B Ward

Imperial College London, Londinium, England, United Kingdom

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Publications (7)44.15 Total impact

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    ABSTRACT: Biomarkers in breast cancer to monitor minimal residual disease have remained elusive. We hypothesized that genomic analysis of circulating free DNA (cfDNA) isolated from plasma may form the basis for a means of detecting and monitoring breast cancer. We profiled 251 genomes using Affymetrix SNP 6.0 arrays to determine copy number variations (CNVs) and loss of heterozygosity (LOH), comparing 138 cfDNA samples with matched primary tumor and normal leukocyte DNA in 65 breast cancer patients and eight healthy female controls. Concordance of SNP genotype calls in paired cfDNA and leukocyte DNA samples distinguished between breast cancer patients and healthy female controls (P < 0.0001) and between preoperative patients and patients on follow-up who had surgery and treatment (P = 0.0016). Principal component analyses of cfDNA SNP/copy number results also separated presurgical breast cancer patients from the healthy controls, suggesting specific CNVs in cfDNA have clinical significance. We identified focal high-level DNA amplification in paired tumor and cfDNA clustered in a number of chromosome arms, some of which harbor genes with oncogenic potential, including USP17L2 (DUB3), BRF1, MTA1, and JAG2. Remarkably, in 50 patients on follow-up, specific CNVs were detected in cfDNA, mirroring the primary tumor, up to 12 yr after diagnosis despite no other evidence of disease. These data demonstrate the potential of SNP/CNV analysis of cfDNA to distinguish between patients with breast cancer and healthy controls during routine follow-up. The genomic profiles of cfDNA infer dormancy/minimal residual disease in the majority of patients on follow-up.
    Genome Research 02/2012; 22(2):220-31. · 14.40 Impact Factor
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    ABSTRACT: The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors. Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA). The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047). Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer.
    British Journal of Cancer 12/2011; 106(2):375-82. · 5.08 Impact Factor
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    ABSTRACT: Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20-25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer. Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control. We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry. The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer.
    British Journal of Cancer 03/2011; 104(8):1342-8. · 5.08 Impact Factor
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    ABSTRACT: Studies of EGFR expression in breast cancer have shown inconsistent results due in part to a large range of methods used. Anti-EGFR therapy trials have often not used patient selection because of this. We describe the use of the CellSearch system (Veridex LLC, NJ, USA) to enumerate and measure EGFR expression on the surface of circulating tumor cells (CTCs), derived from the peripheral blood of individuals with metastatic breast cancer over time. The CellSearch system was used to quantify CTCs and EGFR measurement was performed on all samples. The specificity of EGFR phenotyping was further examined by spiking with cell lines with increased and low (or absent) levels of EGFR expression using the CellSearch system to enrich and phenotype the CTCs. Serial samples were obtained from 33 individuals with metastatic breast cancer. CTCs derived from these individuals had consistent levels of EGFR expression at different time points, and none of the patients 'switched' from a positive to negative EGFR phenotype or vice versa. The specificity of EGFR phenotyping by the CellSearch system was verified by staining of EGFR only being present in a high EGFR expressing EGFR cell line (MDA-MB-468), as confirmed by Western blotting. Measurement of EGFR on the surface of CTCs, derived from individuals with metastatic breast cancer patients is possible using the CellSearch system and showed consistent positivity over time. The use of this system will now be validated in a prospective study aiming to identify patients for anti-EGFR therapy based on the expression profile of CTCs.
    Pharmacogenomics 01/2009; 10(1):51-7. · 3.86 Impact Factor
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    ABSTRACT: The purpose of this study was to determine whether primary breast cancer patients showed evidence of circulating tumour cells (CTCs) during follow-up as an alternative to monitoring disseminated bone marrow tumour cells (DTCs) by immunocytochemistry and reverse transcriptase (RT)-PCR for the detection of micrometastases. We planned to compare CTC and DTC frequency in low-risk and high-risk patients. We identified two cohorts of primary breast cancer patients who were at low (group II, T(1)N(0), n=18) or high (group III, >3 nodes positive (with one exception, a patient with two positive nodes) n=33) risk of relapse who were being followed up after primary treatment. We tested each cohort for CTCs using the CellSearch system on 1-7 occasions and for DTCs by immunocytochemistry and RT-PCR on 1-2 occasions over a period of 2 years. We also examined patients with confirmed metastatic disease (group IV, n=12) and 21 control healthy volunteers for CTCs (group I). All group I samples were negative for CTCs. In contrast, 7 out of 18 (39%) group II primary patients and 23 out of 33 (70%) group III patients were positive for CTCs (P=0.042). If we count only samples with >1 cell as positive: 2 out of 18 (11%) group II patients were positive compared with 10 out of 33 (30%) in group III (P=0.174). In the case of DTCs, 1 out of 13 (8%) group II patients were positive compared with 19 out of 27 (70%) in group III (P<0.001). Only 10 out of 33 (30%) patients in group III showed no evidence of CTCs in all tests over the period of testing, compared with 11 out of 18 (61%) in group II (P=0.033). A significant proportion of poor prognosis primary breast cancer patients (group III) have evidence of CTCs on follow-up. Many also have evidence of DTCs, which are more often found in patients who were lymph node positive. As repeat sampling of peripheral blood is more acceptable to patients, the measurement of CTCs warrants further investigation because it enables blood samples to be taken more frequently, thus possibly enabling clinicians to have prior warning of impending overt metastatic disease.
    British Journal of Cancer 12/2008; 100(1):160-6. · 5.08 Impact Factor
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    Breast Cancer Research 01/2007; 9:1-1. · 5.33 Impact Factor
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    Breast Cancer Research 01/2005; 7:1-2. · 5.33 Impact Factor

Publication Stats

113 Citations
44.15 Total Impact Points

Institutions

  • 2005–2011
    • Imperial College London
      • Department of Medicine
      Londinium, England, United Kingdom
  • 2008
    • Imperial College Healthcare NHS Trust
      • Division of Medical Oncology
      Londinium, England, United Kingdom