Replication Study for the Association of Five SNPs Identified by GWAS and Trastuzumab-Induced Cardiotoxicity in Japanese and Singaporean Cohorts.

Trastuzumab (herceptin) is an effective drug for human epidermal growth factor receptor type 2 (HER2)-positive cancer. However, cardiotoxicity remains a serious complication. In our previous genome-wide association study (GWAS), we identified potential associations for five single nucleotide polymorphisms (SNPs) with trastuzumab-induced cardiotoxicity in a Japanese population. To validate this association, here we performed replication studies using Japanese and Singaporean case-control cohorts (Japan: 6 cases and 206 controls; Singapore: 22 cases and 178 controls). Although none of the SNPs showed a statistically significant association with trastuzumab-induced cardiotoxicity, we show that three (rs8032978, rs7406710 and rs9316695) and four (rs8032978, rs7406710, rs28415722 and rs11932853) SNPs had an effect in the same direction in the Japanese and the Singaporean cohort, respectively, as that in our previous study. Combining the previous study with the current replication studies, we find a strong association for two SNPs, rs8032978 and rs7406710, with trastuzumab-induced cardiotoxicity (Pcombined = 4.92 × 10-5 and 5.50 × 10-5, respectively). These data suggest that rs8032978 and rs7406710 could be predictive markers of trastuzumab-induced cardiotoxicity in Japanese and Singaporean populations, and support their potential use in clinical risk assessment. These findings offer a first step toward the development of clinically available markers for the potential risk of trastuzumab-induced cardiotoxicity as well as an improved understanding of the pathogenesis of this complication.

Determination of novel CYP2D6 haplotype using the targeted sequencing followed by the long-read sequencing and the functional characterization in the Japanese population.

Next-generation sequencing (NGS) has identified variations in cytochrome P450 (CYP) 2D6 associated with drug responses. However, determination of novel haplotypes is difficult because of the short reads generated by NGS. We aimed to identify novel CYP2D6 variants in the Japanese population and predict the CYP2D6 phenotype based on in vitro metabolic studies. Using a targeted NGS panel (PKSeq), 990 Japanese genomes were sequenced, and then novel CYP2D6 haplotypes were determined. Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl-tamoxifen 4-hydroxylation were calculated by in vitro metabolic studies using cDNA-expressed CYP2D6 proteins. After determination of the CYP2D6 diplotypes, phenotypes of the individuals were predicted based on the in vitro metabolic activities. Targeted NGS identified 14 CYP2D6 variants not registered in the Pharmacogene Variation Consortium (PharmVar) database. Ten novel haplotypes were registered as CYP2D6*128 to *137 alleles in the PharmVar database. Based on the Vmax/Km value of each allele, *128, *129, *130, *131, *132, and *133 were predicted to be nonfunctional alleles. According to the results of the present study, six normal metabolizers (NM) and one intermediate (IM) metabolizers were designated as IM and poor metabolizers (PM), respectively. Our findings provide important insights into novel haplotypes and haplotypes of CYP2D6 and the effects on in vitro metabolic activities.

Pharmacogenetics for severe adverse drug reactions induced by molecular-targeted therapy.

Molecular-targeted drugs specifically interfere with molecules that are frequently overexpressed or mutated in cancer cells. As such, these drugs are generally considered to precisely attack cancer cells, thereby inducing fewer adverse drug reactions (ADRs). However, molecular-targeted drugs can still cause characteristic ADRs that, although rarely severe, can be life-threatening. Therefore, it is becoming increasingly important to be able to predict which patients are at risk of developing ADRs after treatment with molecular-targeted therapy. The emerging field of pharmacogenetics aims to better distinguish the genetic variants associated with drug toxicity and efficacy to improve the selection of therapeutic strategies for each genetic profile. Here, we provide an overview of the current reports on the relationship between genetic variants and molecular-targeted drug-induced severe ADRs in oncology.

Serial circulating tumour DNA analysis for locally advanced rectal cancer treated with preoperative therapy: prediction of pathological response and postoperative recurrence.

BACKGROUND: The "watch-and-wait" approach is a common treatment option amongst patients with locally advanced rectal cancer (LARC). However, the diagnostic sensitivity of clinical modalities, such as colonoscopy and magnetic resonance imaging to determine pathological response, is not high. We analysed the clinical utility of circulating tumour DNA (ctDNA) of patients with LARC to predict response to preoperative therapy and postoperative recurrence. METHODS: A serial ctDNA analysis of 222 plasma samples from 85 patients with LARC was performed using amplicon-based deep sequencing on a cell-free DNA panel covering 14 genes with over 240 hotspots. RESULTS: ctDNA was detected in 57.6% and 22.3% of samples at baseline and after preoperative treatment, respectively, which was significantly different (P = 0.0003). Change in ctDNA was an independent predictor of complete response to preoperative therapy (P = 0.0276). In addition, postoperative ctDNA and carcinoembryonic antigen (CEA) were independent prognostic markers for risk of recurrence after surgery (ctDNA, P = 0.0127 and CEA, P = 0.0105), with a combined analysis having cumulative effects on recurrence-free survival (P = 1.0 × 10-16). CONCLUSIONS: Serial ctDNA analysis may offer clinically useful predictive and prognostic markers for response to preoperative therapy and postoperative recurrence in patients with LARC.

Clinical utility of circulating tumor DNA for colorectal cancer.

Colorectal cancer (CRC) is currently the most common type of cancer in Japan, and its prognosis has improved because of development of diagnosis and advancement in treatments including surgery and chemotherapy. However, because of intratumor heterogeneity and clonal evolution, tumors often develop resistance to treatment. Genotyping tumor tissue in search of somatic genetic alterations for actionable information has become routine examination in clinical practice. However, the inherent molecular heterogeneity of metastatic tumors and the ability of cancer genomes to dynamically evolve are not properly captured by tissue specimens only. Circulating tumor DNA (ctDNA) carrying tumor-specific genetic or epigenetic alterations is released into the circulation from tumor cells undergoing apoptosis or necrosis. Analysis of ctDNA has the potential to change clinical practice by exploiting blood rather than tissue, as a source of information. Here, we provide an overview of the characteristics of ctDNA and focus on detection methods for ctDNA, and the feasibility of use of ctDNA to monitor tumor dynamics for patients with colorectal cancer.

A Genome-Wide Association Study Identifies Five Novel Genetic Markers for Trastuzumab-Induced Cardiotoxicity in Japanese Population.

Trastuzumab has been administered to patients with human epidermal growth factor receptor 2 (HER2)-positive cancer, however, the cardiotoxicity is identified as one of the life-threatening toxicities. Clinically useful biomarker for trastuzumab-induced cardiotoxicity has been expected to be developed. To identify a novel genetic marker(s) determining the risk of trastuzumab-induced cardiotoxicity, we performed a first genome-wide association study (GWAS) in Japanese population. We enrolled 481 patients who had been treated with trastuzumab and carried out a GWAS using 11 cases (with cardiotoxicity) and 257 controls (without cardiotoxicity). Top 100 single nucleotide polymorphisms (SNPs) which revealed the smallest p values in GWAS (p = 7.60 × 10-7 - 2.01 × 10-4) were further examined using replication samples consisted of 14 cases and 199 controls. The combined analysis of the GWAS and replication study indicated possible association of five loci with trastuzumab-induced cardiotoxicity (rs9316695 on chromosome 13q14.3, rs28415722 on chromosome 15q26.3, rs7406710 on chromosome 17q25.3, rs11932853 on chromosome 4q25, and rs8032978 on chromosome 15q26.3, Pcombined = 6.00 × 10-6, 8.88 × 10-5, 1.07 × 10-4, 1.42 × 10-4, 1.60 × 10-4, respectively). Furthermore, we developed a risk prediction model for trastuzumab-induced cardiotoxicity using the five marker SNPs. The incidence of trastuzumab-induced cardiotoxicity in patients with risk score ≥5 was significantly higher (42.5%) compared to that in patients with score ≤ 4 (1.8%) (p = 7.82 × 10-15, odds ratio = 40.0). These findings suggest the potential to improve the ability of physicians to avoid the trastuzumab-induced cardiotoxicity for patients with HER2-positive cancer.

Breast cancer: The translation of big genomic data to cancer precision medicine.

Cancer is a complex genetic disease that develops from the accumulation of genomic alterations in which germline variations predispose individuals to cancer and somatic alterations initiate and trigger the progression of cancer. For the past 2 decades, genomic research has advanced remarkably, evolving from single-gene to whole-genome screening by using genome-wide association study and next-generation sequencing that contributes to big genomic data. International collaborative efforts have contributed to curating these data to identify clinically significant alterations that could be used in clinical settings. Focusing on breast cancer, the present review summarizes the identification of genomic alterations with high-throughput screening as well as the use of genomic information in clinical trials that match cancer patients to therapies, which further leads to cancer precision medicine. Furthermore, cancer screening and monitoring were enhanced greatly by the use of liquid biopsies. With the growing data complexity and size, there is much anticipation in exploiting deep machine learning and artificial intelligence to curate integrative "-omics" data to refine the current medical practice to be applied in the near future.

Whole exome sequencing to identify genetic markers for trastuzumab-induced cardiotoxicity.

Although trastuzumab-induced cardiotoxicity is an important determinant to limit the use of this drug, the molecular mechanism of risk for this toxicity is not well understood. To identify genetic variants determining the risk of trastuzumab-induced cardiotoxicity, we carried out whole exome sequencing of germline DNA samples from 9 patients with trastuzumab-induced cardiotoxicity, and conducted a case-control association study of 2258 genetic variants between 9 cases (with trastuzumab-induced cardiotoxicity) and general Japanese population controls registered in the Human Genetic Variation Database (HGVD). The top variant which showed the lowest P-value in the screening study was rs139503277 in PHD Finger Protein 3 (Pmin = .00012, odds ratio [OR] = 51.23). To further validate the result of screening study, we carried out a replication study of 10 variants showing Pmin < .001 in the screening study using 234 independent patients treated with trastuzumab, including 10 cases and 224 controls (without trastuzumab-induced cardiotoxicity). In the replication study, we observed that three variants had an effect in the same direction as in the screening study (rs78272919 in exon 2 of Keratin 15, rs5762940 in exon 2 of zinc and ring finger 3, and rs139944387 in exon 44 of Eyes shut homologs [EYS]). A combined result of the screening and the replication studies suggested an association of a locus on chromosome 6q12 with trastuzumab-induced cardiotoxicity (rs139944387 in EYS, combined Pmin = .00056, OR = 13.73). This finding provides new insights into personalized trastuzumab therapy for patients with human epidermal growth factor receptor 2 (HER2)-positive cancer.

Cross-sectional analysis of BioBank Japan clinical data: A large cohort of 200,000 patients with 47 common diseases.

BACKGROUND: To implement personalized medicine, we established a large-scale patient cohort, BioBank Japan, in 2003. BioBank Japan contains DNA, serum, and clinical information derived from approximately 200,000 patients with 47 diseases. Serum and clinical information were collected annually until 2012. METHODS: We analyzed clinical information of participants at enrollment, including age, sex, body mass index, hypertension, and smoking and drinking status, across 47 diseases, and compared the results with the Japanese database on Patient Survey and National Health and Nutrition Survey. We conducted multivariate logistic regression analysis, adjusting for sex and age, to assess the association between family history and disease development. RESULTS: Distribution of age at enrollment reflected the typical age of disease onset. Analysis of the clinical information revealed strong associations between smoking and chronic obstructive pulmonary disease, drinking and esophageal cancer, high body mass index and metabolic disease, and hypertension and cardiovascular disease. Logistic regression analysis showed that individuals with a family history of keloid exhibited a higher odds ratio than those without a family history, highlighting the strong impact of host genetic factor(s) on disease onset. CONCLUSIONS: Cross-sectional analysis of the clinical information of participants at enrollment revealed characteristics of the present cohort. Analysis of family history revealed the impact of host genetic factors on each disease. BioBank Japan, by publicly distributing DNA, serum, and clinical information, could be a fundamental infrastructure for the implementation of personalized medicine.

Overview of BioBank Japan follow-up data in 32 diseases.

BACKGROUND: We established a patient-oriented biobank, BioBank Japan, with information on approximately 200,000 patients, suffering from any of 47 common diseases. This follow-up survey focused on 32 diseases, potentially associated with poor vital prognosis, and collected patient survival information, including cause of death. We performed a survival analysis for all subjects to get an overview of BioBank Japan follow-up data. METHODS: A total of 141,612 participants were included. The survival data were last updated in 2014. Kaplan-Meier survival analysis was performed after categorizing subjects according to sex, age group, and disease status. Relative survival rates were estimated using a survival-rate table of the Japanese general population. RESULTS: Of 141,612 subjects (56.48% male) with 1,087,434 person-years and a 97.0% follow-up rate, 35,482 patients died during follow-up. Mean age at enrollment was 64.24 years for male subjects and 63.98 years for female subjects. The 5-year and 10-year relative survival rates for all subjects were 0.944 and 0.911, respectively, with a median follow-up duration of 8.40 years. Patients with pancreatic cancer had the least favorable prognosis (10-year relative survival: 0.184) and patients with dyslipidemia had the most favorable prognosis (1.013). The most common cause of death was malignant neoplasms. A number of subjects died from diseases other than their registered disease(s). CONCLUSIONS: This is the first report to perform follow-up survival analysis across various common diseases. Further studies should use detailed clinical and genomic information to identify predictors of mortality in patients with common diseases, contributing to the implementation of personalized medicine.

Overview of the BioBank Japan Project: Study design and profile.

BACKGROUND: The BioBank Japan (BBJ) Project was launched in 2003 with the aim of providing evidence for the implementation of personalized medicine by constructing a large, patient-based biobank (BBJ). This report describes the study design and profile of BBJ participants who were registered during the first 5-year period of the project. METHODS: The BBJ is a registry of patients diagnosed with any of 47 target common diseases. Patients were enrolled at 12 cooperative medical institutes all over Japan from June 2003 to March 2008. Clinical information was collected annually via interviews and medical record reviews until 2013. We collected DNA from all participants at baseline and collected annual serum samples until 2013. In addition, we followed patients who reported a history of 32 of the 47 target diseases to collect survival data, including cause of death. RESULTS: During the 5-year period, 200,000 participants were registered in the study. The total number of cases was 291,274 at baseline. Baseline data for 199,982 participants (53.1% male) were available for analysis. The average age at entry was 62.7 years for men and 61.5 years for women. Follow-up surveys were performed for participants with any of 32 diseases, and survival time data for 141,612 participants were available for analysis. CONCLUSIONS: The BBJ Project has constructed the infrastructure for genomic research for various common diseases. This clinical information, coupled with genomic data, will provide important clues for the implementation of personalized medicine.

VAV3 mediates resistance to breast cancer endocrine therapy.

INTRODUCTION: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. METHODS: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. RESULTS: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. CONCLUSIONS: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.

Genome-wide association study for intracranial aneurysm in the Japanese population identifies three candidate susceptible loci and a functional genetic variant at EDNRA.

Aneurysmal subarachnoid hemorrhage (aSAH) is the most serious subtype of stroke. Genetic factors have been known to play an important role in the development of intracranial aneurysm (IA), some of which further progress to subarachnoid hemorrhage (SAH). In this study, we conducted a genome-wide association study (GWAS) to identify common genetic variants that are associated with the risk of IA, using 1383 aSAH subjects and 5484 control individuals in the Japanese population. We selected 36 single-nucleotide polymorphisms (SNPs) that showed suggestive association (P <1 × 10(-4)) in the GWAS as well as additional 7 SNPs that were previously reported to be associated with IA, and further genotyped an additional set of 1048 IA cases and 7212 controls. We identified an SNP, rs6842241, near EDNRA at chromosome 4q31.22 (combined P-value = 9.58 × 10(-9); odds ratio = 1.25), which was found to be significantly associated with IA. Additionally, we successfully replicated and validated rs10757272 on CDKN2BAS at chromosome 9p21.3 (combined P-value = 1.55 × 10(-7); odds ratio = 1.21) to be significantly associated with IA as previously reported. Furthermore, we performed functional analysis with the associated genetic variants on EDNRA, and identified two alleles of rs6841581 that have different binding affinities to a nuclear protein(s). The transcriptional activity of the susceptible allele of this variant was significantly lower than the other, suggesting that this functional variant might affect the expression of EDNRA and subsequently result in the IA susceptibility. Identification of genetic variants on EDNRA is of clinical significance probably due to its role in vessel hemodynamic stress. Our findings should contribute to a better understanding of physiopathology of IA.

A genome-wide association study identifies locus at 10q22 associated with clinical outcomes of adjuvant tamoxifen therapy for breast cancer patients in Japanese.

Although many association studies of polymorphisms in candidate genes with the clinical outcomes of breast cancer patients receiving adjuvant tamoxifen therapy have been reported, genetic factors determining individual response to tamoxifen are not fully understood. To identify genetic polymorphisms associated with clinical outcomes of patients with tamoxifen treatment, we conducted a genome-wide association study (GWAS). We studied 462 Japanese patients with hormone receptor-positive, invasive breast cancer receiving adjuvant tamoxifen therapy. Of them, 240 patients were analyzed by genome-wide genotyping using the Illumina Human610-Quad BeadChips, and two independent sets of 105 and 117 cases were used for replication studies. In the GWAS, we detected significant associations with recurrence-free survival at 15 single-nucleotide polymorphisms (SNPs) on nine chromosomal loci (1p31, 1q41, 5q33, 7p11, 10q22, 12q13, 13q22, 18q12 and 19p13) that satisfied a genome-wide significant threshold (log-rank P= 2.87 × 10(-9)-9.41 × 10(-8)). Among them, rs10509373 in C10orf11 gene on 10q22 was significantly associated with recurrence-free survival in the replication study (log-rank P= 2.02 × 10(-4)) and a combined analysis indicated a strong association of this SNP with recurrence-free survival in breast cancer patients treated with tamoxifen (log-rank P= 1.26 × 10(-10)). Hazard ratio per C allele of rs10509373 was 4.51 [95% confidence interval (CI), 2.72-7.51; P= 6.29 × 10(-9)]. In a combined analysis of rs10509373 genotype with previously identified genetic makers, CYP2D6 and ABCC2, the number of risk alleles of these three genes had cumulative effects on recurrence-free survival among 345 patients receiving tamoxifen monotherapy (log-rank P= 2.28 × 10(-12)). In conclusion, we identified a novel locus associated with recurrence-free survival in Japanese breast cancer patients receiving adjuvant tamoxifen therapy.

Common variant near the endothelin receptor type A (EDNRA) gene is associated with intracranial aneurysm risk.

The pathogenesis of intracranial aneurysm (IA) formation and rupture is complex, with significant contribution from genetic factors. We previously reported genome-wide association studies based on European discovery and Japanese replication cohorts of 5,891 cases and 14,181 controls that identified five disease-related loci. These studies were based on testing replication of genomic regions that contained SNPs with posterior probability of association (PPA) greater than 0.5 in the discovery cohort. To identify additional IA risk loci, we pursued 14 loci with PPAs in the discovery cohort between 0.1 and 0.5. Twenty-five SNPs from these loci were genotyped using two independent Japanese cohorts, and the results from discovery and replication cohorts were combined by meta-analysis. The results demonstrated significant association of IA with rs6841581 on chromosome 4q31.23, immediately 5' of the endothelin receptor type A with P = 2.2 × 10(-8) [odds ratio (OR) = 1.22, PPA = 0.986]. We also observed substantially increased evidence of association for two other regions on chromosomes 12q22 (OR = 1.16, P = 1.1 × 10(-7), PPA = 0.934) and 20p12.1 (OR = 1.20, P = 6.9 × 10(-7), PPA = 0.728). Although endothelin signaling has been hypothesized to play a role in various cardiovascular disorders for over two decades, our results are unique in providing genetic evidence for a significant association with IA and suggest that manipulation of the endothelin pathway may have important implications for the prevention and treatment of IA.

A genome-wide association study of chemotherapy-induced alopecia in breast cancer patients.

INTRODUCTION: Chemotherapy-induced alopecia is one of the most common adverse events caused by conventional cytotoxic chemotherapy, yet there has been very little progress in the prevention or treatment of this side effect. Although this is not a life-threatening event, alopecia is very psychologically difficult for many women to manage. In order to improve the quality of life for these women, it is important to elucidate the molecular mechanisms of chemotherapy-induced alopecia and develop ways to effectively prevent and/or treat it. To identify the genetic risk factors associated with chemotherapy-induced alopecia, we conducted a genome-wide association study (GWAS) using DNA samples from breast cancer patients who were treated with chemotherapy. METHODS: We performed a case-control association study of 303 individuals who developed grade 2 alopecia, and compared them with 880 breast cancer patients who did not show hair loss after being treated with conventional chemotherapy. In addition, we separately analyzed a subset of patients who received specific combination therapies by GWASs and applied the weighted genetic risk scoring (wGRS) system to investigate the cumulative effects of the associated SNPs. RESULTS: We identified an SNP significantly associated with drug-induced grade 2 alopecia (rs3820706 in CACNB4 (calcium channel voltage-dependent subunit beta 4) on 2q23, P = 8.13 × 10(-9), OR = 3.71) and detected several SNPs that showed some suggestive associations by subgroup analyses. We also classified patients into four groups on the basis of wGRS analysis and found that patients who classified in the highest risk group showed 443 times higher risk of antimicrotubule agents-induced alopecia than the lowest risk group. CONCLUSIONS: Our study suggests several associated genes and should shed some light on the molecular mechanism of alopecia in chemotherapy-treated breast cancer patients and hopefully will contribute to development of interventions that will improve the quality of life (QOL) of cancer patients.

Genome-wide association study of chemotherapeutic agent-induced severe neutropenia/leucopenia for patients in Biobank Japan.

Chemotherapeutic agents are notoriously known to have a narrow therapeutic range that often results in life-threatening toxicity. Hence, it is clinically important to identify the patients who are at high risk for severe toxicity to certain chemotherapy through a pharmacogenomics approach. In this study, we carried out multiple genome-wide association studies (GWAS) of 13 122 cancer patients who received different chemotherapy regimens, including cyclophosphamide- and platinum-based (cisplatin and carboplatin), anthracycline-based (doxorubicin and epirubicin), and antimetabolite-based (5-fluorouracil and gemcitabine) treatment, antimicrotubule agents (paclitaxel and docetaxel), and topoisomerase inhibitors (camptothecin and etoposide), as well as combination therapy with paclitaxel and carboplatin, to identify genetic variants that are associated with the risk of severe neutropenia/leucopenia in the Japanese population. In addition, we used a weighted genetic risk scoring system to evaluate the cumulative effects of the suggestive genetic variants identified from GWAS in order to predict the risk levels of individuals who carry multiple risk alleles. Although we failed to identify genetic variants that surpassed the genome-wide significance level (P < 5.0 × 10(-8) ) through GWAS, probably due to insufficient statistical power and complex clinical features, we were able to shortlist some of the suggestive associated loci. The current study is at the relatively preliminary stage, but does highlight the complexity and problematic issues associated with retrospective pharmacogenomics studies. However, we hope that verification of these genetic variants through local and international collaborations could improve the clinical outcome for cancer patients.

Important and critical scientific aspects in pharmacogenomics analysis: lessons from controversial results of tamoxifen and CYP2D6 studies.

Tamoxifen contributes to decreased recurrence and mortality of patients with hormone receptor-positive breast cancer. As this drug is metabolized by phase I and phase II enzymes, the interindividual variations of their enzymatic activity are thought to be associated with individual responses to tamoxifen. Among these enzymes, CYP2D6 is considered to be a rate-limiting enzyme in the generation of endoxifen, a principal active metabolite of tamoxifen, and the genetic polymorphisms of CYP2D6 have been extensively investigated in association with the plasma endoxifen concentrations and clinical outcome of tamoxifen therapy. In addition to CYP2D6, other genetic factors including polymorphisms in various drug-metabolizing enzymes and drug transporters have been implicated to their relations to clinical outcome of tamoxifen therapy, but their effects would be small. Although the results of association studies are controversial, accumulation of the evidence has revealed us the important and critical issues in the tamoxifen pharmacogenomics study, namely the quality of genotyping, the coverage of genetic variations, the criteria for sample collection and the source of DNAs, which are considered to be common problematic issues in pharmacogenomics studies. This review points out common critical issues in pharmacogenomics studies through the lessons we have learned from tamoxifen pharmacogenomics, as well as summarizes the results of pharmacogenomics studies for tamoxifen treatment.

A genome-wide association study identifies four genetic markers for hematological toxicities in cancer patients receiving gemcitabine therapy.

OBJECTIVE: Genetic factors are thought to be one of the causes of individual variability in the adverse reactions observed in cancer patients who received gemcitabine therapy. However, genetic factors determining the risk of adverse reactions of gemcitabine are not fully understood. PATIENTS AND METHODS: To identify a genetic factor(s) determining the risk of gemcitabine-induced leukopenia/neutropenia, we conducted a genome-wide association study, by genotyping over 610 000 single nucleotide polymorphisms (SNPs), and a replication study in a total of 174 patients, including 54 patients with at least grade 3 leukopenia/neutropenia and 120 patients without any toxicities. RESULTS: We identified four loci possibly associated with gemcitabine-induced leukopenia/neutropenia [rs11141915 in DAPK1 on chromosome 9q21, combined P=1.27×10, odds ratio (OR)=4.10; rs1901440 on chromosome 2q12, combined P=3.11×10, OR=34.00; rs12046844 in PDE4B on chromosome 1p31, combined P=4.56×10, OR=4.13; rs11719165 on chromosome 3q29, combined P=5.98×10, OR=2.60]. When we examined the combined effects of these four SNPs, by classifying patients into four groups on the basis of the total number of risk genotypes of these four SNPs, significantly higher risks of gemcitabine-induced leukopenia/neutropenia were observed in the patients having two and three risk genotypes (P=6.25×10, OR=11.97 and P=4.13×10, OR=50.00, respectively) relative to patients with zero or one risk genotype. CONCLUSION: We identified four novel SNPs associated with gemcitabine-induced severe leukopenia/neutropenia. These SNPs might be applicable in predicting the risk of hematological toxicity in patients receiving gemcitabine therapy.

Dose-adjustment study of tamoxifen based on CYP2D6 genotypes in Japanese breast cancer patients.

CYP2D6 is a key enzyme responsible for the metabolism of tamoxifen to active metabolites, endoxifen, and 4-hydroxytamoxifen. The breast cancer patients who are heterozygous and homozygous for decreased-function and null alleles of CYP2D6 showed lower plasma concentrations of endoxifen and 4-hydroxytamoxifen compared to patients with homozygous-wild-type allele, resulting in worse clinical outcome in tamoxifen therapy. We recruited 98 Japanese breast cancer patients, who had been taking 20 mg of tamoxifen daily as adjuvant setting. For the patients who have one or no normal allele of CYP2D6, dosages of tamoxifen were increased to 30 and 40 mg/day, respectively. The plasma concentrations of tamoxifen and its metabolites were measured at 8 weeks after dose-adjustment using liquid chromatography-tandem mass spectrometry. Association between tamoxifen dose and the incidence of adverse events during the tamoxifen treatment was investigated. In the patients with CYP2D6*1/*10 and CYP2D6*10/*10, the mean plasma endoxifen levels after dose increase were 1.4- and 1.7-fold higher, respectively, than those before the increase (P < 0.001). These plasma concentrations of endoxifen achieved similar level of those in the CYP2D6*1/*1 patients receiving 20 mg/day of tamoxifen. Plasma 4-hydroxytamoxifen concentrations in the patients with CYP2D6*1/*10 and CYP2D6*10/*10 were also significantly increased to the similar levels of the CYP2D6*1/*1 patients according to the increasing tamoxifen dosages (P < 0.001). The incidence of adverse events was not significantly different between before and after dose adjustment. This study provides the evidence that dose adjustment is useful for the patients carrying CYP2D6*10 allele to maintain the effective endoxifen level.