7R Allele Research Articles

Association between dopamine D4 receptor polymorphism and age related changes in brain glucose metabolism.

Aging is associated with reductions in brain glucose metabolism in some cortical and subcortical regions, but the rate of decrease varies significantly between individuals, likely reflecting genetic and environmental factors and their interactions. Here we test the hypothesis that the variant of the dopamine receptor D4 (DRD4) gene (VNTR in exon 3), which has been associated with novelty seeking and sensitivity to environmental stimuli (negative and positive) including the beneficial effects of physical activity on longevity, influence the effects of aging on the human brain. We used positron emission tomography (PET) and [18F]fluoro-D-glucose (18FDG) to measure brain glucose metabolism (marker of brain function) under baseline conditions (no stimulation) in 82 healthy individuals (age range 22–55 years). We determined their DRD4 genotype and found an interaction with age: individuals who did not carry the 7-repeat allele (7R−, n = 53) had a significant (p<0.0001) negative association between age and relative glucose metabolism (normalized to whole brain glucose metabolism) in frontal (r = −0.52), temporal (r = −0.51) and striatal regions (r = −0.47, p<0.001); such that older individuals had lower metabolism than younger ones. In contrast, for carriers of the 7R allele (7R+ n = 29), these correlations with age were not significant and they only showed a positive association with cerebellar glucose metabolism (r = +0.55; p = 0.002). Regression slopes of regional brain glucose metabolism with age differed significantly between the 7R+ and 7R− groups in cerebellum, inferior temporal cortex and striatum. These results provide evidence that the DRD4 genotype might modulate the associations between regional brain glucose metabolism and age and that the carriers of the 7R allele appear to be less sensitive to the effects of age on brain glucose metabolism.

Abstract 2372: Predictive markers in patients with non-small cell lung cancer with pemetrexed treatment.

Abstract Background: This study is desiged to evaluate biomarkers that are correlated with the efficacy of pemetrexed in patients with non-small-cell lung cancer (NSCLC). Methods: Patients with stage III or IV NSCLC who received pemetrexed monotherapy after failure of one or more chemotherapy regimen between January 2003 and August 2011 were included. Polymorphism of reduced folate carrier gene (SLC19A, rs1051298), 5,10-methylenetetrahydroflate reductase (MTHFR, rs1801133) and thymidylate synthase (TS, rs45445694, rs16430) gene were analyzed by PCR and direct sequencing method using peripheral blood. Results: Total 123 patients were included. The median age was 61 years (range 35-82). Fifty eight (47.2%) patients were female. Predominant histology was adenocarcinoma (92.7%). Metastatic diseases were 114 (80.5%) patients. Patients received one or more lines of systemic therapy before pemetrexed therapy, one line in 53 (43.1%) patients, two in 51(41.5%), three in 14 (11.02%), and four in 4 (3.3%). Median 4 cycles of chemotherapy were given (range 1-44). Response rate was 22.7% (CR, n=2, PR, n=26) and stable disease was detected in 54 patients (43.9%). Median follow up duration was 28.7months (range 4.07-84.33months). Median progression free survival (PFS) was 4.2 months (95% CI: 2.9-5.6) and median overall survival (OS) was 18.9 months (95% CI: 13.3-24.6). Clinical factors like, sex, histology of tumor (adenocarcinoma vs, nonadenocarcinoma), differentiation, smoking status and EGFR mutation status (active mutation vs. other mutation vs. wild type) did not have any significant difference on response and PFS. Only polymorphisms of TS promoter enhancer region (TSER) were related with PFS. 2R/2R repeat in the TSER (2R/2R vs 2R/3R, 3R/3R 1.0m vs 4.3m, p&amp;lt;0.01) and CC homozygote variant in second repeat of the 3R allele (CC vs GC, GG 2.8m vs 5.0m, p=0.04) had poor PFS. All polymorphism did not have effect on response rate and overall survival. Conclusions: This study suggests that polymorphisms of TSER associated PFS in pemetrexed treatment. Further prospective studies will be needed to validate these polymorphism as a useful biomarker of pemetrexed treatment in NSCLC patients. Citation Format: Hiun S. Chae, Yoon Ho Ko, Eun Kyoung Jeon, Sook Hee Hong, Jeong-Oh Kim, Seung Joon Kim, Kyo Young Lee, Young Kyoon Kim, HoonKyo Kim, Seok young Park, In Sook Woo, Jin Hyoung Kang. Predictive markers in patients with non-small cell lung cancer with pemetrexed treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2372. doi:10.1158/1538-7445.AM2013-2372

Prognostic significance of thymidylate synthase polymorphisms in rectal cancer patients treated with neoadjuvant chemoradiotherapy

There is a lack of prognostic factors of preoperative chemoradiation for locally advanced rectal cancer. Thymidylate synthase (TS) is the most important target of 5-fluorouracil; three main genetic polymorphisms of TS have been described. We analysed the prognostic value of these in patients with locally advanced rectal cancer treated with fluoropyrimidine-based chemoradiation. Ninety-nine patients treated between November 2001 and March 2009 were included. All were treated by radiotherapy (5040 cGy) and concomitant fluoropyrimidine-based chemotherapy. Three polymorphisms were analysed: (i) a double (2R) or triple (3R) repeat of a 28 base pair (bp) tandem sequence upstream of the ATG codon initiation site in the 5'-terminal regulatory region, (ii) a functional G > C single nucleotide polymorphism present in the second repeat of the 3R alleles and (iii) a 6 bp deletion at nucleotide 1494 in the 3'-untranslated region. DNA was extracted from paraffin-embedded core biopsies taken from the tumour and the genotype was analysed using polymerase chain reaction restriction fragment length polymorphism. The 6 bp polymorphism was significantly associated with disease-free survival (+ 6 bp/+ 6 bp vs-6 bp/-6 bp, P = 0.032 logistic regression). No differences were found in disease-free survival according to the other polymorphisms studied. No relationship was observed between the different TS genotypes and pathological regression. The study suggests that the TS 6 bp polymorphism may be a predictor of disease-free survival in patients with locally advanced rectal cancer treated with fluoropyrimidine-based chemoradiation.

Dopaminergic D4 receptor polymorphism among 24 populations of India: an anthropological insight

Various studies have tried to elucidate the genomics underlying the ethnic diversity of Indian populations. The 48-basepair variable number tandem repeat (VNTR) of the DRD4 gene, besides its clinical importance, has been a subject of interest for understanding human evolutionary history worldwide. In spite of its highly polymorphic nature it has not been much explored by anthropologists, especially in the Indian context. In the present study, an attempt was made to infer the allelic variation of the repeat allele among 24 Indian ethnic groups belonging to different ethnic and linguistic groups along with different geographic affiliations. Only the 2R, 3R, 4R, 5R, 6R, and 7R repeat alleles were found in the studied populations, with 4R being the most frequent followed by 2R. All the other repeat alleles (3R, 5R, 6R, and 7R) occurred at very low frequency. Based on the 4R and 5R frequencies, the study reveals a common genetic substratum of the Indian population as already proposed by earlier studies. Further, the study also highlights the inflow of genes from the north.

The novel allele (3R) of the VNTR polymorphism in the XRCC5 promoter region dramatically decreases the gene expression

Polymorphism of variable number of tandem repeats (VNTR) in the promoter region of X-ray repair cross-complementing 5 (MIM: 194364, XRCC5; rs6147172) was reported. The aim of the present study is to evaluate the influence of this polymorphism on XRCC5 mRNA levels. Genotypes of XRCC5 VNTR were determined by high resolution of melting analysis (HRMA). The quantitative XRCC5 mRNA expression (compared to ß-actin expression) among 0R/1R, 1R/2R, and 1R/3R genotypes was investigated. There was a negative correlation between the overall number of tandem repeats and XRCC5 expression (r=−0.965, df=7, P<0.001). The mRNA level of XRCC5 decreased as function of number of tandem repeats. The 3R allele of the VNTR polymorphism in the XRCC5 promoter region dramatically decreases the gene expression.

Thymidylate synthase gene (TYMS) polymorphisms in sporadic and hereditary breast cancer

BackgroundBreast cancer (BC) is a genetic disorder characterized by growth and proliferation of breast cells in a disorderly. In Brazil, there are approximately 49.240 new cases of BC, every year. The BC etiology is still poorly understood. The BC can be sporadic (SBC) or hereditary (HBC). Recent studies have correlated gene polymorphisms with the BC, such as alterations in thymidylate synthase gene (TYMS), which are used to improve diagnosis and prevention of the disease. Polymorphisms in the TYMS gene 5’-UTR region, usually present reps double (2R) and/or triple (3R). Studies have shown that homozygous 3R/3R is overexpressed compared with 2R/2R genotype, and these polymorphic variations may contribute to individual susceptibility to the development of BC. In this context, the objective of this study was to evaluate the frequency of the TYMS 2R and 3R polymorphisms, comparing genotypic and allelic distribution with SBC and HBC patients.MethodsIn this study we included a total of 204 subjects, 70 with BC (33 with SBC, and 37 with HBC) and 134 healthy subjects (controls). The Polymerase Chain Reaction was the method used.ResultsResults demonstrated a high frequency of the 3R allele at BC, SBC, and HBC groups. The frequency of genotype 2R/3R was significantly higher in BC group. This work showed association between the 2R/3R variants (OR = 4.14, CI95% = 1.77-9.71) in the development of SBC, and 2R/2R (OR = 0.233, CI95% = 1.63-7.65) and 2R/3R (OR = 3.53, CI95% = 0.06-0.81) for developing HBC. To BC, there was association with the genotype 2R/3R (OR: 3.79, CI95% = 2.03-7.08).ConclusionOur results show relation to the development of BC in association with the analyzed polymorphisms.

DRD4 gene and obsessive compulsive disorder: Do symptom dimensions have specific genetic correlates?

The dopamine D4 receptor (DRD4) is a promising candidate gene in obsessive compulsive disorder (OCD). A 48-bp variable number of tandem repeats (VNTR) sequence in exon 3 has been studied previously, and alleles containing 2-11 repeats (2R-11R) have been identified. We investigated the association of DRD4 VNTR polymorphism with OCD and its relationship with various clinical parameters (age of onset, gender, family history, co-morbidity, factor-analyzed symptom dimensions and insight). One hundred and seventy three South Indian OCD patients (DSM-IV) recruited from a specialty OCD clinic were evaluated using the Yale-Brown obsessive compulsive scale (YBOCS), YBOCS item-11 for insight, Mini International Neuropsychiatric Interview (MINI) plus, tic disorder subsection of the MINI-KID and Clinical Global Impression scale. 201 healthy controls were evaluated using MINI plus. All subjects were genotyped for the DRD4 VNTR polymorphism. Genotype frequencies did not deviate significantly from the Hardy-Weinberg equilibrium. Case-control association analysis revealed that the 7R allele frequency was significantly greater in OCD patients than controls. This difference was restricted to the women subsample when performing the gender sub-analysis. Among other clinical variables examined, factor 3 (symmetry) was associated with presence of 2R allele. Linear regression analysis confirmed the association of symmetry dimension with the 2R allele (Beta=0.23, t=2.96, p=0.004, CI=0.19-0.95). Our data provides further evidence that DRD4 VNTR polymorphism is associated with OCD. Furthermore, the presence of the 2R allele was significantly associated with the symmetry dimension. This dimension may represent a more homogeneous subtype of OCD with a genetic etiology.

Variación de alelos del gen receptor de dopamina DRD4 en escolares chilenos de diferente origen étnico y su relación con riesgo de déficit atencional/hiperactividad

Worldwide diversity of alleles of D4 receptor gene (DRD4), linked to attention deficit hyperactivity disorder (ADHD), is mostly the result of length and single nucleotide polymorphisms in a 48-bp tandem repeat (VNTR). Alleles containing from two (2R) to eleven (11R) repeats have been identified. The most common are 4R, 7R and 2R.To study the association of ADHD risk with DRD4 genotypes in Chilean students.ADHD risk data were obtained through the abbreviated Conner's Scale for School Teachers in 66 Aymara children (11 cases and 55 controls), 91 Rapa-Nui children (60 cases ad 31 controls) and 96 children from a mixed urban population from Santiago (51 cases and 45 controls). DNA extracted from saliva was amplified by polymerase chain reaction (PCR) to genotype the DRD4 VNTR.The distribution of DRD4 alleles reveals that, beneath the 4R allele, 7R exhibits the second highest frequencies in Aymara and Santiago children. In Polynesian children, 2R ranks after 4R. A statistically significant association between ADHD risk and 2R/4R genotype was identified in Polynesian children (p < 0.05; odds ratio = 3.7).Different DRD4 genotypes are associated with ADHD phenotype in Chilean populations, probably as a consequence of their initial colonization history.

Correlation between thymidylate synthase gene polymorphisms and efficacy of pemetrexed in advanced non-small cell lung cancer

One of the target genes of pemetrexed (PEM), thymidylate synthase (TS), has been shown to have a close association with its efficacy. TS gene polymorphisms have been shown to be associated with the efficacy of antifolate treatment in enteron tumors. The purpose of this study was to investigate the clinical significance of TS gene polymorphisms in patients with advanced NSCLC receiving PEM-based treatment. The variable nucleoid tandem repeat in the 5′-UTR region was amplified and detected using fluorescently labeled multiplex short tandem repeat polymerase chain reaction. The polymorphism in the 3′-UTR region of the TS gene was detected using the Taqman probe. Efficacy of PEM was assessed according to the Response Evaluation Criteria in Solid Tumors, version 1.1. None of the genotypes were associated with gender, smoking status and age. Disease control rate (DCR), objective response rate (ORR) and progression-free survival (PFS) were similar between patients harboring 2R and 3R alleles (PFS, p=0.518; DCR, p=0.631; ORR, p=0.541), as well as those with a 6-bp insertion and 6-bp deletion (PFS, p=0.776; DCR, p=0.626; ORR, p=0.330). To study the combined effect of TS polymorphisms, the study population was divided into three groups: 2R&6 del, 2R&6 ins and 3R&6 del. No significant differences were observed among the different groups according to DCR (p=0.517), ORR (p=0.611) and PFS (p=0.938). In conclusion, polymorphisms of the TS gene do not appear to be a prognostic marker for advanced NSCLC patients receiving PEM-based treatment.

Introducing a novel allele for the polymorphism of variable number of tandem repeats in the promoter region of XRCC5

Polymorphism of variable number of tandem repeats (VNTR) in the promoter region of X-ray repair cross-complementing 5 (XRCC5; MIM: 194364, rs6147172) has been reported. The main aim of the present study is to introduce a novel allele for the VNTR polymorphism in the promoter region of XRCC5. The participants of the present study were of 535 (140 males, 395 females), unrelated, adult, healthy Iranian blood donors (Caucasians/Muslims). Genotypes of XRCC5 VNTR were determined by a high resolution melting analysis, and confirmed by DNA sequencing. Based on the sequencing of new bands upper than the 2R allele band, a novel allele was introduced (named 3R allele). The promoter region of XRCC5 contains several copies of Sp1 recognition cis regulatory elements. The novel 3R allele is capable of expanding the number of cis regulatory elements to eight. The prevalence of the 0R, 1R, 2R and 3R alleles in our sample was 0.0645, 0.5439, 0.3794 and 0.0122, respectively. The study group was at the Hardy–Weinberg equilibrium for the genotypic frequencies (χ2=3.95, df=6, P=0.73). It is suggested that the prevalence of the novel allele (3R allele) among European populations may be higher than its prevalence among Iranians.

Polymorphisms in the Thymidylate Synthase Gene and Risk of Colorectal Cancer

To evaluate the relationship between polymorphisms (28 bp repeated sequences in 5'-UTR and 6-bp ins/ del in 3'-UTR) in then thymidylate synthetase gene (TS) and risk of colorectal, colon and rectal cancers, we conducted a case-control study with 315 cases of colorectal cancer and 439 population-based controls in Jiangsu province, China. TS genotypes were identified using PCR-RFLP (restriction fragment length polymorphism) methods. Odds ratios (ORs) were estimated with an unconditional logistic regression model. We found that the distributions of 5'-UTR genotypes in TS were significantly different between controls and male colon cases (χ2 =8.25, P = 0.016). Compared with 3R/3R genotype, individuals with the 2R allele were at an increased risk of colon cancer (age-, BMI-, smoking- and alcohol drinking-adjusted OR=1.98, 95%CI: 1.11-3.53) among men. In contrast, the 6-bp ins/del polymorphism at the TS 3'- UTR did not influence risk of the colorectal, colon and rectal cancers. When combined genotypes for both TS 5'-UTR and 3'-UTR polymorphisms were evaluated, individuals with the 5'-UTR 2R allele had a OR of 3.61 (95%CI: 1.38-9.49) for colon cancer among men with the 3'-UTR -6bp/-6bp genotype. These results show that the polymorphism of the 28 bp repeated sequences in TS 5'-UTR could influence susceptibility to colon cancer and that there was a coordinated effect between TS 3'-UTR and 5'-UTR polymorphisms in increasing risk of colon cancer among Chinese men.

Dopamine Genes (DRD2/ANKK1-TaqA1 and DRD4-7R) and Executive Function: Their Interaction with Obesity

Obesity is a multifactorial disease caused by the interaction between genotype and environment, and it is considered to be a type of addictive alteration. The A1 allele of the DRD2/ANKK1-TaqIA gene has been associated with addictive disorders, with obesity and with the performance in executive functions. The 7 repeat allele of the DRD4 gene has likewise been associated with the performance in executive functions, as well as with addictive behaviors and impulsivity. Participants were included in the obesity group (N = 42) if their body mass index (BMI) was equal to or above 30, and in the lean group (N = 42) if their BMI was below 25. The DRD2/ANKK1-TaqIA and DRD4 VNTR polymorphisms were obtained. All subjects underwent neuropsychological assessment. Eating behavior traits were evaluated. The ‘DRD2/ANKK1-TaqIA A1-allele status’ had a significant effect on almost all the executive variables, but no significant ‘DRD4 7R-allele status’ effects were observed for any of the executive variables analyzed. There was a significant ‘group’ x ‘DRD2/ANKK1-TaqIA A1-allele status’ interaction effect on LN and ‘group’ x ‘DRD4 7R-allele status’ interaction effect on TMT B-A score. Being obese and a carrier of the A1 allele of DRD2/ANKK1-TaqIA or the 7R allele of DRD4 VNTR polymorphisms could confer a weakness as regards the performance of executive functions.

Association of the TYMS 3G/3G genotype with poor response and GGH 354GG genotype with the bone marrow toxicity of the methotrexate in RA patients

Gamma-glutamyl hydrolase (GGH), cyclin D1 (CCND1) and thymidylate synthase (TS) genes encode enzymes that are involved in methotrexate (MTX) action. In a group of 184 RA patients treated with MTX, we have investigated whether selected polymorphisms in these genes modulate MTX efficacy and/or have impact on adverse drug effects (ADEs). The efficacy of the MTX therapy has been estimated using the disease activity score in 28 joints (DAS28-ESR) based on EULAR criteria and relative DAS28 values (rDAS28). All adverse drug events were recorded. Patients were genotyped for selected polymorphisms of the GGH (-354 G > T and 452 C > T), CCND1 (870 A > G) and TYMS (variable number of tandem repeats, VNTR, and G to C substitution of triple repeat, 3R allele) gene. Association studies have been performed between obtained genotypes and the efficacy and toxicity of MTX. According to the EULAR response criteria, 146 RA patients (79.3 %) were classified as responders (good/moderate response) and 38 (20.7 %) as non-responders (poor response). Higher frequency of the TYMS 3 G/3 G genotype has been found among non-responders as compared to individuals with remaining genotypes (p = 0.02). ADEs were recorded in 53 patients. Among those patients eight experienced bone marrow toxicity, all of them carried GGH -354GG genotype (p = 0.003). No other significant association were observed. The 3 G/3 G genotype of the TYMS gene may indicate predisposition of poor response to MTX and GG genotype of GGH -354 T > G polymorphism may have high predictive value for myelosuppression in RA patients.

Molecular Variation at the SLC6A3 Locus Predicts Lifetime Risk of PTSD in the Detroit Neighborhood Health Study

Recent work suggests that the 9-repeat (9R) allele located in the 3′UTR VNTR of the SLC6A3 gene increases risk of posttraumatic stress disorder (PTSD). However, no study reporting this association to date has been based on population-based samples. Furthermore, no study of which we are aware has assessed the joint action of genetic and DNA methylation variation at SLC6A3 on risk of PTSD. In this study, we assessed whether molecular variation at SLC6A3 locus influences risk of PTSD. Participants (n = 320; 62 cases/258 controls) were drawn from an urban, community-based sample of predominantly African American Detroit adult residents, and included those who had completed a baseline telephone survey, had provided blood specimens, and had a homozygous genotype for either the 9R or 10R allele or a heterozygous 9R/10R genotype. The influence of DNA methylation variation in the SLC6A3 promoter locus was also assessed in a subset of participants with available methylation data (n = 83; 16 cases/67 controls). In the full analytic sample, 9R allele carriers had almost double the risk of lifetime PTSD compared to 10R/10R genotype carriers (OR = 1.98, 95% CI = 1.02–3.86), controlling for age, sex, race, socioeconomic status, number of traumas, smoking, and lifetime depression. In the subsample of participants with available methylation data, a significant (p = 0.008) interaction was observed whereby 9R allele carriers showed an increased risk of lifetime PTSD only in conjunction with high methylation in the SLC6A3 promoter locus, controlling for the same covariates. Our results confirm previous reports supporting a role for the 9R allele in increasing susceptibility to PTSD. They further extend these findings by providing preliminary evidence that a “double hit” model, including both a putatively reduced-function allele and high methylation in the promoter region, may more accurately capture molecular risk of PTSD at the SLC6A3 locus.

Gene polymorphisms of folate metabolizing enzymes and susceptibility to lung adenocarcinoma in Serbia.

e12020 Background: Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) are important enzymes of the folate metabolism and are suggested as new prognostic factors for lung cancer. Cytosine to thymine transition at nucleotide 677 (C677T) leads to reduced MTHFR activity. The TS promoter has a tandem repeat polymorphism (2R, 3R) and a guanine to cytosine transition in the 3R allele related to TS protein expression. The aim of this study was to analyze the association of MTHFR and TS polymorphisms with lung adenocarcinoma in Serbia. Methods: A case-control study including 55 late-stage lung adenocarcinoma patients and 53 healthy subjects was performed. Restriction fragment length polymorphism analysis was used for MTHFR and TS genotyping. Depending on the presence of high (3RG) or low (2R, 3RC) expression alleles, TS functional groups were subclassified into HH (3RG/3RG), HL (2R/3RG, 3RG/3RC) and LL (2R/2R, 2R/3RC, 3RC/3RC) groups. Descriptive analyses included genotype and allelic frequencies; the odds ratio (OR) and 95 % confidence interval (CI) were calculated as an estimate of relative risk. Significance was considered for p &lt; 0.05. Results: The distribution of the MTHFR variants in patients vs. controls was 61.8 % vs. 24.5 % for CC, 32.7 % vs. 62.3 % for CT and 5.5 % vs. 13.2 % for TT. A significant difference in CC vs. TT+CT MTHFR genotype distribution was observed between patients and controls (χ2 = 13.79; OR = 4.98; 95 % CI, 2.14 – 11.61). There was no significant association between the TS polymorphisms and the risk of lung adenocarcinoma occurrence, but in the CT+TT MTHFR subgroup, a nonsignificant difference in LL vs. HL+HH TS genotype distribution was observed between patients and controls (χ2 = 0.04; OR = 1.07; 95% CI, 0.31 – 3.67). Conclusions: A significant corelation between the CC MTHFR genotype and lung adenocarcinoma occurence in Serbia was found. Also, there was no significant correlation with lung adenocarcinoma occurence in the CT+TT MTHFR subgroup for carriers of the LL TS genotype. As this study was performed on a relatively small sample size further large case-control studies including analysis of gene-gene interactions with genes coding for other folate metabolism enzymes is needed.

Thymidylate synthase genotype specific dosing of capecitabine: Proof of concept phase I study.

2551 Background: Thymidylate synthase (TYMS) is the target of fluoropyrimidines (FP). TYMS 3R3R is the predominant genotype in East Asian (EA) patients and is associated with increased TYMS expression, reduced FP related toxicity and relative FP resistance. Dose finding studies for FP were developed in Caucasians, a population that typically harbors TYMS 2R2R and 2R3R genotypes. We hypothesize the recommended phase II dose (RP2D) of capecitabine is higher in 3R3R and similar for 2R allele carriers compared to the FDA approved dose. Objectives 1) To determine the maximal tolerated dose (MTD) and RP2D of capecitabine in EA patients with advanced stage malignancy 2) To determine the safety and toxicity of this regimen and 3) To perform plasma pharmacokinetics (PK) of capecitabine and its metabolites. Methods: EA patients with advanced stage cancer were prospectively allocated into two cohorts: Group A (3R3R) and Group B (2R3R, 2R2R). In each cohort, dose escalation followed a standard phase I 3+3 design. Additional patients were treated at the R2P2 dose level (DL). Initial dose was 1250/m2 bd for 14 days q3w (DL 1) with 125 mg/m2 bd increments subsequently. Pharmacokinetics (PK) of capecitabine and its metabolites were performed using a LC-MS/MS method. Results: 23 patients (Group A=18; group B=5) received 94 cycles (median 2.5, range 1-8). Median age was 58 (range 34-74) years. Median turnaround time for TYMS genotyping was 1 day. In group A, grade 3-4 DLTs were seen in 3 patients: diarhoea, neutropenic fever, hand-foot syndrome, and mucositis. MTD was at DL 4 (1625mg/m2 bd) and the RP2D was 1500mg/m2 bd (DL 3). DL 3 was expanded to n=9. At DL 3, day 1 mean (± SD) capecitabine and 5FU Cmax was 12.3 ± 9.9 and 0.91 ± 0.73 µg/mL, respectively and AUC0 – t was 9.84 ± 5.53 and 1.21 ± 0.54 h*µg/mL, respectively. Compared with DL1, Cmax for capecitabine and 5FU was 2 and 2.02 fold higher and AUC 0-t was 1.49 and 1.59 fold higher at DL3. Accrual in group B was ceased at DL2 due to lack of patient enrolment; no DLT was seen. By ROC analysis, day 1 5FU AUC0 – t of 1.74 h*µg/mL predicted for DLT (p=0.022, sensitivity 100%, specificity 90%). Conclusions: In EA patients with TSER 3R3R, the RP2D was 1500 mg/m2 bd. The D1 5FU AUC0 – t at RP2D was 59% more than the FDA approved dose (DL1).

Abstract 1874: The role of functional polymorphisms in folate metabolism on 6-mercaptopurine-mediated hematological toxicity in children with acute lymphoblastic leukemia

Abstract Purpose: Defective S-methylation of 6-mercarptopurine (6-MP) either due to reduced activity of thiopurine s-methyltransferase (TPMT) or due to reduced synthesis of S-adenosyl methionine (SAM) induces hematological toxicity in children undergoing 6-MP treatment for acute lymphoblastic leukemia (ALL). Recently, we have demonstrated the genetic variants of TMPT results in hematological toxicity when on 6-MP. In the current study, we have aimed to explore the role of seven functional polymorphisms in folate metabolism as pharmacogenomic determinants of 6-MP mediated toxicity. Experimental Procedures: In 126 children with ALL being treated with 6-MP, Glutamate carboxypeptidase II (GCPII) C1561T, Reduced folate carrier 1 (RFC1) G80A, cytosolic serine hydroxymethyl transferase (cSHMT) C1420T, Thymidylate synthase (TYMS) 3′-UTR ins6/del6, Methylene tetrahydrofolate reductase (MTHFR) C677T, Methionine synthase (MTR) A2756G polymorphisms were analysed by PCR-RFLP method while TYMS 5′-UTR 28 bp tandem repeat analysis was done by PCR-AFLP method. Summary: Univariate analysis showed positive association of GCPII C1561T (r= 0.23) and cSHMT C1420T (r=0.15) variants with hematological toxicity. Multi dimensionality reduction analysis confirmed synergetic gene-gene interaction between these two loci (×2=5.9, p=0.02) apart from showing an interesting tri-variate interaction i.e, RFC1 G80A/cSHMT C1420T/TYMS 5′-2R3R. RFC1 G80A showed additive interaction with cSHMT while TYMS 2R alleles showed counteracting interaction. Toxicity grade prediction model was developed using multiple linear regression approach, which showed moderate predictability of aberrations in folate metabolism and TPMT*3C (Area under the receiver operating characteristic curve=0.69) thus explaining 36% variation in toxicity. Conclusion: Aberrations in folate metabolism and TPMT*3C contribute to 36% 6-MP mediated toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1874. doi:1538-7445.AM2012-1874

Multifactorial pharmacogenetic analysis in colorectal cancer patients receiving 5‐fluorouracil‐based therapy together with cetuximab–irinotecan

To examine the predictive value of gene polymorphisms potentially linked to toxicity, clinical response, time to progression and overall survival, following cetuximab-tegafur-uracil (UFT)-irinotecan therapy. Fifty-two patients with advanced colorectal cancer were enrolled in an ancillary pharmacogenetic study of the phase II CETUFTIRI trial. Treatment consisted of 21 day cycles of cetuximab (day 1-day 8-day 15, 250 mg m(-2) week(-1) following a 400 mg m(-2) initial dose) together with irinotecan (day 1, 250 mg m(-2)) and UFT-folinic acid (days 1-14, 250 mg m(-2) day(-1) UFT, 90 mg day(-1) folinic acid). Analysed gene polymorphisms (blood DNA) were as follows: EGFR (CA repeats in intron 1, -216G>T, -191C>A), EGF (61A>G), FCGR2A (131Arg>His), FCGR3A (158Phe>Val), UDP-glycosyltransferase1-polypeptide A1 (TA repeats), TYMS (28 bp repeats, including the G>C mutation on the 3R allele, 6 bp deletion in 3' UTR) and MTHFR (677C>T, 1298A>C). Maximum toxicity grade was linked to EGFR-191C>A polymorphism, with 71.1% grade 3-4 toxicity in CC patients vs. 28.6% in other patients (P= 0.010). A tendency to a better response was observed in patients bearing the TYMS 3RG allele (P= 0.029) and those bearing the FCGR3A 158Val genotype (P= 0.020). The greater the score of favourable TYMS and FCGR3A genotypes, the better the response rate (P= 0.009) and the longer the overall survival (P= 0.007). In multivariate analysis, the score of favourable genotypes was a stronger survival predictor than the performance status. Present data suggest the importance of FCGR3A 158Phe>Val and TYMS 5' UTR polymorphisms in responsiveness and survival of patients receiving cetuximab-fluoropyrimidine-based therapy.

Influence of folate pathway polymorphisms on high-dose methotrexate-related toxicity and survival in childhood acute lymphoblastic leukemia

The prediction of high-dose methotrexate (HD-MTX) toxicity is a key issue in the individualization of treatment in childhood acute lymphoblastic leukemia (ALL). Our aim was to evaluate the influence of MTX pathway polymorphisms on HD-MTX treat- ment outcome in children with ALL. In total, 167 children with ALL were genotyped for methylenetetrahydrofolate dehydrogenase (MTHFD1) 1958G > A, methylenetetrahydrofolate reductase (MTHFR) 677C > T and 1298A > C and thymidylate synthase (TYMS) 2R > 3R polymorphisms. The MTHFD1 1958A allele significantly reduced the odds of hepatotoxicity (adjusted p = 0.009), while the TYMS 3R allele significantly reduced the odds of leukocytopenia and thrombocytopenia (adjusted p = 0.005 and adjusted p = 0.002, respectively). MTHFR polymorphisms did not influence HD-MTX-related toxicity, but a significant effect of MTHFR 677C > T–TYMS 2R > 3R and MTHFD1 1958G > A–MTHFR 677C > T interactions on HD-MTX-related toxicity was observed. None of the investigated polymorphisms influenced survival. Our study suggests an important role of polymorphisms and gene–gene interactions within the folate pathway in HD-MTX-related toxicity in childhood ALL.

Thymidylate synthase enhancer region polymorphism not related to susceptibility to acute lymphoblastic leukemia in the Kashmir population

Thymidylate synthase (TS) is a crucial enzyme in folate metabolism and plays a vital role in DNA synthesis and repair. The most common polymorphism in TS is a unique double (2R) or triple (3R) 28-bp tandem repeat sequence in the enhancer region of the TS gene (TSER). This genetic variation in TSER has been widely investigated and has been implicated as a risk factor for the development of various cancers, including acute lymphoblastic leukemia. It has also been found to influence sensitivity to anti-cancer drugs, such as methotrexate. We evaluated this polymorphism in acute lymphoblastic leukemia patients in the Kashmir population. In order to determine whether a double (2R2R) versus a triple (3R3R) 28-bp tandem repeat in the TSER modulates risk for acute lymphoblastic leukemia, 72 acute lymphoblastic leukemia cases and 144 age and gender matched, unrelated healthy individuals from the Kashmir region of India were evaluated for this polymorphism by PCR and direct sequencing. We found the frequency of the TS 2R allele to be 32.6 and 26.0%, in cases and controls, respectively. The TS 2R/2R genotype was found to be present in 15.27% of the cases and 9.72% of the controls, the 2R/3R variant in 34.72% of the cases and 32.63% of the controls, and the 3R/3R genotype in 50.0% of the cases and 57.63% of the controls. There was a significant association between the TS 2R/2R genotype and gender of acute lymphoblastic leukemia patients with males harboring the 2R/2R genotype exhibiting a higher risk of developing acute lymphoblastic leukemia than females (P = 0.009) We concluded that the TSER polymorphism appears not to be a risk factor for susceptibility to acute lymphoblastic leukemia in the Kashmir population.