M E Dolan

The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response

The NIH Pharmacogenetics Research Network (PGRN) is a collaborative group of investigators with a wide range of research interests, but all attempting to correlate drug response with genetic variation. Several research groups concentrate on drugs used to treat specific medical disorders (asthma, depression, cardiovascular disease, addiction of nicotine, and cancer), whereas others are focused on specific groups of proteins that interact with drugs (membrane transporters and phase II drug‐metabolizing enzymes). The diverse scientific information is stored and annotated in a publicly accessible knowledge base, the Pharmacogenetics and Pharmacogenomics Knowledge base (PharmGKB). This report highlights selected achievements and scientific approaches as well as hypotheses about future directions of each of the groups within the PGRN. Seven major topics are included: informatics (PharmGKB), cardiovascular, pulmonary, addiction, cancer, transport, and metabolism.

Cancer Pharmacoethnicity: Ethnic Differences in Susceptibility to the Effects of Chemotherapy

A long-term goal of pharmacogenomics research is the design of individualized therapy based on the genomic sequence of the patient, in order to maximize response and minimize adverse drug reactions. Pharmacoethnicity, or ethnic diversity in drug response or toxicity, is becoming increasingly recognized as an important factor accounting for interindividual variation in anticancer drug responsiveness. Although pharmacoethnicity is determined by genetic and nongenetic factors, there is rapidly accumulating clinical evidence about ethnic differences in the frequencies of polymorphisms within many of the important cancer drug-related genes. This article reviews the current clinical evidence for ethnic differences in anticancer drug disposition and sensitivity while highlighting the challenges, and potential solutions, to acquiring such knowledge. The discovery of “ethnic-specific genetic signatures,” representing unique sets of drug susceptibility-governing polymorphisms, may be the outcome of such work. Ultimately, such understanding will further the lofty goal of individualization of chemotherapy based on a persons unique genetic make-up to improve the tolerability and effectiveness of chemotherapy for all patients.

O6-benzylguanine in humans: metabolic, pharmacokinetic, and pharmacodynamic findings.

PURPOSE O6-Benzylguanine is a potent inactivator of the DNA-repair protein, O6-alkylguanine-DNA alkyl-transferase (AGT), that enhances sensitivity to nitrosoureas in tumor-cell lines and tumor-bearing animals. The objective of this study was to determine the pharmacokinetics and metabolic fate of O6-Benzylguanine in humans and its effect on AGT activity in peripheral-blood mononuclear cells (PBMCs). PATIENTS AND METHODS Twenty-five cancer patients were treated with O6-Benzylguanine at a dose level of 10, 20, 40, and 80 mg/m2 intravenously (IV) over 1 hour. Plasma and urine samples were collected and analyzed for O6-Benzylguanine and O6-Benzyl-8-oxoguanine concentrations. AGT activity in PBMCs was determined up to 2 weeks postinfusion. RESULTS There was no toxicity attributable to O6-Benzylguanine alone at all doses tested. O6-Benzylguanine rapidly disappeared from plasma and was converted to a major metabolite, O6-Benzyl-8-oxoguanine. The half-life of O6-Benzyl-8-oxoguanine increased with dose from 2.8 to 9.2 hours at doses of 10 and 80 mg/m2, respectively. The maximum concentration Cmax and area under the concentration-time curve (AUC) for O6-Benzyl-8-oxoguanine were, respectively, 2.2- and 12- to 29-fold greater than those of O6-Benzylguanine. At all doses, depletion of AGT activity was observed in lymphocytes with a return to baseline by 1 week posttreatment. CONCLUSION This study demonstrates that administration of O6-Benzylguanine to humans results in a rapid conversion to O6-Benzyl-8-oxoguanine, which follows nonlinear kinetics. Both compounds contribute to an effective depletion of AGT activity in lymphocytes; however, prolonged depletion of AGT activity is likely due primarily to the effect of O6-Benzyl-8-oxoguanine.

Pharmacodynamics of fluorouracil-based induction chemotherapy in advanced head and neck cancer.

PURPOSE AND METHODS To optimize the biochemical modulation of fluorouracil (5-FU), we administered the pure I-stereoisomer of leucovorin (LV) as a 132-hour continuous intravenous infusion (CIV) with cisplatin 100 mg/m2, 5-FU 640 mg/m2/d as a 120-hour CIV, and interferon alfa-2b (IFN) at 2 MU/m2/d for 6 days for three cycles (I-PFL-IFN). Pharmacologic parameters included morning (AM) and afternoon (PM) plasma concentrations of 5-FU, LV and its active metabolite 5-methyl tetrahydrofolate (MTHF), and dihydropyrimidine dehydrogenase (DPD) activity in peripheral mononuclear cells. RESULTS Eighty-nine patients were treated (86 stage IV). Neutropenia and mucositis were the most common toxicities. Sixty-six percent achieved a complete remission (CR). There was a trend for higher PM versus AM 5-FU concentrations (median, 1.64 v 1.51 mumoles/L; P = .08), but not for LV plus MTHF (P = .66). The mean +/- SD DPD activity was 0.21 +/- 0.14 nmol/min/mg and did not correlate with plasma concentrations of 5-FU or LV plus MTHF or clinical toxicities. Higher PM 5-FU concentrations correlated with worse leukopenia (P = .04) and severity of mucositis (P = .04). PM 5-FU concentration was higher in women than in men (P = .07), with no apparent difference in severity of toxicities. The maximum 5-FU concentration was higher in CR than non-CR patients (median, 2.01 v 1.54 mumoles/L; P = .02) and higher in women than men who achieved a CR (median, 2.77 v 1.91 mumoles/L; P = .03). No correlation of CR with dose-intensity was found. CONCLUSION L-PFL-IFN is active in stage IV head and neck cancer. 5-FU concentration is a significant predictor of toxicity. In women, optimization of response outcome requires a higher 5-FU concentration. Individualized 5-FU dosing to obtain higher 5-FU plasma concentrations may be indicated.

Heritable and non-genetic factors as variables of pharmacologic phenotypes in lymphoblastoid cell lines

Publicly available genetic and expression data on lymphoblastoid cell lines (LCLs) make them a unique resource for understanding the genetic underpinnings of pharmacological outcomes and disease. LCLs have been used for pharmacogenomic discovery and validation of clinical findings associated with drug response. However, variation in cellular growth rate, baseline Epstein–Barr virus (EBV) copy number and ATP levels can all be confounders in such studies. Our objective is to better define confounding variables that affect pharmacological end points in LCLs. To this end, we evaluated the effect of these three variables on drug-induced cytotoxicity in LCLs. The drugs evaluated included daunorubicin, etoposide, carboplatin, cisplatin, cytarabine, pemetrexed, 5′-deoxyfluorouridine, vorinostat, methotrexate, 6-mercaptopurine, and 5-fluorouracil. Baseline ATP or EBV copy number were not significantly correlated with cellular growth rate or drug-induced cytotoxicity. In contrast, cellular growth rate and drug-induced cytotoxicity were significantly, directly related for all drugs except vorinostat. Importantly, cellular growth rate is under appreciable genetic influence (h2=0.30–0.39) with five suggestive linkage regions across the genome. Not surprisingly, a percentage of SNPs that significantly associate with drug-induced cytotoxicity also associate with cellular growth rate (P⩽0.0001). Studies using LCLs for pharmacologic outcomes should therefore consider that a portion of the genetic variation explaining drug-induced cytotoxicity is mediated via heritable effects on growth rate.

Integration of Cell Line and Clinical Trial Genome-Wide Analyses Supports a Polygenic Architecture of Paclitaxel-Induced Sensory Peripheral Neuropathy

Purpose: We sought to show the relevance of a lymphoblastoid cell line (LCL) model in the discovery of clinically relevant genetic variants affecting chemotherapeutic response by comparing LCL genome-wide association study (GWAS) results to clinical GWAS results. Experimental Design: A GWAS of paclitaxel-induced cytotoxicity was conducted in 247 LCLs from the HapMap Project and compared with a GWAS of sensory peripheral neuropathy in patients with breast cancer (n = 855) treated with paclitaxel in the Cancer and Leukemia Group B (CALGB) 40101 trial. Significant enrichment was assessed by permutation resampling analysis. Results: We observed an enrichment of LCL cytotoxicity-associated single-nucleotide polymorphisms (SNP) in the sensory peripheral neuropathy-associated SNPs from the clinical trial with concordant allelic directions of effect (empirical P = 0.007). Of the 24 SNPs that overlap between the clinical trial (P < 0.05) and the preclinical cytotoxicity study (P < 0.001), 19 of them are expression quantitative trait loci (eQTL), which is a significant enrichment of this functional class (empirical P = 0.0447). One of these eQTLs is located in RFX2, which encodes a member of the DNA-binding regulatory factor X family. Decreased expression of this gene by siRNA resulted in increased sensitivity of Neuroscreen-1(NS-1; rat pheochromocytoma) cells to paclitaxel as measured by reduced neurite outgrowth and increased cytotoxicity, functionally validating the involvement of RFX2 in nerve cell response to paclitaxel. Conclusions: The enrichment results and functional example imply that cellular models of chemotherapeutic toxicity may capture components of the underlying polygenic architecture of related traits in patients. Clin Cancer Res; 19(2); 491–9. ©2012 AACR.

PACdb: a database for cell-based pharmacogenomics

We have developed Pharmacogenomics And Cell database (PACdb), a results database that makes available relationships between single nucleotide polymorphisms, gene expression, and cellular sensitivity to various drugs in cell-based models to help determine genetic variants associated with drug response. The current version also supports summary analysis on differentially expressed genes between the HapMap samples of European and African ancestry, as well as queries for summary information of correlations between gene expression and pharmacological phenotypes. At present, data generated on the following anticancer agents are included: carboplatin, cisplatin, etoposide, daunorubicin, and cytarabine (Ara-C). The database is also available to assist in the investigation of the effects of potential confounding variables (e.g. cell proliferation rate) in lymphoblastoid cell lines. PACdb will be regularly updated to include more drugs and new datasets (e.g. baseline microRNA levels). PACdb will be linked into PharmGKB to benefit the next wave of pharmacogenetic and pharmacogenomic discovery.

Genome-wide Meta-analysis Identifies Variants Associated with Platinating Agent Susceptibility Across Populations

Platinating agents are used in the treatment of many cancers, yet they can induce toxicities and resistance that limit their utility. Using previously published and additional world population panels of diverse ancestry totaling 608 lymphoblastoid cell lines (LCLs), we performed meta-analyses of over 3 million single-nucleotide polymorphisms (SNPs) for both carboplatin- and cisplatin-induced cytotoxicity. The most significant SNP in the carboplatin meta-analysis is located in an intron of NBAS (neuroblastoma amplified sequence; P=5.1 × 10−7). The most significant SNP in the cisplatin meta-analysis is upstream of KRT16P2 (P=5.8 × 10−7). We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Most of the variants that associate with platinum-induced cytotoxicity are polymorphic across multiple world populations; therefore, they could be tested in follow-up studies in diverse clinical populations. Seven genes previously implicated in platinating agent response, including BCL2 (B-cell CLL/lymphoma 2), GSTM1 (glutathione S-transferase mu 1), GSTT1, ERCC2 and ERCC6, were also implicated in our meta-analyses.

FstSNP-HapMap3: a database of SNPs with high population differentiation for HapMap3.

The International HapMap Project has recently made available genotypes and frequency data for phase 3 (NCBI build 36, dbSNPb129) of the HapMap providing an enriched genotype dataset for approximately 1.6 million single nucleotide polymorphisms (SNPs) from 1,115 individuals with ancestry from parts of Africa, Asia, Europe, North America and Mexico. In the present study, we aim to facilitate pharmacogenetics studies by providing a database of SNPs with high population differentiation through a genomewide test on allele frequency variation among 11 HapMap3 samples. Common SNPs with minor allele frequency greater than 5¢ from each of 11 HapMap3 samples were included in the present analysis. The population differentiation is measured in terms of fixation index (Fst), and the SNPs with Fst values over 0.5 were defined as highly differentiated SNPs. Our tests were carried out between all pairs of the 11 HapMap3 samples or among subgroups with the same continental ancestries. Altogether we carried out 64 genomewide Fst tests and identified 28,215 highly differentiated SNPs for 49 different combinations of HapMap3 samples in the current database. Availability http://FstSNP-hapmap3.googlecode.com/

Chemotherapeutic-induced apoptosis: a phenotype for pharmacogenomics studies.

Aim To determine whether cellular apoptosis is a suitable phenotypic trait for pharmacogenomics studies by evaluating caspase 3/7-mediated activity in lymphoblastoid cell lines after treatment with six chemotherapeutic agents: 5′-deoxyfluorouridine, pemetrexed, cytarabine, paclitaxel, carboplatin, and cisplatin. Materials and methods Using monozygotic twin pair and sibling pair lymphoblastoid cell lines, we identified conditions for measurement of caspase 3/7 activity in lymphoblastoid cell lines. Genome-wide association studies were performed with over 2 million single nucleotide polymorphisms (SNPs) and cisplatin-induced apoptosis in HapMap CEU cell lines (n=77). Results Although treatment with 5′-deoxyfluorouridine and pemetrexed for up to 24 h resulted in low levels of apoptosis or interindividual variation in caspase-dependent cell death; paclitaxel, cisplatin, carboplatin, and cytarabine treatment for 24 h resulted in 9.4-fold, 9.1-fold, 7.0-fold, and 6.0-fold increases in apoptosis relative to control, respectively. There was a weak correlation between caspase activity and cytotoxicity (r2=0.03–0.29) demonstrating that cytotoxicity and apoptosis are two distinct phenotypes that may produce independent genetic associations. Estimated heritability (h2) for apoptosis was 0.57 and 0.29 for cytarabine (5 and 40 &mgr;mol/l, respectively), 0.22 for paclitaxel (12.5 nmol/l), and 0.34 for cisplatin (5 &mgr;mol/l). In the genome-wide association study using the HapMap CEU panel, we identified a significant enrichment of cisplatin-induced cytotoxicity SNPs within the significant cisplatin-induced apoptosis SNPs and an enrichment of expression quantitative trait loci (eQTL). Among these eQTLs, we identified several eQTLs with known function related to apoptosis and/or cytotoxicity. Conclusion Our study identifies apoptosis as a phenotype for pharmacogenomic studies in lymphoblastoid cell lines after treatment with paclitaxel, cisplatin, carboplatin, and cytarabine that may have utility for discovering biomarkers to predict response to certain chemotherapeutics.