Risk factors for serious adverse effects of thiopurines in patients with Crohn's disease.

PURPOSE: Thiopurines are effective in attaining and maintaining remission in patients with inflammatory bowel diseases (IBD). The major drawback of these drugs are their serious adverse effects (SAE), highlighting the importance of preemptive identification of patients at risk. We aimed to examine whether gene polymorphisms in GSTM1, GSTT1 and TPMT, combined with various clinical parameters, can predict thiopurine induced SAE. METHODS: A retrospective cohort of 176 Crohn's Disease (CD) patients treated with thiopurines (131 with 6MP and 45 with azathioprine) was genotyped for common polymorphisms in GSTM1, GSTT1 and TPMT. Clinical data including SAE, age, ethnicity, gender and smoking status were extracted from patient charts. SAEs evaluated were myelosuppression, hepatotoxicity and pancreatitis. Associations between demographic, clinical, and genetic variables and thiopurine induced SAE were assessed. RESULTS: Twenty-four patients (14%) developed SAE, revealing a significant association between thiopurine induced SAE and GSTM1-null genotype (P=0.05), older age (P=0.016) and active smoking status (P=0.043) and SAE. On multi-variant analysis, past or current smokers were at increased risk for developing thiopurine related SAE (OR 2.915, CI 95%: 1.199- 7.084), specifically pancreatitis (p<0.001). No association was found between TPMT or GSTT1 polymorphisms and the development of SAE. CONCLUSIONS: Active smoking and GSTM1-null genotype appear to be risk factors for thiopurine induced SAEs (i.e. myelosuppression, hepatotoxicity and pancreatitis) in patients with CD. Corroboration of these associations in larger cohorts is warranted.

Population distribution of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C risk alleles for methotrexate toxicity in Israel.

Methylenetetrahydrofolate reductase (MTHFR) is a central regulatory enzyme in the folate pathway. Two non-synonymous single nucleotide polymorphisms in MTHFR, C677T (rs1801133) and A1298C (rs1801131) have been associated with reduced MTHFR enzyme activity. These polymorphisms, especially C677T, appear to be linked with methotrexate-related toxicity, particularly hepatotoxicity; thus, pretreatment identification of individuals carrying these polymorphisms may be of clinical relevance. The purpose of this study was to determine the frequency and distribution of MTHFR polymorphic variants, known to functionally impair MTHFR activity, in the highly heterogeneous Israeli population. MTHFR genotyping was carried out in the representatives of three major demographic groups in Israel by PCR-restriction fragment length polymorphism and high-resolution melting. The relative distribution of variant alleles 677T and 1298C was found to be similar in individuals of Jewish, Druze and Arab Moslem descent (p = 0.09). However, Ashkenazi Jews displayed a 1.9-fold higher frequency of variant 677T and a 1.8-fold lower frequency of variant 1298C compared to non-Ashkenazi Jews (p < 0.001). Distinct differences in the relative frequencies of both polymorphisms were also found between Ashkenazi Jews and Druze (p < 0.01 for C677T, p < 0.01 for A1298C) or Ashkenazi Jews and Arab Moslem (p < 0.01 for C677T, p < 0.05 for A1298C). These data underscore the importance of geographic genetic analysis for a better understanding of human pharmacotherapy and personalized medicine.

Pharmacokinetic and pharmacogenetic analysis of oral busulfan in stem cell transplantation: prediction of poor drug metabolism to prevent drug toxicity.

High dose busulfan (BU) has become a mainstay in conditioning regimens for hematopoietic stem cell transplantation (HSCT), despite its unpredictable response, narrow therapeutic index and severe toxicity. The present study provides an integration of pharmacokinetic and genetic data of 63 adults with acute myeloid leukemia (AML) preconditioned for HSCT with high dose oral BU, with the aim of defining biomarkers predictive of poor BU metabolism. BU area under the concentration time curve (AUC) demonstrated that 76% of the patients achieved target AUC; 24% required dose modification. The main findings of this study were: (1) AML patients carrying the GSTP1 rs1695 variant allele were at risk of developing supra-therapeutic BU-AUC due to reduced BU clearance. (2) Combined polymorphisms in GSTM1 and ABCB1 were associated with BU clearance and AUC rates. In conclusion, GST and ABCB1 genotyping may assist care-givers in personalizing BU dosage with less trial-and-error and may enable preemptive identification of patients at risk for BU toxicity.

Molecular morphometric analysis shows relative intra-tumoural homogeneity for KRAS mutations in colorectal cancer.

KRAS mutation status has a significant role determining anti-epidermal growth factor receptor (anti-EGFR) treatment response in colon carcinoma patients. Malignant transformation is a dynamic process and therefore, it is conceivable that, at a certain point, the tumor cells' mass might be heterogeneous for particular mutations. Therefore, the fraction of tumor cells carrying a particular mutation may be more relevant for treatment than the simple determination of presence or absence of mutation. The purpose of this study is to assess whether or not KRAS mutation status is heterogeneous and, if so, to what extent in colon carcinoma samples. Deoxyribonucleic acid was extracted from formalin-fixed paraffin-embedded samples of colon carcinoma and analyzed for the presence of KRAS mutation. The relative fraction of mutated versus wild-type KRAS alleles was evaluated by real-time polymerase chain reaction. Additionally, the relative fraction of cancer cells in the tissue sample was evaluated using computer assisted morphometric analysis. Using this data, we calculated the fraction of mutation containing cells in the samples. Colon carcinoma (169 cases) were analyzed, and a KRAS mutation was found in 75 cases (44%), of which 42 were available for morphometric analysis. In 41 (97.6%) of these cases, the fraction of mutation containing tumor cells was 50% or higher, indicating the absence of significant KRAS mutation status heterogeneity. There was a strong positive correlation (R = 0.66, P < 0.0001) between the fraction of mutated KRAS alleles and the fraction of cancer cells in the samples. The strong positive correlation between the fraction of mutated KRAS alleles and the fraction of cancer cells in the samples indicate homogeneity of KRAS mutation status in colorectal carcinoma.

Non-Jewish Israeli IBD patients have significantly higher glutathione S-transferase GSTT1-null frequency.

BACKGROUND: The involvement of oxidant/antioxidant imbalance in the development of inflammatory bowel disease (IBD) is well documented. Two members of the glutathione S-transferase (GST) family of enzymes, GSTM1 and GSTT1, known to take part in cellular protection against electrophiles, demonstrate common deletion variants (termed null) associated with impaired enzyme function. AIM: To evaluate the effect of GSTM1/GSTT1 genotype on IBD susceptibility in a Israeli cohort and to study the correlation between GSTM1/GSTT1 genotype, smoking status, and a variety of clinical characteristics of IBD. METHODS: A cohort of 606 Israeli IBD patients (453 with Crohn's disease [CD] and 153 with ulcerative colitis [UC]) and 528 ethnically matched healthy controls were genotyped for the null variants of GSTM1 and GSTT1. In patients, phenotype-genotype correlations were examined. RESULTS: Ethnic stratification of healthy controls revealed a higher frequency of GSTT1-null in Jewish and Arab Moslem individuals compared to Druze individuals (P < 0.0005), but no difference in GSTM1-null was found. Comparing IBD patients (both CD and UC) to healthy controls revealed a pattern of lower GSTM1-null and higher GSTT1-null frequencies, which reached significance in Arab Moslem patients. No association was found between NOD2/CARD15 mutation carriage and GSTM1/GSTT1 genotype. No statistically significant association was found between GSTT1-null or GSTM1-null, smoking status, and other phenotypes of CD/UC. CONCLUSIONS: GSTT1-null appears to be associated with IBD, while GSTM1-null appears to be conversely associated with IBD. No association was found between GSTT1-null or GSTM1-null and specific IBD phenotypes.

Influence of glutathione S-transferase A1, P1, M1, T1 polymorphisms on oral busulfan pharmacokinetics in children with congenital hemoglobinopathies undergoing hematopoietic stem cell transplantation.

BACKGROUND: Busulfan (BU), often used in high dose for myeloablation before hematopoietic stem cell transplantation (HSCT), has been implicated in certain HSCT toxicities, including the occurrence of hepatic veno-occlusive disease (HVOD). In addition to weight and age, gene polymorphisms in specific members of the glutathione-transferase (GST) gene family (A1, P1, M1, and T1), involved in BU metabolism, may play a role in the wide inter-patient variability in systemic BU concentrations. PROCEDURE: The present study integrated clinical data regarding the occurrence of HVOD, graft versus host disease (GVHD), BU pharmacokinetics and GSTA1, GSTP1, GSTM1, and GSTT1 genotypes of 18 children who received BU in their pre-HSCT conditioning regimen. The children were all treated for congenital hemoglobinopathies and were all of Arab Moslem descent. RESULTS: The data demonstrate an association between GSTA1 and GSTP1 genotypes and BU-maximal concentration (C(max)) (P = 0.01, P = 0.02, respectively), area under the concentration-time curve (AUC) (P = 0.02, P = 0.01, respectively) and oral BU clearance/kg body weight (P < 0.02, P = 0.08, respectively). GSTM1-null individuals demonstrated lower BU-AUC/Kg compared to GSTM1-positive individuals. In addition, an association between GVHD and GSTM1-null genotype was found. CONCLUSIONS: GSTA1, GSTP1, and GSTM1 genotyping prior to HSCT in children with congenital hemoglobinopathies may allow better prediction of oral BU kinetics and the need for BU dose adjustment, as well as prediction of transplant related toxicity such as GVHD, thereby improving clinical outcome.

Distribution of CYP2C9 and VKORC1 risk alleles for warfarin sensitivity and resistance in the Israeli population.

PURPOSE: This study was designed to delineate the relative frequency of CYP2C9 and VKORC1 polymorphisms known to affect warfarin response in the highly heterogeneous Israeli population. METHODS: Frequencies of CYP2C9 allelic variants CYP2C9*2, CYP2C9*3 and of VKORC1 single nucleotide polymorphisms (snps) -1639G>A and D36Y were determined in genomic DNA of 438 healthy unrelated Israeli volunteers of Jewish, Druze and Arab Moslem descent, using allele specific PCR-RFLP. Genotyping results obtained were confirmed by probe free High Resolution Melt (HRM) Technology. RESULTS: Arab Moslems had a higher frequency of warfarin "sensitive" CYP2C9*2, CYP2C9*3 and VKROC1 -1639G>A alleles (0.21, 0.07 and 0.58, respectively) than both Jews (0.13, 0.11 and 0.57, respectively) and Druze (0.12, 0.06 and 0.53, respectively). Statistically significant differences were found in CYP2C9*2 between Druze and Moslems (p=0.01) and between Jews and Moslems (p=0.016) and in CYP2C9*3 between Druze and Jews (p=0.0086). VKORC1(-1639G>A) was the major gene polymorphism associated with warfarin sensitivity in all 3 subpopulations. In contrast, the warfarin "resistant" VKORC1 D36Y allele was very rare in the Israeli population (0-0.015). The results presented demonstrate that allelic variants in CYP2C9 and VKORC1 are very common in Israel with approximately 95% of Jews, approximately 84% of Druze and approximately 91% of Arab Moslems manifesting at least one known warfarin "sensitive" or "resistant" allele. CONCLUSIONS: Individualized genotype based warfarin therapy is highly relevant in the Israeli population due to the high incidence of genetic variations associated with warfarin sensitivity in all 3 non-mixing subpopulations tested.

LNA-based PCR clamping enrichment assay for the identification of KRAS mutations.

INTRODUCTION: KRAS mutations in colon carcinomas are associated with lack of response to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo genetic testing to be eligible for treatment. Several methods for KRAS mutation analysis exist, but many are not sensitive enough to detect a mutation in samples with low fraction of malignant cells. In the present study, we developed a KRAS mutations detection method that is both simple and sensitive. METHODS: Using a locked nucleic acid (LNA) containing oligonucleotide, we developed a PCR clamping method that preferentially amplifies the mutated over wild type KRAS. We evaluated the sensitivity of this method using serial dilutions of plasmids containing wild-type and mutated KRAS fragments. Additionally, KRAS mutation status was evaluated on 60 archived tissue samples of colon carcinoma, and compared to direct sequencing and high resolution melting (HRM) methods. RESULTS: The PCR clamping method could detect as little as 1% mutated DNA in the sample analyzed. Of the 29 KRAS mutations identified by the PCR clamping method, only 23 (79%) were identified by standard direct sequencing. The results of PCR clamping correlated with HRM results. CONCLUSIONS: LNA based PCR clamping method is a simple and highly sensitive method for the detection of KRAS mutations.