Scott, Lucy C.
Biomarker development for gastrointestinal and ovarian cancer: a proteomic approach.
PhD thesis, University of Glasgow.
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The development of new biomarkers for cancer patients would be advantageous in population screening for the early detection of cancers, pathological diagnosis, assessment of prognosis, tailoring treatment to individuals, and assessment of treatment response. With this in mind different proteomic approaches were used to identify biomarkers which could potentially aid prognosis and predict response in gastrointestinal and ovarian cancer.
Raf Kinase Inhibitor Protein (RKIP) was originally purified from bovine brain extracts and named phosphatidylethanolamine-binding protein (PEBP). It has subsequently been shown to be a widely expressed and highly conserved protein. Several recent studies have suggested that RKIP may suppress metastasis in melanoma, prostate, and breast cancer, as reduction or loss of RKIP expression was observed in metastatic cell lines and metastatic tissue. In this part of the project RKIP expression was assessed by immunohistochemistry in tissue microarrays (TMA) from patients with colorectal and ovarian cancer. The results confirmed the findings of earlier studies and suggest that the level of RKIP expression is significantly and inversely associated with metastatic disease and can predict the risk of metastatic relapse in patients with no evidence of metastases at presentation. The level of RKIP expression as a prognostic factor was independent of sex, age, tumour site, mitotic index, lymphovascular invasion and tumour stage.
Cytokeratin 18 (CK18) is an epithelial-specific cytokeratin that undergoes cleavage by caspases during apoptosis. Measurement of caspase-cleaved (CK18-NE) or total cytokeratin 18 (CK18) from epithelial-derived tumours could be a simple, non-invasive way to monitor or predict responses to treatment. Soluble plasma CK18-NE and CK18 were measured by ELISA from 73 patients with advanced gastrointestinal adenocarcinomas before treatment and during chemotherapy, as well as 100 healthy volunteers. Both CK18-NE and total CK18 plasma levels were significantly higher in patients compared to the healthy volunteers (p=0.015, p<0.001). The total CK18 baseline plasma levels prior to treatment were significantly higher (p=0.009) in patients who develop progressive disease than those who achieve partial response or stable disease and this correlation was confirmed in an independent validation set. The peak plasma levels of CK18 occurring in any cycle following treatment were also found to be associated with tumour response, but peak levels of CK18-NE did not reach significance (p=0.01, and p=0.07, respectively).
A surface-enhanced laser desorption-ionisation mass spectrometry (SELDI-MS) pilot study on serum from 8 oesophageal cancer patients and 8 healthy volunteers revealed a novel biomarker, ~4kDa, downregulated in patients (p=0.012). An expanded 30 tumour/normal study was performed for validation which confirmed the down-regulation of this potential biomarker (p<0.0001). Attempts to identify tentatively suggested that the peptide may be inter-alpha-trypsin inhibitor heavy chain H4 precursor, which was interesting as a cleavage fragment of inter-alpha -trypsin inhibitor heavy chain H4 had been previously found to be up-regulated in patients with ovarian cancer, and down-regulated in patients with breast cancer. However, it was not possible to confidently confirm this identification. In a further part of this study, haptoglobin was found to be significantly more abundant in the serum from patients with oesophageal cancer compared to healthy volunteers. It was straightforward to isolate and identify and would be amenable to immunoassay as there are good antibodies available for confirmation.
In conclusion, with the current lack of effective markers of metastatic relapse in colorectal cancer, a straightforward test like RKIP expression in the primary tumour may be a very cost-effective way to identify which patients may derive greater benefit from adjuvant treatment and closer post-operative surveillance, and in patients with advanced gastrointestinal malignancy levels of plasma CK18 are a potential marker of tumour response.
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