Study on the use of potential prognostic parameters in breast cancer patients

by Hu, Xichun

Abstract (Summary)
(Uncorrected OCR) ABSTRACT OF THESIS ENTITLED Study on the use of potential prognostic parameters in breast cancer patients Submitted by Xichun Hu For the degree of Doctor of Philosophy At the University of Hong Kong In March 2001 Breast cancer remains one of the commonest malignancies worldwide. Metastasis is the leading cause of treatment failure and death for breast cancer patients. The commonly used tumor markers in peripheral blood cannot meet the requirements in the treatment of breast cancer patients. The aims of my Ph.D. project were to assess candidate tumor markers in peripheral blood. CEA, CA15.3 and TPS serum levels were evaluated in 128 breast cancer patients. Correlation with tumor characteristics and disease free survival and overall survival was analyzed. The patients with advanced stages and extensive nodal metastasis had significantly higher CEA and CA15.3 levels than the other patients (P < 0.001 and P < 0.05 respectively). For disease free survival, univariate analyses showed its correlation with nodal metastasis and c-erbB-2 and multivariate analyses i confirmed their value and found age and CEA level to be two other independent prognostic factors. For overall survival, univariate analyses demonstrated its correlation with nodal metastasis, c-erbB-2 and estrogen receptor status. Multivariate analyses revealed nodal metastasis status to be the only important variable. The role of promoter methylation of p16 and E-cadherin genes was also investigated in 36 patients. Methylation-specific PCR (MSP) assay was done in the free plasma DNA samples and matched primary tumors. The percentage of hypermethylated p16 gene was 11.1% (4/36) in primary tumor DNA and 8.3% (3/36) in plasma DNA, while the percentage of hypermethylated E-cadherin gene was 25% (9/36) and 19.4% (7/36) respectively. None of the 25 cases without molecular events in primary tumor DNA was found to be positive in their plasma samples. The presence of hypermethylated p16 gene in plasma DNA demonstrated significant correlation with status of nodal metastasis (P = 0.012). E-cadherin promoter methylation status was associated with its expression in primary tumors as detected with western blotting assay. The presence of circulating breast cancer cells was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. This assay was evaluated in in vitro experiments and in patients with breast cancer and benign breast disease. The low detection limit for cytokeratin (CK) 19 and CK 20 of this assay with ethidium bromide gel analysis was one MCF-7 cell among 105 HL-60 leukemic cells, whereas the low detection limit for ?human chorionic gonadotropin (?hCG) was one among 106 cells. For the benign cases, 10% (3/30) were positive for CK19 and all were negative for CK20 and ?hCG, whereas 9.72% (7/72), 2.78% (2/72) and 12.5% (9/72) of the malignant cases were positive for CK19, CK20 and ?hCG respectively. The detection rate of circulating breast cancer cells was unchanged when CK19 was combined with CK20, but it increased to 18.1% (13/72) when the marker was combined with ?hCG. Significant difference was observed for ?hCG between benign cases and affected patients with stage II, III and IV disease (P = 0.026). With this assay, fine needle aspiration and operation were shown to shed tumor cells into ii the circulation. The quantification of circulating breast cancer cells was done on the enriched cells by magnetic-activated cell separation (MACS) enrichment system. The combination of MACS with immunocytochemistry (ICC) or flow cytometry (FCM) was evaluated step by step in the detection of tumor cells in peripheral blood of breast cancer patients or the tumor cells spiked in the normal blood. Enrichment rate of spiked tumor cells was 37 ~ 2300 folds as determined by FCM and was negatively correlated with the ratio of tumor cells to normal nucleated cells (P < 0.05). The positive rate was only 5.6% (2/36) with only ICC and was as high as 38.9% (14/36) with MACS/ICC (P < 0.001). The positive rates in the enriched fraction were 0% (0/4), 33.3% (8/24), 60% (3/5) and 100% (3/3) for the patients with stages I, II, III and IV respectively (P < 0.05). In conclusion, circulating breast cancer cells are present in the blood of breast cancer patients. The presence of these cells is associated with clinical staging. p16 promoter methylation in free plasma DNA is a promising tumor marker. CEA and CA15.3 levels are associated with the prognostic factors related to poor prognosis. Pre-operation CEA level correlates with disease free survival. iii
Bibliographical Information:


School:The University of Hong Kong

School Location:China - Hong Kong SAR

Source Type:Master's Thesis

Keywords:genetic transcription polymerase chain reaction tumor markers diagnostic use breast cancer aspects metastasis


Date of Publication:01/01/2001

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