Kurzfassung

Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. Early detection greatly improves prognosis; however, the invasive, unpleasant and inconvenient nature of current diagnostic procedures limits their applicability. No serum-based test is currently of sufficient sensitivity or specificity for widespread use.
CRC follows a gradual progression from benign polyps through early cancers to late and metastatic cancers (1). Screening programmes for early...Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. Early detection greatly improves prognosis; however, the invasive, unpleasant and inconvenient nature of current diagnostic procedures limits their applicability. No serum-based test is currently of sufficient sensitivity or specificity for widespread use.
CRC follows a gradual progression from benign polyps through early cancers to late and metastatic cancers (1). Screening programmes for early diagnoses have resulted in a reduction in mortality because survival decreases with increasing stage (2,3,4). Endoscopic examination of the colon remains the gold standard for diagnosis; however, this is invasive, unpleasant and carries associated risk of morbidity and mortality. Identification of high-risk patients using a less invasive test would decrease the number of such procedures required.
To date, no blood or stool biomarkers with both high sensitivity and specificity for potentially curable early stage disease have been validated for clinical use (5). At present stage, new tests to recognize colorectal cancer at an early stage are needed. The present project aims at the analysis of samples from noncancer, polyp and CRC specimen by mass spectrometry (MS) and western blot to find new unique biomarkers in human blood plasma proteome for the evaluation of new antibody based blood tests.

To discover new biomarkers for colorectal cancer, we decided to use the blood plasma proteome, because it’s ideal source of diagnostic markers and therapeutic targets for many human diseases. Blood plasma is a circulating representation of all body tissues and of both physiological and pathological processes. For this project, we will collect blood from a cohort of patients with colorectal cancer, with polyps and non-cancer control subjects.
The enormous dynamic range of human blood plasma proteome poses a significant challenge for current MS based proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids, which is an essential aspect for successful biomarker discovery efforts. It is planned, that for a first experiment, in order to test the sensitivity of the mass spectrometry technique, to collect 15 probes and later on to increase the number.

Despite significant recent advances, current proteomics technologies still fall short of being able to reliably detect in blood plasma low ng/ml to sub-ng/ml protein concentrations, a level of detection often required for discovering disease-specific biomarkers.

One of the most commonly applied strategies to potentially alleviate the “masking” effect created by the presence of the highly abundant proteins is to remove them. In the human plasma proteome, 22 proteins are responsible for 99% of the bulk mass of the total protein content in human plasma; this leaves perhaps hundreds of thousands of other proteins in only 1% of the protein mass. As a result, effective strategies for removing these proteins should greatly enhance detection of low abundance proteins within this important plasma proteome.
In this project we want to use a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates about 60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction. These antibody based separation systems have been demonstrated to be highly efficient for removing the specifically targeted proteins as well as being both reproducible and selective.
The proteins in the flow-through and eluted fractions will be digested and analyzed by mass spectrometry to identify proteins responsible for peaks which characterise the CRC samples and therefore have the potential to function as biomarkers.
At this stage, we are intended to proof the observed markers with western blot technique, to test the biological relevance of the proteins.
Finally, our aim is to concentrate us to the 2-3 most promising proteins to develop a functional blood test, ideally an ELISA.
Taken together, the proposal will allow us to evaluate new blood tests for early detection of CRC and risk assessment. At least a therapy can be started at early stage and so improvement of life-quality of the patients should be expected.






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