(The) impact of circulating tumor DNA in biliary tract cancer under chemotherapy

다국어 초록 (Multilingual Abstract)

Biliary tract cancer (BTC) is a highly aggressive cancer with a very poor prognosis. In general, the incidence of BTC is higher in Eastern countries than in Western countries. BTC was once considered a geographically region-specific disease. However, according to recent reports, the incidence of BTC has increased globally. Most patients with BTC were first diagnosed at the advanced stage because the disease is usually asymptomatic during the early stage. Tissue biopsy is the current gold standard for cancer diagnosis, but this invasive technique has challenges. Despite the increased incidence rate and poor prognosis of BTC, understanding this disease is still not satisfactory. To discover actionable target genes and monitor the drug response of patients, we enrolled unresectable BTC patients (n = 41), and circulating-tumor DNA (ctDNA) from plasma samples was collected at multiple timepoints while patients received chemotherapy (pre-1st chemotherapy, pre-2nd chemotherapy, pre-4th chemotherapy, and progression disease). All samples were deep sequenced with a large panel containing 531 pan-cancer genes. We identified highly observed variants, such as TP53, ARID2, KRAS, ARID1A, PDE4DIP, ARID1B, CHD4, FAT1, PIK3CA, SPEN, APC, ATM, ATR, ERBB4, FGFR2, and IDH1. In addition, copy number alterations (CNAs) of MYC, ERBB2, CDKN2A, GATA4, ARID2, MDM2, PIK3R3, CDK12, and EGFR were observed. Key pathways and genes were curated from the literature and detected single nucleotide variants (SNVs) were categorized by them. Epigenetic regulation, TP53 signaling, the PI3K/AKT/mTOR and RAS/RAF/ERK pathways, DNA damage, angiogenesis, and DNA repair were highly ranked. TP53, ARID2, and PTPRT frequently occurred under chemotherapy. In particular, the PTPRT mutation remarkably increased in a cohort with progression disease as compared with that of cohorts at other timepoints. The survival rate of BTC patients with a low tumor mutation burden (TMB) was higher than that of the high TMB patient group. Also, a new threshold by delta blood TMB (dTMB) showed potential as a marker for diagnosis. In the present study, we suggested the advantages of cell-free DNA (cfDNA)-targeted sequencing and discussed candidates of precision therapy and understanding molecular profiling of BTC patients under chemotherapy.

목차 (Table of Contents)

• ABSTRACT 1
• I. INTRODUCTION 3
• II. MATERIALS AND METHODS 10
• 1. Study samples 10
• 2. Genomic DNA extraction 13
• 3. Circulating tumor DNA extraction 13
• 4. TMB500 panel 14
• 5. Targeted sequencing 14
• A. DNA fragmentation 14
• B. End repair and A-tailing 15
• C. Adaptor ligation 15
• D. Pre-PCR 16
• E. Hybridization capture-based target enrichment 16
• F. Post-PCR 17
• G. Sequencing 18
• 6. Data processing and variant calling 18
• 7. Variant interpretation 18
• 8. Assessment of concordance rate between tissue and cfDNA 19
• 9. Estimation of microsatellite instability 19
• 10. Estimation of tumor mutation burden 19
• 11. Pathway analysis 20
• III. RESULTS 21
• 1. Sequencing quality 21
• 2. High concordance rate between tissue NGS and cfDNA NGS 22
• 3. Spectrum of cfDNA somatic variants in biliary tract cancer 24
• 4. Comparison of the number of patients with differentially mutated genes between timepoints 27
• 5. Enrichment pathway of significant variants 29
• 6. The PTPRT gene contributes to poor prognosis of BTC patients 35
• 7. Platinum drug resistance genes affects prognosis of BTC patients 36 8. Change of pathway frequency in non-persistent PDR patients 38
• 9. PI3K and STAT3 pathways affect the prognosis of BTC patients 40
• 10. TMB is a potential prognostic marker 42
• 11. Comparison of the cfDNA amount at each timepoint with clinical features 48
• IV. DISCUSSION 50
• V. CONCLUSION 59
• REFERENCES 60
• APPENDICES 68
• ABSTRACT(IN KOREAN) 72

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