 43.9%-77.6%) among patients with the CEP72 TT genotype compared with 23.4% (95% CI, 17.4%-29.4%) among patients with the CC/CT genotype (P<.001). The risk of developing neuropathy was significantly higher in patients with the CEP72 TT genotype (hazard ratio for TT vs CT/CC, 3.58; 95% CI, 2.1-6.1) in a Cox regression model adjusting for cumulative vincristine dose and genetically determined ancestry (eTable 7 in the Supplement). This CEP72 SNP was not significantly associated with the incidence of vincristine-induced neuropathy among patients randomized to treatment with the higher (experimental) dose of vincristine (eFigure 5 in the Supplement).
By fitting an accelerated failure time model22 with CEP72 genotype, cumulative vincristine dose, and genetically determined ancestry as explanatory variables, the estimated ratio of median time to the first episode of neuropathy in patients with a TT genotype compared with patients with a CT or CC genotype was approximately 20% shorter in TT patients of any race and at any cumulative vincristine dose level (P = 6.19×10−6 for the regression coefficient of the SNP) (eTable 8 in the Supplement). Without adjusting for covariates, among those who developed grade 2 to 4 neuropathy, the average time to development of neuropathy was 225 days (95% CI, 169-281 days) in patients with the CEP72 genotype TT compared with 307 days (95% CI, 244-370 days) in patients with the CT/CC genotype.
In unadjusted analysis comparing the mean grade of neuropathy observed at the first episode (assigning grade 0 to those who did not develop neuropathy) in patients treated with 1.5 mg/m2 of vincristine, the mean grade of neuropathy in white patients with the CEP72 high-risk genotype (TT) was significantly higher (2.7-fold) than the mean grade of neuropathy in patients with the CEP72 CC or CT genotypes (1.23 [95% CI, 0.74-1.72] vs 0.45 [95% CI, 0.3-0.6]; P=.004 by t test). The numbers of patients in other ancestry groups were too small to make meaningful subgroup comparisons. Using the Poisson regression model to assess the severity (grade) of neuropathy at first episode, with genetically defined ancestry and cumulative vincristine dose as covariates in the combined cohort, the average grade (by CTCAE) of neuropathy was 2.4-fold greater (95% CI, 1.59-3.69) in patients homozygous for the CEP72 risk allele (TT) compared with all other patients (P < .0001) (eTable 9 in the Supplement).
Because rs924607 is located in the promoter region of CEP72, we examined CEP72 messenger RNA (mRNA) expression levels for association with rs924607 genotype in HapMap samples (CEU), using the publicly available gene expression data set.24 Patients who were homozygous for the variant T allele had significantly lower expression of CEP72 mRNA compared with those who were heterozygous or homozygous for the wild-type C allele (P = .03) (eFigure 6A in the Supplement). The effect of rs924607 genotype on CEP72 expression was confirmed using a luciferase reporter assay in 2 different human cell lines, documenting significantly lower expression with the T allele compared with the C allele in both SH-SY5Y neuroblastoma cells (eFigure 6B in the Supplement) and Nalm6 leukemia cells (eFigure 6C in the Supplement). The rs924607 T variant creates a consensus binding site for the NKX-6.3 transcription factor (repressor) (eFigure 6, D-H and eTable 10 in the Supplement), markedly enhancing NKX-6.3 binding to the risk allele, leading to lower CEP72 mRNA expression.
The relationship between CEP72 expression and vincristine-induced neurotoxicity was assessed in neurons derived from human induced pluripotent stem cells (iPSCs), revealing significantly greater sensitivity to vincristine when CEP72 expression was reduced (eFigure 7, A-B in the Supplement).
Likewise, reducing expression of CEP72 in 2 human ALL cells lines, Nalm6 (B-cell lineage) (eFigure 7C in the Supplement) and CEM (T-cell lineage) (eFigure 7E), was associated with increased sensitivity to vincristine (eFigure 7, D and F). Moreover, primary leukemia cells from patients with newly diagnosed ALL who were homozygous for the CEP72 risk allele (TT) (mean LC50, 0.9 μM; 95% CI, −0.5 to 2.3 μM) were more sensitive to vincristine compared with patients with either the CT (mean LC50, 2.6 μM; 95% CI, 0.6-4.7 μM) or CC (mean LC50, 11.9 μM; 95% CI, 3.0-20.7 μM) CEP72 genotype (P = .02) (eFigure 7G).
This study found that an inherited variant in the promoter region of the CEP72 gene was associated with a higher incidence and severity of vincristine-related peripheral neuropathy in children with ALL, with the cumulative incidence of neuropathy significantly higher and the mean grade of neuropathy significantly greater in patients homozygous for the CEP72 risk allele (TT) compared with all other patients. The CEP72 gene encodes a centrosomal protein that is essential for microtubule formation, and vincristine exerts its pharmacologic effects by inhibiting microtubule formation. We found that the CEP72 variant associated with vincristine neuropathy (T allele at rs924607) creates a binding site for a transcriptional repressor, leading to lower CEP72 mRNA expression, and we showed that reducing the expression of CEP72 in human iPSC neurons or leukemia cells increased their sensitivity to vincristine. Because vincristine-induced toxicity can be influenced by cumulative dose of vincristine and can differ by race, we included both cumulative dose and ancestry as covariates in our analyses, revealing that the CEP72 rs924607SNP was the only SNP reaching genome-wide significance (<5×10−8) in its association with vincristine-induced neuropathy. The frequency of the risk allele (T) was lower in African American patients compared with other racial groups, consistent with its lower frequency in African ancestry samples in the HapMap collection. This finding is also consistent with the reported lower incidence of vincristine-induced neuropathy in black patients.25 We found that children who inherited 2 copies of the CEP72 risk allele (homozygous for T at rs924607) had a significantly higher incidence of vincristine-induced peripheral neuropathy (60.8% [95% CI, 43.9%-77.6%] in patients with the TT genotype compared with 23.4% [95% CI, 17.4%-29.4] in patients with the CC/CT genotype). We also found that patients with CEP72 TT had increased severity (grade) of toxicity at first episode (2.4-fold [95% CI 1.6-3.7] higher grade of neuropathy for TT vs CT/CC) at any given cumulative dose of vincristine.
Vincristine is widely used to treat leukemia, lymphoma, various solid tumors, and brain tumors in children and adults.26 Vincristine-induced peripheral neuropathy, characterized by sensory and motor dysfunction, causes extensive morbidity and often disrupts treatment. In children with ALL, the reported incidence of vincristine-induced neuropathy differs across treatment protocols, in part because of the grade of toxicity considered,27,28 the vincristine dose and number of doses given,29 and the types of symptoms captured.9 In the current study, patients received uniform treatment according to prospective clinical trial protocols, with neuropathy assessed by NCI criteria to prospectively identify all patients who developed grades 2 to 4 neuropathy during continuation therapy with vincristine. Although a genetic component of vincristine-induced neuropathy would be consistent with previously reported racial differences in the incidence of vincristine-induced neuropathy,25 previous candidate gene studies have not yielded reproducible findings. We therefore took an agnostic genome-wide approach to identify genetic variants influencing vincristine-induced peripheral neuropathy, revealing the previously unrecognized association with CEP72 genotype.
The current study has limitations, including the small sample size (n = 321), the small number of patients homozygous for the CEP72 risk allele (n = 50; approximately 16% of patients), and our assessment of children only in the context of ALL chemotherapy. However, our clinical findings are corroborated by multiple lines of laboratory evidence in human iPSC neurons and leukemia cells and, if verified in additional patient populations, could lead to a new approach for identifying patients at high risk of vincristine-related neuropathy. Because our data suggest that the leukemia cells of patients who are homozygous for the CEP72 risk allele are more sensitive to vincristine, it may be possible to treat these patients with a lower dose of vincristine to decrease the risk or severity of neuropathy without compromising the antileukemic effects of vincristine, a possibility that merits assessment in future clinical trials.
In this preliminary study of children with ALL, an inherited polymorphism in the promoter region of CEP72 was associated with increased risk and severity of vincristine-related peripheral neuropathy. If replicated in additional populations, this finding may provide a basis for safer dosing of this widely prescribed anticancer agent.
Corresponding Author: William E. Evans, PharmD, Hematological Malignancies Program, Department of Pharmaceutical Sciences, St Jude Children’s Research Hospital, 262 Danny Thomas Pl, Memphis, TN 38105 (firstname.lastname@example.org).
Author Contributions: Dr Evans had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Evans, Relling.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Diouf, Crews, Pui, Cheng, Evans.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Pei, Cheng.
Obtained funding: Evans, Relling, Hunger.
Study supervision: Evans.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.
Funding/Support: The study was supported in part by National Institutes of Health grants U01 GM92666 (Drs Pei, Cheng, Yang, Paugh, Loh, Devidas, Relling, and Evans), UO1 GM61393 (Dr Dolan), R01 CA136765 (Dr Dolan), and R37 CA36401 (Drs Relling and Evans), F32 CA165823 (Dr Wheeler); Comprehensive Cancer Center grants CA21765, U10 CA98543 (COG Chair’s grant), U10 CA98413 (COG Statistical Center), and U24 CA114766 (COG Specimen Banking); and by the National Cancer Institute and the American Lebanese Syrian Associated Charities.
Role of the Funder/Sponsor: None of the funding sources played any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication.
Additional Contributions: We thank the St Jude Hartwell Center for Bioinformatics and Biotechnology, the Flow Cytometry and Cell Sorting Shared Resource of the St Jude Children’s Research Hospital Comprehensive Cancer Center, and the following individuals from St Jude Children’s Research Hospital: Kirsten Ness, PT, PhD, for her assistance with preparation of the online-only summary of NCI CTCAE for neuropathy; Jun Yang, PhD, Virginia Perez-Andreu, MD, Nancy Kornegay, MBA, Mark Wilkinson, BS, and Colton Smith, PhD, for their expertise and assistance in specific components of data analysis; Julie Groff, BA, Elizabeth Stevens, and Joshua Stokes, BA, for preparation of figures; and Caleb Simmons, BA, for the preparation of the manuscript. No financial compensation was received for these contributions.