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Mutation analysis of cell-free DNA and single circulating tumor cells in metastatic breast cancer patients with high CTC counts

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Title: Mutation analysis of cell-free DNA and single circulating tumor cells in metastatic breast cancer patients with high CTC counts
Authors: Shaw, JA
Guttery, DS
Hills, A
Fernandez-Garcia, D
Page, K
Rosales, BM
Goddard, KS
Hastings, RK
Luo, J
Ogle, O
Woodley, L
Ali, S
Stebbing, J
Coombes, RC
Item Type: Journal Article
Abstract: Purpose: The purpose of this study was to directly compare mutation profiles in multiple single CTCs and cfDNA isolated from the same blood samples taken from patients with metastaic breast cancer (MBC). We aimed to determine whether cell-free DNA would reflect the heterogeneity observed in 40 single CTCs. Experimental design: CTCs were enumerated by Cellsearch. CTC count was compared with the quantity of matched cfDNA and serum CA15-3 and alkaline phosphatase (ALP) in 112 patients with metastatic breast cancer. In 5 patients with {greater than or equal to}100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumour tissue by targeted next generation sequencing (NGS) of ~2200 mutations in 50 cancer genes. Results: In the whole cohort, total cfDNA levels and cell counts ({greater than or equal to}5 CTCs) were both significantly associated with overall survival, unlike CA15-3 and ALP. NGS analysis of 40 individual EpCAM-positive CTCs from 5 patients with MBC revealed mutational heterogeneity in PIK3CA, TP53, ESR1 and KRAS genes between individual CTCs. In all 5 patients cfDNA profiles provided an accurate reflection of mutations seen in individual CTCs. ESR1 and KRAS gene mutations were absent from primary tumour tissue and therefore likely reflect either a minor sub-clonal mutation or were acquired with disease progression. Conclusion: Our results demonstrate that cfDNA reflects persisting EpCAM-positive CTCs in patients with high CTC counts and therefore may enable monitoring of the metastatic burden for clinical decision-making. Experimental Design: DNA methylation was investigated in independent tumor cohorts using Illumina HumanMethylation arrays and gene expression by Affymetrix arrays and qRT-PCR. The role of Msh homeobox 1 (MSX1) in drug sensitivity was investigated by gene reintroduction and siRNA knockdown of ovarian cancer cell lines. Results: CpG sites at contiguous genomic locations within the MSX1 gene have significantly lower levels of methylation in independent cohorts of HGSOC patients, which recur by 6 months compared with after 12 months (P < 0.05, q < 0.05, n = 78), have poor RECIST response (P < 0.05, q < 0.05, n = 61), and are associated with PFS in an independent cohort (n = 146). A decrease in methylation at these CpG sites correlates with decreased MSX1 gene expression. MSX1 expression is associated with PFS (HR, 0.92; 95% CI, 0.85–0.99; P = 0.029; n = 309). Cisplatin-resistant ovarian cancer cell lines have reduced MSX1 expression, and MSX1 overexpression leads to cisplatin sensitization, increased apoptosis, and increased cisplatin-induced p21 expression. Conclusions: Hypomethylation of CpG sites within the MSX1 gene is associated with resistant HGSOC disease at presentation and identifies expression of MSX1 as conferring platinum drug sensitivity.
Issue Date: 1-Jan-2017
Date of Acceptance: 12-Jun-2016
URI: http://hdl.handle.net/10044/1/34119
DOI: 10.1158/1078-0432.CCR-16-0825
ISSN: 1557-3265
Publisher: American Association for Cancer Research
Journal / Book Title: Clinical Cancer Research
Volume: 22
Issue: 15
Copyright Statement: © 2016 American Association for Cancer Research
Sponsor/Funder: Cancer Research UK
Action Against Cancer
Action Against Cancer
Action Against Cancer
Action Against Cancer
Action Against Cancer
Medical Research Council (MRC)
Action Against Cancer
Medical Research Council (MRC)
Action Against Cancer
Breast Cancer Care & Breast Cancer Now
Action Against Cancer
Action Against Cancer
Cancer Research UK
Eisai Europe Ltd
Action Against Cancer
Imperial College Trust
Imperial College Healthcare NHS Trust- BRC Funding
Action Against Cancer
Action Against Cancer
Action Against Cancer
Action Against Cancer
Pink Ribbon Foundation
Imperial College Trust
Worldwide Cancer Research
Imperial College Trust
Imperial College Healthcare Charity
Breast Cancer Care & Breast Cancer Now
Imperial College Trust
Breast Cancer Research Trust
Funder's Grant Number: 18078
WSCC_P48438
N/A
042016-04
032015-1
082014-3
MR/M018687/1
072016-02
G1100425
n/a
WSCC_P33478
112016-01
082017-01
RM60G0378
2014-004112-11
0714-02
N/A
RDB04 79560
072015-01
N/A
0614-02
n/a
N/A
WSCC_P38089
10-0510
n/a
R59U
2009NovPhD22
N/A
n/a
Keywords: Science & Technology
Life Sciences & Biomedicine
Oncology
DROPLET DIGITAL PCR
PIK3CA MUTATIONS
ESR1 MUTATIONS
PLASMA
THERAPY
RESISTANCE
EVOLUTION
VARIANTS
SURVIVAL
Science & Technology
Life Sciences & Biomedicine
Oncology
INTEGRATIVE GENOMICS VIEWER
DROPLET DIGITAL PCR
PIK3CA MUTATIONS
ESR1 MUTATIONS
LUNG-CANCER
PLASMA DNA
THERAPY
EVOLUTION
VARIANTS
HETEROGENEITY
Adult
Aged
Aged, 80 and over
Biomarkers, Tumor
Breast Neoplasms
Cell Count
Circulating Tumor DNA
DNA Mutational Analysis
Female
High-Throughput Nucleotide Sequencing
Humans
Middle Aged
Mutation
Neoplasm Metastasis
Neoplastic Cells, Circulating
Workflow
Humans
Breast Neoplasms
Neoplasm Metastasis
Cell Count
DNA Mutational Analysis
Mutation
Adult
Aged
Aged, 80 and over
Middle Aged
Female
Neoplastic Cells, Circulating
Workflow
High-Throughput Nucleotide Sequencing
Biomarkers, Tumor
Circulating Tumor DNA
1112 Oncology and Carcinogenesis
Oncology & Carcinogenesis
Publication Status: Published
Online Publication Date: 2016-06-22
Appears in Collections:Department of Surgery and Cancer
Faculty of Medicine