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研究生：	蘇勇曄 Su, Yung-Yeh
論文名稱：	胰臟癌精準醫療及其臨床應用 Precision medicine in pancreatic cancer and its clinical application
指導教授：	沈延盛 Shan, Yan-Shen
學位類別：	博士 Doctor
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2023
畢業學年度：	112
語文別：	英文
論文頁數：	133
中文關鍵詞：	次世代定序、基因多型性、鉑金化療、同源重組修復、癌幹細胞
外文關鍵詞：	Next-generation sequencing, gene polymorphism, platinum-based chemotherapy, homologous recombination, cancer stemness
相關次數：	點閱：80 下載：11
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胰臟癌為先進國家成為十大癌症死因第三或第四名。胰臟癌目前僅有不到十項藥物被證實具有療效。我們假設通過精準醫學可以改善胰臟癌患者的預後。為此，將建立自有的次世代定序（NGS），包括目標定序、全外顯子定序和單細胞RNA定序，然後將自有的NGS用於胰臟癌的轉譯研究。首先專注於UGT1A1多型性與藥物毒性。接著，評估使用內視鏡超音波引導的細針活檢（EUS-FNB）獲得的組織是否適用於次世代定序及單細胞RNA定序。另外亦進行遺傳系(germline)全外顯子定序以探討突變與療效的相關性。最後，探索遺傳系突變、癌症幹細胞和上皮間質轉變之間的相互作用。採用的特定目標定序方式為兩階段放大次世代定序(Two-step PCR Amplicon NGS)。首先，設計目標基因primer時，與傳統PCR類似，但是後端會多一段adaptor序列，如此一來，進行第一階段PCR放大後之的產物尾端將全部帶有adaptor序列，接著將以adaptor序列為primer將用以分辨檢體來源的index barcode以及後續上機定序所需使用的Illumina P5/P7 序列進行第二階段PCR，之後的產物及稱為次世代定序的文庫，可接著上機。上機後產生的序列將儲存成FASTQ檔案。全外顯子定序的方式則是先將DNA打斷成300 base pair 附近的大小，接著進行末端修補(end repair)、A尾(A tailing)後一樣連接上adaptor/index barcode，接著使用全外顯子的探針抓取全外顯子後進行定序。單細胞定序則是先將細胞鏈結溶解成游離單細胞後，以油滴包埋的技術使每個油滴只包含一顆細胞後進行文庫製備。分析接受nal-IRI治療的患者之UGT1A1的基因多型性(UGT1A1*6, UGT1A1*28)後，發現約9.3%患者帶有兩個allele變異，其發生嚴重白血球低下的機率高達73.3%，相較於只帶有0-1個變異的患者只有38.1%會產生此副作用（p=0.012）。在EUS研究中，紅色組織的總DNA量顯著高於白色組織（2.99微克比0.70微克）。 KRAS突變等位基因頻率在白色和紅色組織之間高度一致（Spearman相關係數r=0.66，P<0.0001），證實了紅色組織和白色組織一樣可用於次世代定序。在單細胞分析研究中，早期和晚期胰臟癌共歸納出15個主要細胞型。癌細胞-4的比例在晚期明顯較高。差異表達基因分析顯示，在癌細胞-4中，UBE2C是最高表達的基因。在TCGA PAAD中， UBE2C高表達的胰腺癌具有顯著較差的存活。關於T2212研究，共有55名患者參與，其中27名被隨機分配接受modified FOLFIRINOX，28名接受GOFL，中位存活期分別為19.6個月及17.9個月。對於730例胰臟癌的治療分析，包括159名可切除的胰臟癌患者，有46人接受新輔助化療、113人直接手術，中位存活期分別為35.3個月和21.7個月。對於遺傳系全外顯子定序研究， 527名患者定序後，104人(19.7%)帶有遺傳性癌症相關基因突變，包含約10%含有同源重組基因突變，此外，分析320名有接受標準化療的晚期胰臟癌，在32名有同源重組修復基因突變的患者中，接受第一線鉑金類化療的中位存活期為26.1個月，接受第一線非鉑金類化療的中位存活期則僅9.6個月(P=0.001)。對於癌幹細胞的研究，癌症幹細胞與同源重組基因突變幾乎不會同時存在，因此可將患者分為三組，即無癌幹細胞/有同源重組基因突變、無癌幹細胞/無同源重組基因突變和有癌幹細胞，相應的中位存活期分別為15.9個月、11.8個月和6.6個月（P=0.00096）。在過去的五年裡，我們通過建立自家高成本效益基因檢測，確保了精確的基因數據定序和品質控制。其中第一個研究主題為台灣UGT1A1多型性，幫助醫師通過劑量調整來降低治療毒性。也剖析了台灣人胰臟癌的遺傳性基因圖譜，為精準醫療提供研究資源。並發現癌幹細胞與同源重組基因突變幾乎不會同時存在，更新了我們對胰臟癌的理解。總結，自行開發的基因檢測在解決臨床問題和基因研究方面具有顯著的貢獻，使我們能在胰臟癌轉譯研究領域佔有一席之地。

Pancreatic ductal adenocarcinoma (PDAC) is currently the third or fourth leading cause of cancer death in developed countries. Less than 10 drugs are approved in PDAC. We hypothesize that we may improve the prognosis of PDAC patients through precision medicine. We establish our in-house next generation sequencing (NGS) pipeline, including target sequencing, whole exome sequencing (WES) and single cell RNA sequencing (scRNA-seq). We first apply in-house NGS to study UGT1A1 polymorphism. Next, we examined the feasibility of using endoscopic ultrasound-guided biopsy tissue for NGS and scRNA-seq. Then we performed germline WES to investigate the correlation between germline mutations and treatment outcomes. Finally, we explored the interplay among germline mutations and cancer stem cell (CSC). We employed a two-step PCR amplicon NGS method for targeted sequencing. The design of primers was similar to traditional PCR except for an additional adaptor sequence. In the first PCR, the products had the adaptor sequence. In the second PCR, the adaptor sequence was used as a primer to attach the index barcode and the Illumina P5/P7 sequences required for subsequent sequencing. The resulting products, known as the NGS library, was loaded onto the sequencing machine and results were stored as FASTQ files. For WES, the DNA was initially fragmented into sizes around 300 base pairs. Subsequently, end repair and A-tailing were performed, followed by the attachment of an adaptor/index barcode. Finally, exome was captured using probes. In scRNA-seq, first tissue was dissociated into individual cells and constructed as library using the oil droplets technique. In the UGT1A1 cohort, the frequencies of homozygosity/double heterozygosity (UGT1A1*6/6, UGT1A1*28/28, or UGT1A1*6/*28) was 9.3%. Patients with homozygosity/double heterozygosity had higher incidence of grade ≥3 neutropenia (73.3%) than others (38.1%) (p=0.012). In the EUS cohort, the DNA amount was higher in red tissue (2.99 µg) than white tissue (0.70 µg). The mutation allele frequency of KRAS was highly concordant between white and red tissue (Spearman correlation coefficient r=0.66, P<0.0001). In scRNA-seq, 15 major cell subtypes were identified across early and late stage. The proportion of cancer cells cluster-4 was higher in late stage. Differentially expressed genes analysis showed UBE2C was the most highly expressed gene in cancer cells cluster-4. In TCGA PAAD dataset, UBE2C high expression PDAC had significantly poor survival. For T2212 study, 55 patients were enrolled, with 27 randomized to FOLFIRINOX and 28 to GOFL with a median overall survival (OS) of 19.6 and 17.9 months, respectively. For the cross sectional study, 159 patients with resectable PDAC, 46 underwent neoadjuvant chemotherapy and 113 underwent upfront surgery. The corresponding median OS was 35.3 months and 21.7 months, respectively. For WES cohort, 104 of 527 patients harbored homologous recombination gene mutations (gHRmut). Patients with gHRmut treated with 1L platinum had better median OS (26.1 months) than those treated with 1L non-platinum (9.6 months)(P=0.017). For CSC research, we found CSC phenotype tends to be mutually exclusive with gHRmut. Thus, patients were categorized into three groups, CSC-negative/gHRmut, CSC-negative/gHRwt, and CSC-positive groups and the corresponding median OS was 15.9 months, 11.8 months and 6.6 months, respectively (P=0.00096). Over the past five years, we established a cost-effective in-house NGS pipeline, ensuring data sequencing and quality control. We addressed UGT1A1 polymorphisms in Taiwan, aiding physicians in managing treatment toxicities. We uncovered the genetic landscape of PDAC in the Taiwanese population. The finding of mutual exclusivity between CSC phenotype and gHRmut reshaped our understanding of PDAC. In conclusion, our in-house NGS pipeline significantly contributes to clinical problem-solving and genetic research.

Background	16
1	Diagnosis of pancreatic cancer	16
2	Prognosis of pancreatic cancer	16
2.1	Low resectibility	17
2.2	Early metastases	18
2.3	Drug resistance	19
3	Prognosis of pancreatic cancer in Taiwan	20
4	Technique aspect of precision medicine	20
4.1	Overview of first and next generation sequencing	20
4.2	Single-cell RNA sequencing	21
4.3	Genetic information came from early stage PDAC	21
4.4	Accessibility of NGS in Taiwan	22
5	Precision medicine in pancreatic cancer	23
5.1	Function of UGT1A1	23
5.2	UGT1A1 polymorphisms and irinotecan	24
5.3	Germline mutational landscape in pancreatic cancer	25
5.4	PARP inhibitor in germline BRCA1/2 and HRD	27
5.5	HRD and platinum chemotherapy	30
5.6	CD133/CD44 expression and cancer stemness	31
5.7	Epithelial-mesenchymal transition and GATA6	32
5.8	Interplay among cancer stemness, EMT and DNA repair	33
Hypothesis and Study Aims	34
Methods	36
1	Theme 1-1 To explore the correlation of UGT1A1 polymorphisms and treatment toxicities	36
1.1	Touchdown Polymerase Chain Reaction	36
1.2	Target DNA enrichment and Sequencing	37
1.3	Patients sample for UGT1A1 sequencing	37
1.4	Detection of UGT1A1 polymorphism	38
2	Theme 1-2 To investigate the context of tissue obtained from EUS-guided pancreatic biopsies	39
2.1	DNA extraction	39
2.2	KRAS sequencing	40
3	Theme 1-3 To dissect the complex tumor biology of pancreatic cancer using single-cell RNA sequencing via endoscopic ultrasound-guided fine-needle biopsy	40
3.1	Cell preparation	40
3.2	Library construction of Single-cell RNA sequencing	41
3.3	Single Cell RNA Sequencing data processing and analysis	41
4	Theme 2-1 To explore the treatment outcome of PDAC in Taiwan	42
4.1	Cross-sectional cohort study	42
4.2	TCOG T2212 study	43
4.3	Statistics	44
5	Theme 2-2 To investigate germline mutations of homologous recombination genes and clinical outcomes in pancreatic cancer - a multicenter study in Taiwan	44
5.1	Whole exome Sequencing	44
5.2	Evidence-based annotation	46
5.3	Frequency-based annotation	47
5.4	Functional prediction	48
6	Theme 2-3 To examine the prognostic significance of GATA6 and CD133/CD44 expression, and germline homologous recombination gene mutation in metastatic pancreatic ductal adenocarcinoma	50
6.1	Immunochemical assessment	50
6.2	Cancer stemness	51
6.3	Germline homologous recombination gene	51
Results	52
1	Theme 1-1 To explore the correlation of UGT1A1 polymorphisms and treatment toxicities	52
1.1	Pharmacogenetics distribution of UGT1A1 polymorphismsTables	52
1.2	Adverse events and UGT1A1 polymorphisms	52
1.3	Pharmacokinetics and UGT1A1 polymorphisms	53
2	Theme 1-2 To investigate the context of tissue obtained from EUS-guided pancreatic biopsies	54
2.1	Patient and lesion characteristics of EUS-FNB cohort	54
2.2	EUS-FNB technical aspects	55
2.3	Histologic assessment and diagnostic performance	55
2.4	DNA amount and KRAS mutation allele frequency	56
3	Theme 1-3 To dissect the complex tumor biology of pancreatic cancer using single-cell RNA sequencing via endoscopic ultrasound-guided fine-needle biopsy	57
3.1	Suction versus non-suction techniques	57
3.2	Single-cell transcriptomics	58
4	Theme 2-1 To explore the treatment outcome of PDAC in Taiwan	58
4.1	A Phase II Randomized Trial in Locally Advanced Pancreatic Cancer: TCOG T2212 study	58
4.2	Induction chemotherapy and conversion surgery in locally advanced unresectable pancreatic cancer	63
4.3	The experience of neoadjuvant chemotherapy versus upfront surgery in resectable pancreatic cancer	64
5	Theme 2-2 To investigate germline mutations of homologous recombination genes and clinical outcomes in pancreatic cancer - a multicenter study in Taiwan	66
5.1	Population characteristics	66
5.2	Family cancer history and PDAC diagnosed age	67
5.3	Mutational Landscape	67
5.4	Germline HR genes and treatment outcome	68
6	Theme 2-3 To examine the prognostic significance of GATA6 and CD133/CD44 expression, and germline homologous recombination gene mutation in metastatic pancreatic ductal adenocarcinoma	69
6.1	Baseline characteristics	69
6.2	Cancer stemness and gHRmut predicted survival	70
6.3	Dose intensity and safety profiles	70
Discussion	71
1	Theme 1-1 To explore the correlation of UGT1A1 polymorphisms and treatment toxicities	71
2	Theme 1-2 To investigate the context of tissue obtained from EUS-guided pancreatic biopsies	72
3	Theme 1-3 To dissect the complex tumor biology of pancreatic cancer using single-cell RNA sequencing via endoscopic ultrasound-guided fine-needle biopsy	73
4	Theme 2-1 To explore the treatment outcome of PDAC in Taiwan	74
5	Theme 2-2 To investigate germline mutations of homologous recombination genes and clinical outcomes in pancreatic cancer - a multicenter study in Taiwan	75
6	Theme 2-3 To examine the prognostic significance of GATA6 and CD133/CD44 expression, and germline homologous recombination gene mutation in metastatic pancreatic ductal adenocarcinoma	76
Conclusion and Perspective	77
Reference	80
Figures	92
Tables	113
Related publications	130
                                    

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