Part I: Genome-wide association study of pancreatic cancer in Japanese population

Background

Pancreatic cancer is the fifth leading cause of cancer death with an estimated death of 24,634 patients in Japan in year 2007. Its 5-year survival rate is as low as 6.7%. Since no specific symptom is observed at the early stage, most of the pancreatic cancer patients were diagnosed at their advanced stage with very low possibility of radical cure for the disease.

Previous reports indicated the involvement of both environmental and genetics factors in the etiology of this deleterious disease. Several case-control and cohort epidemiological studies have identified a number of possible risk factors such as smoking, diabetes, chronic pancreatitis, which are likely to predispose individual to the disease. Familial aggregation of the disease has implied the possible involvement of genetic factors in pancreatic cancer; approximately 10% of the patients were reported to have family history and individuals having first-degree relatives with pancreatic cancer revealed 2- to 4- fold higher risk of the disease. These data indicated that genetic factors are likely to play some roles in the development of pancreatic cancer.

Common genetic variations are known to associate with various common diseases and cancers. Single nucleotide polymorphism (SNP) is a type of common genetic variations which occurs in at least 1% of the human genome. The objective of this study is to identify SNPs associated with pancreatic cancer susceptibility in Japanese population through genome-wide association study (GWAS).

Methodology

This study is a collaborative study between The University of Tokyo and National Cancer Center, Japan. A total of 1006 invasive pancreatic ductal adenocarcinoma cases and 5311 controls were recruited. All the samples were genotyped using Illumina HumanHap550v3 or Illumina Human Hap 610 Genotyping BeadChip. For sample quality control, sample with call rate lower than 0.98 were excluded. Additionally, principal component analysis was performed to exclude individual who have admixture genetic component from the major Japanese Hondo cluster. After sample QC, association study was performed on 991 cases and 5209 controls. For SNP quality control, SNPs which have call rate <0.99 in both cases and controls, P-value of Hardy-Weinberg equilibrium test of <1.0x10-6 in control and minor allele frequency of SNP ≦0.01 were excluded from further analysis. A total of 420,236 SNPs on autosomal chromosomes passed the QC filters. Case-control association study was performed by logistic regression analysis after adjustment of age (continuous), sex and smoking status (current/former, never). P-values and OR with 95%CI were calculated for allelic, dominant and recessive models by using PLINK program. We used the minimum P-values obtained from three models to evaluate the statistical significance of the association. Significance threshold was set at P<5.0x10-7. To infer untyped and missing genotypes around the candidate loci, genotype imputation was performed by utilizing a Hidden Markov model programmed in MACH version 1.0. By utilizing the genotype information from the HapMap database, maximum likelihood genotypes for the untyped SNPs were generated. For quality control, imputed SNPs with the estimated r2 of >0.3 were retained.

Results and Discussion

The Q-Q plot for this GWAS based on allelic P-values by logistic regression revealed no significant population stratification with genomic inflation factor λ of 1.026 after sample and SNP QC. Three genomic regions 6p25.3, 12p11.21 and 7q36.2, shown to be significantly associated (P-value<5.0x10-7) with increased risk of pancreatic cancer in Japanese population were successfully identified (Table 1).

The most significantly-associated SNP, rs9502893, is located within a 75-kb linkage disequilibrium (LD) block on chromosome 6p25.3. This LD block includes FOXQ1 (forkhead box (Fox) Q1) gene, which is located 25kb upstream to this marker SNP. Imputation analysis revealed modest association at SNPs located near to or on the FOXQ1 gene suggesting it to be one of the causative genes for pancreatic cancer. FOXQ1 encodes for protein forkhead box (Fox) Q1. The Fox family of transcription factors consists of at least 43 members and mutations in Fox genes can cause significant effects on human common diseases and cancers. A recent study showed that FoxQ1 is overexpressed in pancreatic cancer, suggesting its role in pancreatic tumorigenesis.

The second significantly-associated SNP, rs708224, located in the second intron of the gene BICD1 (Bicaudal-D homolog 1) on chromosome 12p11. The 80-kb LD block showing the association corresponds to the second intron of BICD1 as revealed by the imputation analysis. Bicaudal-D homolog 1 protein plays a role in vacuolar trafficking. A recent study suggested that genetic variations within the BICD1 gene could alter its transcriptional levels and in turn influence telomere length in humans. In addition, several recent studies have documented reduced telomere length in pancreatic ductal adenocarnoma specimens, suggesting telomeric dysfunction in pancreatic cancer cells.

The third locus is marked by rs6464375 in the first intron of DPP6 gene. The SNP indicated suggestive associations only under recessive model. DPP6 encodes protein dipeptidyl-peptidase 6, which binds to specific voltage-gated potassium channels and alters their expression and biophysical properties. A recent study on core signaling pathways in human pancreatic cancers found three somatic mutations in DPP6 among 24 pancreatic cancer samples examined by detailed sequence analyses. This report also suggested that DPP6 might play a crucial role in regulation of invasion of pancreatic cancer cells.

Conclusion

This study represents the first GWAS to identify common genetic variants associated with pancreatic cancer in Japanese population. Genes that identified by this study have been implicated to play important roles in the pathogenesis, telomere dysfunction and invasion of pancreatic cancer cells. Our findings may contribute to a better understanding of pancreatic carcinogenesis.

Part II: Identification of genetic variants associated with cyclophosphamide-induced adverse drug reactions in breast cancer

Background

Cyclophosphamide (CPA) is one of the most widely used anticancer drugs in the treatment of hematological malignancies and a variety of solid tumors including breast cancer. The CPA-based combination treatment has known to be effective for breast cancer, but often causes adverse drug reactions (ADRs), such as leucopenia/neutropenia and gastrointestinal symptoms such as vomiting, anorexia and nausea. Most of the drug-metabolizing enzymes and transporters contain a wide range of genetic polymorphisms, which might cause a large interindividual variability in the plasma concentration of drugs. Furthermore, anticancer therapies are notoriously known to have narrow therapeutic range; higher concentration in patients' body causes toxicity and lower concentration reduces the efficacy of the drugs. Hence, the role of pharmacogenomics which is expected to provide a predictive way for severe drug toxicity is greatly essential. The objective of this study is to discover SNPs associated with CPA-induced ADRs in patients with breast cancer using a case-control association study, focusing on not only the drug-metabolizing enzymes, but also the transporters, which might also play an important role in pharmacokinetics of CPA or its active forms.

Methodology

All the samples of this study were recruited from Biobank Japan, the University of Tokyo. In this study, patients who revealed ≧grade3 leucopenia or neutropenia, or those with ≧grade2 gastrointestinal toxicity induced by CPA combination therapy were defined as cases (ADRs), while controls (non-ADRs) were defined as patients who had shown no toxicity during CPA-based combination therapy. The first exploratory sample set consist of 76 cases (ADRs) and 140 controls (non-ADRs), and an independent second set samples (replication set) consist of 108 ADR cases and 79 non-ADR controls were recruited. A total of 141 SNPs (tagSNPs and functional SNPs) and two deletion polymorphisms in 13 candidate genes, which are involved in activation, detoxification and transportation of CPA (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, ALDH1A1, ALDH3A1, GSTA1, GSTM1, GSTP1, GSTT1, ABCC2, and ABCC4), were genotyped. The selection criteria of the tagSNPs were based on the measures of linkage disequilibrium (LD) with r2 value ≧0.8 and minor allele frequency (MAF) of >10% from the HapMap database. All the SNPs were genotyped using multiplex polymerase chain reaction (PCR)-invader assay or direct sequencing. Case-control association study was evaluated using Cochran-Armitage trend test. Additionally, multiple testing, Bonferroni correction threshold (P-value<0.00035) were applied, to assess the significance level of the association. Subgroup analysis, such as types of ADR developed after receiving CPA combination therapy and types of chemotherapy regimen for breast cancer, were also evaluated.

Results and Discussion

Among the 143 common variations analyzed in this study, one SNP, rs9561778 in ABCC4 is significantly associated with CPA combination therapy induced ADR after applying strict Bonferroni's correction (Table 2). For the subgroup based on types of ADRs, rs9561778 showed association with both the gastrointestinal toxicity and leucopenia/neutropenia, yielding similar trends of odds ratio (Table 2). Additionally, this SNP also revealed significant association with a higher odds ratio with patients treated with the CA(F) (Cyclophosphamide and Anthracyclin with or without 5-fluorouracil) drug regimen (Table 2) in which CA(F) regimen is one of the most major combination therapies for breast cancer.

ABCC4 is a member of the superfamily of ATP-binding cassette (ABC) transporters. A recent study indicated that CPA and/or its active metabolites are the substrates to ABCC4 because the in vitro CPA cytotoxicity was significantly enhanced after addition of ABCC4 inhibitor. Since ABCC4 is known to express in kidney, the expression of ABCC4 in the kidney might play an important role in elimination of CPA and its metabolites from the body and genetic variations within this gene might affect the amount or nature of this transporter, resulting in impairment of excretion and subsequent overdose manifestation. In addition, the expression of ABCC4 in the sinusoidal membrane of hepatocytes might facilitate the secretion of active metabolites of CPA produced in the liver into the systemic circulation. Variants on this gene might cause excess efflux of CPA and its metabolites, which consequently increased systemic drug concentration in the body.

Conclusion

Through candidate gene approach, associations of ABCC4 genotypes and CPA-induced ADRs were identified. Although the association as well as the mechanism to induce ADRs should be further validated by using larger number of samples or by molecular analysis, this study has contributed another piece of puzzle into the mist of prediction system which may help in identifying patients at risk of CPA-induced ADRs and lead to a better prognosis and quality of life for patients with cancer.

Table1: Association study of GWAS of pancreatic cancer in Japanese population

Odds ratios, 95% confidence limits and P-values were obtained using logistic regression analysis according to allelic, dominant and recessive model after adjustment of age, sex and smoking. RAF, risk allele frequency; OR, odds ratio;L95 U95, lower and upper confidence limits; Pmin, minimum P-volue among tnree genetic models.'Position and relative loci (Relativeloc) based on NCB/ Human Genome Build 36.

Table 2: Association study of SNP rs9561778 with CPA combination therapy induced ADRs and its subgroup analysis.

GI*:≧Grade 2 Gastrointestinal toxicity, LN** :≧a-Grades leucopenia or ropenia CAF***C. ;Cyclophosphamide, A. AnihraCyclin (Epirubicin or Adriamyc n), F. 5-fluorouracil

本論文は2章からなり、第一章は膵臓がんの発癌関連遺伝子の同定について述べられており、第二章は乳がん患者におけるシクロヘキサミドによる副作用発症関連遺伝子についての報告である。

第一章では、筆者はすい臓がんの疾患感受性遺伝子の同定目的でゲノムワイド関連解析を行った。すい臓の初期では自覚症状に乏しく、癌患者の多くは発見時には既に進行期となっている。その為、すい臓患者の予後は非常に不良で癌全体の死因の第5位と鳴っている。また膵臓がんの発症機序は殆ど明らかとなっていない事から、疾患感受性遺伝子を同定することで、癌の早期発見に繋がるだけでなく、病気の原因解明に役立つと期待できる。

本研究は東京大学医科学研究所と国立がんセンターとの共同研究で行われている。計1006人のすい臓がん患者と5311名のコントロールについて、55万箇所のSNPのタイピングを行った。Q-Q plotの結果より、用いたサンプルで集団の構造化がないことが確認され、解析結果が信頼できるものであると考えられた。最終的にquality checkを通過した420,236SNPについて年齢、性別、喫煙暦を調整因子としてcase-control相関解析を行った。またその際の有意水準を5x10-7とした。最終的に6p25.3,12p11.21, 7q36.2の3つの領域がすい臓がん発症と強い関連を示した。

もっとも強い関連をしめした6p25.3上のSNPrs9502893はFOXQ1を含む50kbの連鎖不平衡領域内にあった。FOXQ1は大腸癌や膵癌で発現上昇が報告されており、発癌との関与が示唆されている。また欧米人で関連が報告されているSNPの内、13q22.1領域のSNPが日本人サンプルでも関連が明らかとなった。今回の研究は、日本人で始めてのゲノムワイド関連解析であり、膵臓がんの原因解明や予防、早期発見に有用となると期待できる。

第2章では、薬理遺伝学的な解析を行った。筆者は乳がん患者におけるシクロヘキサミドに対する副作用の有無に関連する遺伝子の探索を行った。本研究では、候補遺伝子としてシクロヘキサミドの代謝に関わる13の遺伝子を抽出し、これらの遺伝子領域から141のタグSNP(MAF >0.1, r2>0.8)を選択した。副作用群184名、非副作用群219名の解析の結果、ABCC4遺伝子上のrs9561778が副作用の発症と有意な関連を示した。

ABCC4遺伝子はABCtransporterファミリーに属し、シクロヘキサミドの輸送に関与することがin vitroの結果より示されている。ABCC4は腎臓で高発現している事より、遺伝子型の違いによって腎からの排出が低下し、血中濃度が上昇する結果副作用のリスクが高まると考えられる。実際in silicoの解析により、今回解析したSNPに対し転写因子の結合の程度がアレル間で異なることが示されている。

以上のように候補遺伝子の解析の結果、ABCC4の遺伝子型とシクロヘキサミドによる副作用のリスクが関連を示すことが明らかとなった。膵癌の解析結果と合わせて、遺伝子多型を疾患の発症予測や薬剤の副作用予測に用いることで、疾患の早期発見や有効な治療法の選択が可能となる。今回の研究成果は個別化医療の推進に向けて有用であると考えられる。

なお本論文は、清谷一馬、莚田秦誠、醍醐弥太郎、前佛均、松田浩一、中村祐輔、坂本裕美、吉田輝彦、高橋篤、久保充明、鎌谷直之との共同研究であるが、論文提出者が主体となって分析及び検証を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(生命科学)の学を授与できると認める。