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Somatic mutation profile of epithelial cell adhesion molecule positive single cells from blood of metastatic breast cancer patients
전이 유방암 환자 혈액에서 상피세포 부착 분자 양성 단일세포의 체세포 돌연변이

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Authors
최지혜
Advisor
한원식
Major
의과대학 의학과
Issue Date
2018-02
Publisher
서울대학교 대학원
Keywords
Breast neoplasmCirculating tumor cellNext generation sequencingLiquid biopsy
Description
학위논문 (석사)-- 서울대학교 대학원 : 의과대학 의학과, 2018. 2. 한원식.
Abstract
Abstract
Somatic mutation profile of epithelial cell adhesion molecule positive single cells from blood of metastatic breast cancer patients

Background: Circulating tumor cell (CTC) enumeration provides prognostic information for chemotherapy in metastatic breast cancer. However, due to its rarity and heterogeneity, it is difficult to distinguish true CTCs from normal blood cells and perform genomic analysis on them for use in therapeutic strategies. Most currently available CTC detection systems consist of an enumeration of putative CTCs without further analysis. The aim of this study was to evaluate the feasibility of single cell picking and target sequencing of epithelial cell adhesion molecule (EpCAM)-positive cells for detecting CTCs.
Methods: Whole blood sampled from metastatic breast cancer patients who were newly diagnosed with metastasis or who had disease progression during palliative treatment were used for this study. After applying IsoFlux Circulating Tumor Cell Enrichment Kit (Fluxion, South San Francisco, CA, USA), single CTC candidates were picked from a pool of EpCAM-positive cells. Genomic DNA from the picked cells was whole genome amplified and target sequencing was performed using Ion AmpliSeq™ Cancer Hotspot Panel (Life Technologies, Carlsbad, CA, USA). Target sequencing reads were mapped on human genome reference (hg19) using BWA-MEM (0.7.10). Single nucleotide variants (SNVs) were annotated using dbSNP, Human Variome Project 0.2 and COSMIC databases.
Results: A total of 172 EpCAM-positive cells were selected according to size and EpCAM status from whole blood of 11 patients. The remaining cells were grouped into a pooled sample for each patient. The mean read depth of the target genes was 13455ⅹ. A mean 8.55 mutations as determined by SNVs listed in the COSMIC database but not in dbSNP and Variome Data 0.2 were detected in each patient. Cells with multiple mutated genes, or those with a mutated gene repeatedly observed in another cell from the same patient were judged to be putative CTCs. At least 2 putative CTCs were detected in 7 patients while no CTCs were detected in 2 patients. Mutated genes observed in the putative CTCs were ABL1, AKT1, APC, CDH1, CDKN2A, ERBB2, FGFR3, HRAS, IDH1, JAK2, KDR, NPM1, RB1, RET, SMARCB1, STK11, and TP53.
Conclusions: Potential CTCs were successfully identified by single cell picking and target sequencing of EpCAM-positive cells from whole blood of metastatic breast cancer patients. Unique mutations not detected in other single cells and pooled samples can be used to distinguish putative CTCs from normal cells. Our results implicate an area for research and validation of CK negative subgroups of CTCs.
Language
English
URI
https://hdl.handle.net/10371/142350
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Medicine (의학과)Theses (Master's Degree_의학과)
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