Renee Baak-Pablo

Pharmacogenetic Pathway Analysis for Determination of Sunitinib-Induced Toxicity

PURPOSE To identify genetic markers in the pharmacokinetic and pharmacodynamic pathways of sunitinib that predispose for development of toxicities: thrombocytopenia, leukopenia, mucosal inflammation, hand-foot syndrome, and any toxicity according to National Cancer Institute Common Toxicity Criteria higher than grade 2. PATIENTS AND METHODS A multicenter pharmacogenetic association study was performed in 219 patients treated with single-agent sunitinib. A total of 31 single nucleotide polymorphisms in 12 candidate genes, together with several nongenetic variants, were analyzed for a possible association with toxicity. In addition, genetic haplotypes were developed and related to toxicity. RESULTS The risk for leukopenia was increased when the G allele in CYP1A1 2455A/G (odds ratio [OR], 6.24; P = .029) or the T allele in FLT3 738T/C (OR, 2.8; P = .008) were present or CAG in the NR1I3 (5719C/T, 7738A/C, 7837T/G) haplotype (OR, 1.74; P = .041) was absent. Any toxicity higher than grade 2 prevalence was increased when the T allele of vascular endothelial growth factor receptor 2 1191C/T (OR, 2.39; P = .046) or a copy of TT in the ABCG2 (-15622C/T, 1143C/T) haplotype (OR, 2.63; P = .016) were present. The risk for mucosal inflammation was increased in the presence of the G allele in CYP1A1 2455A/G (OR, 4.03; P = .021) and the prevalence of hand-foot syndrome was increased when a copy of TTT in the ABCB1 (3435C/T, 1236C/T, 2677G/T) haplotype (OR, 2.56; P = .035) was present. CONCLUSION This exploratory study suggests that polymorphisms in specific genes encoding for metabolizing enzymes, efflux transporters, and drug targets are associated with sunitinib-related toxicities. A better understanding of genetic and nongenetic determinants of sunitinib toxicity should help to optimize drug treatment in individual patients.

Activation of tumor-promoting type 2 macrophages by EGFR-targeting antibody cetuximab.

Purpose: In a recent randomized phase III clinical trial in metastatic colorectal cancer patients, the addition of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab to bevacizumab and chemotherapy resulted in decreased progression-free survival, in particular for patients with the high-affinity FcγRIIIA. Experimental Design: The presence of natural killer (NK) cells and type 2 (M2) macrophages in colorectal cancer was determined by immunohistochemistry, using antibodies to lineage-specific markers NKp46 and CD68 with CD163, respectively. Influence of tumor-bound cetuximab on M2 macrophages was carried out in vitro with EGFR-expressing tumor cells and short-term differentiated monocytes from blood donors, who were typed for the FcγRIIIA polymorphism (CD16). Results: Antibody-dependent cellular cytotoxicity by NK cells is generally proposed as one of the antitumor mechanisms of mAbs. We found that CD163-positive M2 macrophages are much more abundant in colorectal carcinomas. In vitro analysis of M2 macrophages revealed high levels of Fc-gamma receptors (FcγR) and PD-L1 and production of IL-10 and VEGF but not IL-12. These anti-inflammatory and tumor-promoting mediators were released upon coculture with EGFR-positive tumor cells loaded with low concentrations of cetuximab. Macrophage activation depended on EGFR expression on the tumor cells, FcγRs, target specificity of the mAb and mobility of antibody complexes. Cetuximab-induced macrophage responses were more pronounced for FCGR3A 158-Val (high-affinity) carriers. Conclusion: These results suggest that tumor-promoting M2 macrophages are activated by the therapeutic mAb cetuximab in the local tumor microenvironment and argue that this immune mechanism should be taken into account for the application of therapeutic antibodies. Clin Cancer Res; 17(17); 5668–73. ©2011 AACR.

Influence of the d3-Growth Hormone (GH) Receptor Isoform on Short-Term and Long-Term Treatment Response to GH Replacement in GH-Deficient Adults

OBJECTIVE Recombinant human GH (rhGH) replacement in adults is aimed at improving signs and symptoms of the adult GH deficiency (GHD) syndrome. In children, a common polymorphism of the GH receptor (exon-3 deletion, d3GHR) increases the response to rhGH replacement. The aim of this study was to assess the effects of this polymorphism on the response to rhGH replacement in adults. DESIGN Prospective intervention with rhGH during 1 yr (n = 99) and in a subset during 5 yr (n = 53). PATIENTS AND METHODS The presence of the d3GHR variant was established in GHD patients and linked to short-term and long-term effects of rhGH replacement on IGF-I, lipid metabolism, anthropometric parameters, and bone mineral density. RESULTS Fifty-five patients had two wild-type alleles (56%), whereas 38 patients (38%) had one allele and six patients (6%) had two alleles coding the d3GHR isoform. During short-term rhGH replacement, the increase in IGF-I was higher in patients bearing at least one d3GHR allele, compared with those with two wild-type alleles (at an identical mean dose of rhGH). The decrease in total cholesterol and low-density lipoprotein cholesterol was lower in the group bearing at least one d3GHR allele, whereas the increase in high-density lipoprotein cholesterol was higher, compared with patients with the wild-type genotype. In contrast, these differential responses of GHR genotype could not be demonstrated during long-term rhGH replacement. CONCLUSION The d3GHR genotype contributes, at least for some parameters, to the interindividual differences in efficacy of short-term, but not long-term, rhGH replacement in adults with GHD.

Genotyping of DNA Samples Isolated from Formalin-Fixed Paraffin-Embedded Tissues Using Preamplification

DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is often fragmented and cross-linked and is therefore difficult to genotype. To enable this source of DNA for genotyping analysis using Taqman probes, we tested whether enrichment of the target genes would increase the amount of available DNA. For enrichment of the target genes, we used preamplification by means of diluted Taqman assays. To establish the appropriateness of preamplification, we used DNA extracted from paraffin-embedded tissue and compared the genotyping results of a series of single nucleotide polymorphisms assessed in DNA samples with and without preamplification. In a subset of patients, DNA was isolated from both blood and FFPE tissue to test the reliability of genotyping results derived after preamplification. We found an increase in call rate after preamplification and a convincing concordance in genotype. Based on our findings, we can safely conclude that preamplification of DNA isolated from paraffin-embedded tissue is a valuable and reliable method to optimize genotyping results.

Association of ABCB1, 5-HT3B Receptor and CYP2D6 Genetic Polymorphisms with Ondansetron and Metoclopramide Antiemetic Response in Indonesian Cancer Patients Treated with Highly Emetogenic Chemotherapy

OBJECTIVE Suboptimal treatment of chemotherapy-induced nausea and vomiting and unsatisfactory response to antiemetic drugs cause impairment of cancer patients daily functioning. This study was aimed to investigate the association of selected germline polymorphisms with ondansetron and metoclopramide response in Indonesian cancer patients treated with highly emetogenic chemotherapy. METHODS We enrolled 202 chemotherapy naïve patients treated with cisplatin at a dosage of ≥50 mg/m(2) as monotherapy or as combined chemotherapy. Ondansetron 8 mg and dexamethasone 8 mg intravenously were the standard antiemetic therapy for prevention of acute chemotherapy-induced nausea and vomiting. Metoclopramide 10 mg orally, three times per day as fixed prescription, was given until 5 days after chemotherapy to prevent delayed chemotherapy-induced nausea and vomiting. Primary and secondary outcomes were the occurrence of chemotherapy-induced nausea and vomiting in the acute and delayed phase. The following single-nucleotide polymorphisms were determined in ABCB1: rs1045642, rs2032582 and rs1128503; in 5-HT3B-R: rs45460698, rs4938058 and rs7943062; and in CYP2D6: rs16947 (CYP2D6 2), rs3892097 (CYP2D6 4) and rs1065852 (CYP2D6 10) using Taqman assays. RESULTS During the acute phase, 21.8 and 30.2% patients experienced Grade 3 and 4 nausea and vomiting, respectively, whereas 38.6% patients experienced nausea and/or vomiting in the delayed phase. Carriers of the CTG haplotype of the ABCB1 gene experienced Grade 3 and 4 chemotherapy-induced nausea and vomiting more often than other haplotypes in the delayed phase (P< 0.05). No associations were found with the 5-HT3B receptor haplotypes and CYP2D6-predicted phenotypes. CONCLUSIONS Our study shows that in Indonesian cancer patients treated with highly cytostatic emetogenic, carriership of the CTG haplotype of the ABCB1 gene is related to an increased risk of delayed chemotherapy-induced nausea and vomiting.

Use of plasmid-derived external quality control samples in pharmacogenetic testing

OBJECTIVES Genetic variation in genes encoding for drug-metabolizing enzymes, drug targets and signaling pathways have proven to contribute significantly to differences in drug response. Pharmacogenetics is now expanding from clinical pharmacological research to its application in clinical practice. Genotyping of patients in a routine clinical setting requires robust and reliable genotyping methods. MATERIALS & METHODS A survey of pharmacogenetic association studies for quality control samples published from 2005 to 2007 in the two most prominent pharmacogenetic journals, and development of plasmid-derived external controls. RESULTS Surveying journals revealed that only a minority of papers report the use of quality controls, and no standard procedures are applied. We established 12 plasmid-derived external controls and applied these in pharmacogenetic testing. CONCLUSION There still is a need for quality control materials, especially for application in pharmacogenetic testing. We hope that our initiative to create plasmid-derived controls will help to facilitate quality in the pharmacogenetic genotyping tests applied in research, as well as in routine patient care.

Genetic risk factors for type 2 diabetes mellitus and response to sulfonylurea treatment.

Objective After the identification of type 2 diabetes mellitus (T2DM) risk alleles from genome-wide association studies, models have been developed to identify subjects at high risk to develop T2DM. We hypothesize that a panel of 20 repeatedly associated T2DM risk alleles influences response to sulfonylureas (SUs). Methods Two hundred and seven incident SU (tolbutamide, glibenclamide, glimepiride, gliclazide) users with T2DM were recruited from four primary care centers. A genetic risk score per patient was calculated based on the number of risk-alleles. With this score, patients were categorized into three predefined genetic risk groups. The effect of the genetic risk group on the achievement of stable SU dose, prescribed stable SU dose, and time to stable SU dose was analyzed. Results Carriers of more than 17 T2DM risk alleles had a 1.7-fold reduced likelihood to achieve stable SU dose (P=0.044). No significant effect of the number of T2DM risk alleles on prescribed dose was found. Carriers of more than 17 T2DM risk alleles showed a marginally significant increased time to stable dose (hazard ratio: 0.81; 95% confidence interval, 0.75–1.01, P=0.058). Conclusion T2DM risk alleles are associated with response to SUs in primary care T2DM patients. This suggests that individualization of T2DM treatment according to genetic profile may be an opportunity to improve clinical outcome.

Alternative methods to a TaqMan assay to detect a tri-allelic single nucleotide polymorphism rs757210 in the HNF1β gene.

Abstract Background: Several studies report difficulties in genotyping HNF1β rs757210 using TaqMan probes. This is possibly due to the tri-allelic nature of this single nucleotide polymorphism (SNP). The aim of the present research was to develop alternative methods for genotyping rs757210. Methods: Pyrosequencing and high resolution melting analysis of small amplicons (HRM) were developed and tested in panels of type 2 diabetes mellitus patients (n=258) and healthy blood donors (n=183). Results were confirmed by Sanger sequencing. Results: With pyrosequencing, allele frequencies for the A, G and C allele of 0.42, 0.56, 0.02 and 0.37, 0.62, 0.01 were established in the panel of type 2 diabetes mellitus patients and healthy blood donors, respectively. Similar results were found using the more routinely available HRM method. Results for pyrosequencing and HRM were in 99.6% concordance. Conclusions: Pyrosequencing and HRM can be used to genotype the tri-allelic SNP rs757210 in the HNF1β gene and have the advantage over the commercially available TaqMan analysis that they can determine the rare C-allele variant.

Clinical validation study of genetic markers for capecitabine efficacy in metastatic colorectal cancer patients

Background and aim Pharmacogenetic studies continue to search for pretreatment predictors of chemotherapeutic efficacy and toxicity in metastatic colorectal cancer. Both genome-wide association studies and candidate gene studies have yielded potential genetic markers for chemosensitivity. We conducted a clinical association study, validating the effect of specific genetic markers cited in recently published papers on the efficacy of the oral 5-fluoro-uracil prodrug capecitabine. Patients and methods Germline DNA was collected for 268 metastatic colorectal cancer patients from the CAIRO trial, a multicenter phase III trial, randomizing between combined or sequential first-line treatment with capecitabine, irinotecan, and oxaliplatin. Genotyping was performed for eight single-nucleotide polymorphisms (SNPs), using high-resolution melting curves. Four SNPs are located in the MTRR gene, and another four SNPs showed significant association with 5-fluoro-uracil cytotoxicity in a recent in-vitro genome-wide association study. The primary endpoint was progression-free survival (PFS); secondary endpoints were objective response and overall survival. Results In patients receiving capecitabine monotherapy, rs4702484, located in ADCY2 and close to MTRR, was associated with slightly reduced PFS for homozygous wild-type patients (CC 6.2 vs. CT 8.0 months; P=0.018). For the other selected genetic markers, we found no association with PFS, overall survival, or radiologic response upon treatment with capecitabine, either in the total study population or in the capecitabine monotherapy subgroup. Conclusion With the exception of rs4702484, we found no evidence of an effect on capecitabine chemosensitivity for any of the studied SNPs. More specifically, variants in methionine synthase reductase (MTRR) are not likely associated with capecitabine efficacy.

Pharmacogenetics in transplant patients: mind the mix.

To the Editor: Several consortia have published guidelines to aid clinicians with the interpretation of pharmacogenetic test results,1,2 and an increasing number of medical centers are implementing prospective genotyping.3 Among these, there are many highly specialized care centers with complex patient populations. These patients may present unexpected challenges, as demonstrated by this case.