Association between the ras p21 oncoprotein in blood samples and breast cancer
Introduction
Breast tumors are known to express mutated tumor suppressor proteins (e.g. p53) and/or to overexpress oncoproteins (e.g. erbB-2, ras p21, cyclin D1). It has been shown for a number of tumor types that overexpressed proteins are often shed by the malignant cells and can be detected in blood plasma samples [1], [2], [3]. This suggests that assays to detect these proteins in blood samples may be useful in the early detection of tumors and in the clinical management and follow-up of cancer patients.
Ras p21 is a membrane-associated, guanine nucleotide-binding protein that is involved in the signal transduction pathways that control cell proliferation [4], [5], [6]. When mutated or overexpressed, ras p21 is oncogenic [4], [7], [8], [9], [10]. The ras gene is rarely mutated in breast tumors, but is overexpressed in 50–60% of breast tumors [5], [10]. Tumors can release ras p21 and elevated ras p21 levels are seen in blood samples from patients with tumors that overexpress ras p21 [11], [12], [13]. In pilot studies of lung, liver and colon cancer, elevated plasma levels of ras p21 in plasma have been seen in cases compared to controls [11], [12], [13].
Although there are no prior studies focusing on breast cancer alone, two larger studies of multiple tumor types have compared blood ras p21 levels in patients with those in healthy controls [14], [15]. In a study of 80 mixed cancer patients and 188 community controls, 67% of the cases and 15.7% of the controls were positive for elevated levels of ras p21 in blood as measured by immunoblot (odds ratio (OR)=11, 95% CI=6–20.6) [14]. The case group included a variety of tumor types, including 21 breast cancer patients, but the results were not listed by tumor type [14]. The investigators also evaluated the reliability of scoring techniques and found that computerized image analysis was much more reliable than scoring the blots with the unaided eye [14]. The second study used an enzyme linked immunosorbent assay (ELISA) and found elevated serum p21 ras in 31 of 93 (33%) patients with various types of cancer compared to one of 58 (2%) controls (OR=28.5, 95% CI=3.8–215.6). The highest serum levels of ras p21 were found in cases of breast and urogenital cancers and lymphoma, but again results were not differentiated by tumor type [15]. Since the prevalence of ras p21 overexpression differs by tumor type, the OR estimates from these studies depend in part on the mix of tumor types in the case group and do not provide information on how useful this marker might be with any single tumor type.
These findings, along with the high prevalence (50–60%) of ras p21 overexpression in breast tumors, indicate that ras p21 levels in blood samples might be able to predict the presence of breast cancer. To explore this hypothesis, a pilot breast cancer case-control study was initiated within a larger epidemiologic study to compare the levels of ras p21 present in blood samples collected from untreated breast cancer cases and controls. A Western blot approach was used instead of the ELISA method to allow for the identification and separate analysis of both normal and mutant ras p21 protein in a single assay. An image analysis protocol was developed to semi-quantitatively score the blots, addressing the measurement issues noted by Weissfeld et al. [14].
Section snippets
Patients
The present study analyzed blood samples from the first 94 subjects enrolled in a hospital-based molecular epidemiologic case-control study begun in 1994 at the Columbia Presbyterian Medical Center (CPMC). In the parent case-control study, none of the women had received treatment for either breast cancer or benign breast disease (BBD) at the time the blood sample was drawn. All surgical patients (cases and BBD controls) were enrolled prior to surgery through the breast clinic and the private
Results
The performance characteristics of the assay were assessed in a number of ways. The scoring procedures were found to be highly reliable, with a Kappa score of 0.60 for the dichotomous scoring and a intra-class correlation coefficient of 0.98 for IPU scoring (see Fig. 2). To assess variability across gels, staining intensity data from the 6 and 24 ng control lanes were analyzed. The 6 and 24 ng control lanes from 11 gels had bands that could be evaluated by image analysis. The staining intensity
Discussion
To our knowledge, this is the first study to compare blood plasma levels of ras p21 in breast cancer cases and controls, adjusting for potential confounding variables. Detectable levels of ras p21 protein in blood samples were found to be associated with breast cancer status after adjustment for potential confounding factors. Our results suggest that the presence of ras p21 in blood is associated with a five fold increased risk of breast cancer. Similar findings have been reported with other
Acknowledgements
We gratefully thank Drs Laverne Mooney and Robin Whyatt for their invaluable advice and assistance in designing and conducting this study. We would also like to thank Claribel Blake, Amy Della Rocca and Surah Grumet for their assistance in patient recruitment, and Sonia Lyn, Stan Cho and Jen Russ for their help in manuscript preparation. We thank Dr Alex Halim for his thoughtful comments. This manuscript was supported by grants from the Department of the Army, US Army Medical Research and
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