臺灣大腸直腸癌的全外顯子定序研究

Abstract

研究背景與動機 根據台灣衛生福利部國民健康署的資料，大腸直腸癌在台灣是最常見的癌症之一現居所有癌症發生率的第二名，死亡率則在癌症相關疾病中排名第三。現今台灣關於大腸直腸癌在基因方面的研究大多是對特定基因組進行分析，但這種研究沒有全外顯子定序方法所分析的結果全面，因此本研究旨在使用台大醫院在大腸直腸癌患者中收集到的全外顯子定序的資料來進行分析，以建構台灣患者的基因突變圖譜及找到特有突變事件，同時也會分析台灣病患的突變特徵。此外，研究中亦會使用全外顯子定序分析過程中產生的檔案來進行拷貝數變異分析以更全面的觀察到台灣大腸直腸癌患者不同染色體區域的拷貝數增益或缺失的情況。 研究方法 研究中所使用的資料來自於台大醫院從209位大腸直腸癌病患中所採集的全外顯子定序資料來進行分析，使用的背景基因體版本為hg19。過程中首先將使用BWA-MEM、Samtools、MarkDuplicates、ApplyBQSR等工具將讀段進行比對並排序，並移除重複讀段及使用常見變異資料庫來進行校正。接著，使用GATK的LearnReadOrientationModel、GetPileupSummaries、CalculateContamination等工具判斷是否有假陽性的情形，並使用Mutect2進行正常樣本與腫瘤樣本的配對變異檢測。最後用FilterMutectCalls與SelectVariants將不合格的突變進行過濾。在突變特徵使用的是SigProfilerExtractor來進行分析。此外，在拷貝數變異的過程中，則是用CollectReadCounts、DenoiseReadCounts及ModelSegments等工具先擷取出區間的讀段數量，再使用正常樣本的參考面板將腫瘤樣本的檔案進行標準化及降噪，最後再找出可能的拷貝數變異片段。 研究結果 在COSMIC v70中關於大腸直腸癌收錄筆數≧100的20個常見突變事件中，研究中所捕捉到的常見大腸直腸癌的突變事件共有15個。包含KRAS（rs112445441_p.G13D、rs12193529_p.G12V/ p.G12D、rs12193530_p.G12V/p.G12S）、TP53（rs28934574_p.R282W、rs28934576_p.R273L、rs121913343_ p.R273C、rs11540652_ p.R248Q、rs28934575_ p.G245S、rs28934578_ p.R175H）、PIK3CA（rs12193213_ p.E542K、rs12193279_ p.H1047R）、APC（rs12193332_ p.R1450X）、BRAF（rs113488022_ p.V600E）。因此在本研究中共捕捉到了75%的常見突變事件。此外，在KRAS、APC、OR2T2、KRT76、ACVR2A、PLEKH6、RNF43、DOCK3上有10個突變事件是在COSMIC v70中關於大腸直腸癌收錄筆數較少，但在研究樣本中突變比例較高的突變事件，可能是台灣大腸直腸癌病患特有的突變事件。 在突變特徵的結果中，主要涵蓋的有年齡、DNA聚合酶ε催化次單元突變、DNA錯配修復缺陷、微衛星不穩定性、DNA聚合酶δ1基因突變、同源重組為基礎的DNA損傷修復功能缺陷、馬兜鈴酸暴露，這些應該是台灣大腸直腸癌病患較常見的致癌原因。 拷貝數變異的結果中，第7、8、13、18、20號染色體有較高的樣本變異比例，其中在第8號染色體前段及第18號染色體為缺失，第7、8後半段、13、20號染色體為增益，且這些區域在僅留下拷貝數≧2.5或拷貝數≦1.5的結果時也同樣明顯。而在僅留下拷貝數≧3或拷貝數≦1的結果時，第13及20號染色體仍有相對較高的樣本增益比例。 討論與結論 研究結果中涵蓋了75%的常見大腸直腸癌突變，發現了10個可能是台灣大腸直腸癌病患特有的突變事件，並根據突變特徵的結果找出了可能的致癌原因。同時，在拷貝數變異分析中找出了台灣大腸直腸癌患者有較明顯拷貝數變異的區域，期望這些結果能在大腸直腸癌患者的治療成效與預後上有所助益。

Background and Motivation According to data from the Health Promotion Administration of Taiwan's Ministry of Health and Welfare, colorectal cancer is one of the most common cancers in Taiwan, currently ranking second in incidence among all cancers and third in cancer-related mortality. Most genetic studies on colorectal cancer in Taiwan to date have focused on specific gene sets; however, such studies lack the comprehensiveness of whole-exome sequencing (WES). Therefore, this study aims to analyze WES data collected from colorectal cancer patients at National Taiwan University Hospital to identify the mutational landscape and unique mutation events specific to Taiwanese patients. In addition, the study will also investigate the mutational signatures present in these patients. Furthermore, copy number variation (CNV) analysis will be conducted using data generated during the WES process to provide a more comprehensive understanding of chromosomal gains and losses in different regions among Taiwanese colorectal cancer patients. Research Methods The data used in this study were obtained from WES of 209 colorectal cancer patients at National Taiwan University Hospital, using the hg19 reference genome. First aligning and sorting the sequencing reads using tools such as BWA-MEM, Samtools, MarkDuplicates, and ApplyBQSR. Duplicated reads were removed, and data were calibrated using common variant databases. Next, using tools such as GATK's LearnReadOrientationModel, GetPileupSummaries, and CalculateContamination were used to identify potential false positives. Variants were then detected by pairing normal and tumor samples using Mutect2. FilterMutectCalls and SelectVariants were applied to filter out variants that did not meet quality standards. For mutation signatures, the analysis was performed using SigProfilerExtractor. In addition, for CNV analysis, tools such as CollectReadCounts, DenoiseReadCounts, and ModelSegments were employed to first extract the read count data from genomic regions. Tumor samples were normalized and denoised using reference panels from normal samples, and potential CNV segments were identified Research Results Among the 20 most common mutations in colorectal cancer recorded in COSMIC v70 with ≥100 entries, 15 common mutations were captured in this study. These include KRAS（rs112445441_p.G13D、rs12193529_p.G12V/ p.G12D、rs12193530_p.G12V/p.G12S）、TP53（rs28934574_p.R282W、rs28934576_p.R273L、rs121913343_ p.R273C、rs11540652_ p.R248Q、rs28934575_ p.G245S、rs28934578_ p.R175H）、PIK3CA（rs12193213_ p.E542K、rs12193279_ p.H1047R）、APC（rs12193332_ p.R1450X）、BRAF（rs113488022_ p.V600E）. Thus, 75% of the common mutations were captured in this study. Additionally, 10 mutations with higher variant frequencies in the study samples were found in KRAS, APC, OR2T2, KRT76, ACVR2A, PLEKH6, RNF43, and DOCK3. These mutations were less frequently recorded in COSMIC v70 for colorectal cancer, suggesting they may be unique to Taiwan colorectal cancer patients . The mutation signature results primarily reflect mutational processes associated with aging, mutations in the catalytic subunit of DNA polymerase epsilon (POLE), DNA mismatch repair deficiency (MMRd), microsatellite instability (MSI), mutations in the DNA polymerase delta 1 (POLD1) gene, defects in homologous recombination-based DNA damage repair (HR repair), and exposure to aristolochic acid. These are likely the major carcinogenic mechanisms of colorectal cancer in Taiwanese patients. Based on the CNV results, chromosomes 7, 8, 13, 18, and 20 showed a higher proportion of samples with variations. Specifically, losses were observed on the proximal region of chromosome 8 and on chromosome 18, while gains were observed on the distal region of chromosome 8, as well as on chromosomes 7, 13, and 20. These regions remained prominent even when applying a stricter threshold of copy number ≥2.5 or ≤1.5. Furthermore, under a more stringent threshold of copy number ≥3 or ≤1, chromosomes 13 and 20 still exhibited relatively high frequencies of copy number gains. Discussion and Conclusion The study covered 75% of common colorectal cancer mutations and identified 10 potentially Taiwan-specific mutations. Based on the mutation signature analysis, possible carcinogenic mechanisms were inferred. Through copy number variation analysis, we identified genomic regions with notable alterations in Taiwanese colorectal cancer patients. These findings may provide insights that contribute to enhancing treatment efficacy and prognosis.