Hyun-Jung Cho

Factors affecting the interindividual variability of warfarin dose requirement in adult Korean patients

INTRODUCTION Warfarin, a commonly prescribed anticoagulant, exhibits large interindividual and interethnic differences in the dose required for its anticoagulation effect. Asian patients require a much lower maintenance dose compared with Caucasians; the explanation for these differences remains unknown. METHODS We analyzed five single nucleotide polymorphisms of the vitamin K epoxide reductase complex subunit 1 gene (VKORC1) and the *3 variant of cytochrome P450 (CYP)2C9, as well as the plasma warfarin concentration, in 108 Korean patients with atrial fibrillation. RESULTS Genotypic frequencies of VKORC1 +1173CT and CYP2C9*1/*3 were 17.6 and 10.2%, respectively, in the study population; VKORC1 +1173CC and CYP2C9*3/*4 were detected in one patient each. Patients carrying at least one copy of the VKORC1 +1173C allele, or the H7 (group B) haplotype, required a significantly higher warfarin dose (n = 20; 5.5 +/- 1.7 mg/day) than those homozygous for the +1173T allele, or the H1 (group A) haplotype, (3.8 +/- 1.2 mg/day; p < 0.001). There were statistically significant differences in warfarin dose between the CYP2C9*1/*1 (4.3 +/- 1.6 mg/day; p < 0.001) and those with the other two genotypes including CYP2C9*1/*3 and CYP2C9*3/*4 (2.7 +/- 0.9 mg/day). The multiple regression analysis revealed that the VKORC1 genotype (r2 = 0.197; p < 0.001), the age when warfarin started (r2 = 0.09; p < 0.001), body surface area (r2 = 0.041; p = 0.004) and CYP2C9 genotype (r2 = 0.029; p = 0.014) were factors associated with the daily dose of warfarin required. CONCLUSION In the present study, we found that the VKORC1 polymorphism had a dominant genetic influence on interindividual variability for warfarin dose in Korean patients. It explained approximately 32% of the overall variability in warfarin dose requirements given all of the variables studied. Thus, analysis of the VKORC1 genotypes may be important to guide warfarin dose selection and allow personalized warfarin treatment.

GSTM1, GSTT1 and GSTP1 polymorphisms in the Korean population.

The isoenzymes of the glutathione s transferase (GST) family play a vital role in phase II of biotransformation of many substances. Using a multiplex polymerase chain reaction and a direct sequencing analysis, the frequencies of GSTM1, GSTT1, and GSTP1 polymorphisms were evaluated in 1,051 Korean male subjects. We found that 53.8% of the individuals had the GSTM1 null genotype and 54.3% had the GSTT 1 null genotype. The genotypic distribution of GSTP1 was Ile105/Ile105 in 68.4%, Ile105/Val105 in 29.1% and Val105/Val105 in 2.5%. The most frequently observed combination of GSTM1, GSTP1 and GSTT1 genotypes was Null type/Ile105/Ile105/Null type, while the combination of Non-null type/Val105/Val105/Non-Null type was not observed. We found that the genotype distributions of three GST isoenzymes in the Koreans are similar to those reported in Asians and previously reported Koreans. We believe our results, which are represented by a large population, are reliable estimates of the frequencies of the polymorphic GST alleles in the Koreans and will help future researches on GST polymorphisms.

Thymidylate synthase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) polymorphisms in the korean population for prediction of 5-fluorouracil-associated toxicity

The important cellular proteins for 5-fluorouracil (5-FU) metabolism are the major target enzymes, thymidylate synthase, and the rate-limiting enzyme in the degradation pathway, dihydropyrimidine dehydrogenase. Adverse drug reactions to 5-FU-based chemotherapy have been reported to be in part the result of polymorphisms in the thymidylate synthase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) genes. Therefore, we examined the type and frequency of polymorphisms in the TYMS and DPYD genes in 100 healthy Korean individuals and compared these findings with 21 patients with colorectal cancer who had a grade 3 or greater toxic response to 5-FU treatment. Genotyping analysis of the promotor enhancer region (TSER) and the 3′-untranslated region (3′-UTR) of the TYMS gene as well as haplotype analysis were conducted in all 121 study participants. For the TSER and the 3′-UTR of the TYMS gene, similar genotypes and allele frequencies were observed in control subjects and patients. For the haplotype analysis of the single nucleotide polymorphism G > C at the 12th nucleotide of the second repeat of the 3R allele of the TSER, different haplotype frequencies were noted in comparisons between the two groups; in addition, we found that the 3RC-del 6bp was significantly associated with severe toxicity with 5-FU treatment. Extensive polymorphisms in the DPYD gene were observed; in addition, four polymorphisms were related to the known DPYD allelic variants or to allelic variants that alter protein structure, among which the most common polymorphism was 1627A > G, observed in 20.5% of all alleles. The 496A > G allele and a novel 1774C > T allele were identified in two patients. The DPYD*2A allele, causing exon 14 skipping, was not identified in the study group. The findings, from Korean patients with colon cancer, suggest that polymorphisms of the DPYD gene are not associated with an increased risk for toxic response to 5-FU. These findings suggest that there may be an important relationship between the TYMS haplotypes examined and 5-FU toxicity. The novel variant in the DPYD gene, identified in this study, should be further investigated to confirm its functional significance. A large sample is required before DPYD or TYMS genotyping could be used as markers for individualized treatment of patients with colorectal cancer.

A Novel LZAP-binding Protein, NLBP, Inhibits Cell Invasion

LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-κB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-κB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-κB signaling, and increasing stability of the LZAP protein.

Glutathione-S-transferase genotypes influence the risk of chemotherapy-related toxicities and prognosis in Korean patients with diffuse large B-cell lymphoma

Polymorphisms in detoxification enzymes of the glutathione S-transferase (GST) family are associated with treatment response, resistance, and drug-related toxicity, all of which affect final clinical outcome. In this study, we investigated the influence of the genetic polymorphisms GSTM1, GSTT1, and GSTP1 on treatment response in 94 Korean patients with de novo diffuse large B-cell lymphoma, who had received rituximab plus cyclophosphamide/doxorubicin/vincristine/prednisone (R-CHOP) as a front-line regimen. Deletions of the GSTM1 and GSTT1 genes were detected using a multiplex polymerase chain reaction technique, and the functional GSTP1 polymorphism, Ile(105)Val, was genotyped using the TaqMan assay. The treatment response rate did not differ according to GST polymorphisms. Patients with the GSTT1-null genotype, however, showed more frequent grade III-IV chemotherapy-related toxicities, including leukocytopenia [odds ratio (OR)=3.1; 95% confidence interval (95%CI), 1.2-8.0; P=0.025], fever (OR=5.3; 95% CI, 1.4-19.7; P=0.009), and mucositis (OR=4.6; 95% CI, 1.4-15.1; P=0.012). Patients with the GSTM1/T1 double-null genotype had more grade III-IV thrombocytopenia (OR=7.8; 95% CI, 1.5-41.1; P=0.002) compared to those with other genotypes. In male patients, the GSTM1/T1 double-null genotype was associated with a shorter event-free survival period (P=0.02). This study suggests that GSTT1 deletion may significantly increase the risk of drug-related toxicity after R-CHOP chemotherapy in patients with DLBCL, and is associated with worse prognosis in males.

Development and Comparison of a Warfarin-Dosing Algorithm for Korean Patients With Atrial Fibrillation

BACKGROUND The pharmacokinetics and pharmacodynamics of warfarin are affected by polymorphisms in the genes coding for cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1). OBJECTIVE The objective of this study was to develop a pharmacogenetic dosing algorithm for warfarin in Korean patients with atrial fibrillation and to compare it with the published pharmacogenetic dosing algorithms for accuracy to predict warfarin maintenance dose. METHODS Clinical and genetic data from 130 Korean patients with atrial fibrillation (mean [SD] age: 66.2 [13.3] years; gender, male/female: 86/44; mean body weight: 66.6 [11.6] kg) were used to create a dosing algorithm, which was validated against an independent group of patients (n = 108; mean age: 67.4 [10.1] years; gender, male/female: 69/39; mean body weight: 66.0 [10.9] kg). Validation cohort data for the 12 previously published dosing algorithms incorporating CYP2C9 and VKORC1 genotype information were also applied. RESULTS A multivariate regression model including the variables of age, VKORC1 and CYP2C9 genotype, body surface area, and statin status produced the best model for estimating the warfarin dose (R(2) = 0.62). Among the 12 algorithms that were compared, the predicted doses using algorithms derived from both the Swedish Warfarin Genetics (WARG) study and the Korean population study showed the best correlation with actual warfarin doses. Comparing the percentage of patients whose predicted dosages were within 20% of actual dosages, these algorithms showed similar overall performance. CONCLUSIONS This study derived and validated a multivariate regression model for daily warfarin dose requirements in Korean patients with atrial fibrillation. As no algorithm could be considered the best for all dosing ranges, it may be important to consider the characteristics or limitations of each dosing algorithm and the nature of a population in choosing the most appropriate pharmacogenetic dosing.

Hydrothermal synthesis of anatase nanocrystals with lattice and surface doping tungsten species

We have synthesized anatase nanocrystals of 8–15 nm containing lattice and surface doping tungsten through hydrothermal reactions. We used phosphotungstate–titania nanocomposites with various compositions as the reagents. The homogeneous distribution of tungsten in the reagent solids appears to strongly influence the synthesis results. X-Ray diffraction and Raman spectroscopy data showed that the samples contained brookite impurities when the tungsten content was low (up to W/(W + Ti) = 3.7%) but pure anatase was obtained when the tungsten content was high (up to W/(W + Ti) = 26%). The tungsten content in the anatase nanocrystals is reflected in the lattice parameters, the Ti(W)–O bond distances, the Raman peak positions and peak widths. Tungsten atoms are present as lattice doping and surface coating (wolframyl group). The relative proportion between these species changes with the tungsten content: at low tungsten-content, the lattice doping tungsten is the dominating species that expands the lattice. Around the critical concentration of W/(W + Ti) = 13%, the amount of surface wolframyl group increases while the lattice doping appears to have reached a saturation. A formation mechanism that explains most of the observations was proposed.

Alpha-Smooth Muscle Actin (ACTA2) Is Required for Metastatic Potential of Human Lung Adenocarcinoma

Purpose: Metastatic relapse of primary lung cancer leads to therapeutic resistance and unfavorable clinical prognosis; therefore, identification of key molecules associated with metastatic conversion has significant clinical implications. We previously identified a link between early brain metastasis of lung adenocarcinoma and amplification of the α-smooth muscle actin (ACTA2) gene. The aim of present study was to investigate the prognostic and functional significance of ACTA2 expression in cancer cells for the metastatic potential of lung adenocarcinomas. Experimental Design: ACTA2 expression was analyzed in tumor cells from 263 patients with primary lung adenocarcinomas by immunohistochemistry, and was correlated with clinicopathologic parameters. The expression of ACTA2 in human lung adenocarcinoma cells was modulated with short hairpin RNAs (shRNA) and siRNAs specifically targeting ACTA2. Results: The patients with lung adenocarcinomas with high ACTA2 expression in tumor cells showed significantly enhanced distant metastasis and unfavorable prognosis. ACTA2 downregulation remarkably impaired in vitro migration, invasion, clonogenicity, and transendothelial penetration of lung adenocarcinoma cells without affecting proliferation. Consistent with the in vitro results, depletion of ACTA2 in human lung adenocarcinoma PC14PE6 cells significantly reduced their metastatic potential without altering their tumorigenic potential. Expression of c-MET and FAK in lung adenocarcinoma cells was also reduced by ACTA2-targeting siRNAs and shRNAs, and was accompanied by a loss of mesenchymal characteristics. Conclusions: These findings indicate that ACTA2 regulates c-MET and FAK expression in lung adenocarcinoma cells, which positively and selectively influence metastatic potential. Therefore, ACTA2 could be a promising prognostic biomarker and/or therapeutic target for metastatic lung adenocarcinoma. Clin Cancer Res; 19(21); 5879–89. ©2013 AACR.

Identification of de novo BSCL2 Ser90Leu mutation in a Korean family with silver syndrome and distal hereditary motor neuropathy

Mutations in the Berardinelli–Seip congenital lipodystrophy (BSCL2) gene have been identified in families with distal hereditary motor neuropathy (dHMN) and in families with SPG17‐linked Silver syndrome. We studied the first Korean families with clinical features resembling classic Silver syndrome and dHMN type V. Direct sequencing analysis of the BSCL2 gene revealed a Ser90Leu mutation in the proband, a younger sister, and one of two sons of the proband. The clinical patterns in this family include presentation with lower‐limb and hand‐muscle involvement early in the disease course as well as the presence of Babinski signs with nonprogressive mild spastic paraparesis, resembling classic Silver syndrome and dHMN type V. This study reaffirms the clinical phenotype of the disorders associated with a BSCL2 Ser90Leu mutation and describes a genetically proven family with Silver syndrome and dHMN type V in Asia. Muscle Nerve, 2007

Patient-Derived Xenografts from Non–Small Cell Lung Cancer Brain Metastases Are Valuable Translational Platforms for the Development of Personalized Targeted Therapy

Purpose: The increasing prevalence of distant metastases from non–small cell lung cancer (NSCLC) indicates an urgent need for novel therapeutic modalities. Brain metastasis is particularly common in NSCLC, with severe adverse effects on clinical prognosis. Although the molecular heterogeneity of NSCLC and availability of various targeted agents suggest personalized therapeutic approaches for such brain metastases, further development of appropriate preclinical models is needed to validate the strategies. Experimental Design: We established patient-derived xenografts (PDX) using NSCLC brain metastasis surgical samples and elucidated their possible preclinical and clinical implications for personalized treatment. Results: NSCLC brain metastases (n = 34) showed a significantly higher successful PDX establishment rate than primary specimens (n = 64; 74% vs. 23%). PDXs derived from NSCLC brain metastases recapitulated the pathologic, genetic, and functional properties of corresponding parental tumors. Furthermore, tumor spheres established in vitro from the xenografts under serum-free conditions maintained their in vivo brain metastatic potential. Differential phenotypic and molecular responses to 20 targeted agents could subsequently be screened in vitro using these NSCLC PDXs derived from brain metastases. Although PDX establishment from primary NSCLCs was significantly influenced by histologic subtype, clinical aggressiveness, and genetic alteration status, the brain metastases exhibited consistently adequate in vivo tumor take rate and in vitro tumor sphere formation capacity, regardless of clinical and molecular conditions. Conclusions: Therefore, PDXs from NSCLC brain metastases may better represent the heterogeneous advanced NSCLC population and could be utilized as preclinical models to meet unmet clinical needs such as drug screening for personalized treatments. Clin Cancer Res; 21(5); 1172–82. ©2014 AACR.