Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre-clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI-TOF-MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre-albumin) and C3a-desArg by MS/MS and were further validated by antibody-based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut-off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a-desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.
Colorectal (CRC) malignancies rank world wide at the third place for tumor diseases and account for an annual mortality rate of 492 000 cases. Although several molecular events are known to be involved in the transition from normal tissue to adenoma and finally to undifferentiated carcinoma it remains a challenge to discover new and more reliable biomarkers for diagnosis, prognosis and prediction of outcome. Towards this end a study was designed to identify potential biomarkers which are associated with the molecular events leading from epithelial adenoma to the early stages of carcinoma. A new biomarker discovery strategy was developed to combine the cell specificity and selectivity of laser capture microdissection (LCM) with the resolution power and sensitivity of liquid-chromatography (LC)-matrix-assisted-laser-desorption/ionization mass spectrometry (LC-MALDI-MS). We carefully selected a group of closely matched patients (n = 10 for each group) afflicted with epithelial adenoma (high dysplasia) or early stages of carcinoma (stage I) and used the derived normal as well as the matched tumor tissue samples to reveal protein expression differences. According to this LC-MALDI-MS strategy microdissected cells (around 10 000 cells) were lysed and extracted proteins were digested with Trypsin. Obtained peptides were separated by capillary reversed phase HPLC (Agilent). The resulting LC-fractions (300) were spotted on prespotted AnchorChip targets (PAC, Bruker) and tryptic fragments subsequently detected by reflector MALDI-MS (ultraflex III, Bruker) measurements. This generated between 5000-7000 ion signals ranging from m/z 800 to 4000. Differential peptide analysis was then performed with the goal to discover robust and significant expression differences between patient groups. Therefore, only m/z ions displaying a minimum twofold difference and a p-value of 0.01 between the tissue samples were considered for further analysis. The selected peptides were subsequently fragmented by MS/MS experiments to reveal their primary sequence and protein identity. Our targeted biomarker discovery approach resulted in the identification of more than 30 biomarker candidates which act in diverse cellular functions and can now be linked to early events (e.g. adenoma vs. normal tissue) or later events (e.g. carcinoma vs. normal) of tumor progression. Currently these biomarkers are validated using antibody based assays to further analyze their potential as markers in a clinical setting. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4577.
As there is a great interpatient variability in drug response caused by the individual geno- and phenotype the choice of optimal chemotherapy should be tailored for each patient. Intrinsic drug resistance is one of the main reasons for therapeutic ineffectiveness of first line therapy and recurrence in colorectal cancer. To advance individualized chemotherapy and understanding of drug resistance mechanisms, predictive biomarkers which indicate individual drug response of patients are urgently needed. The prediction of response to first line therapy will identify subgroups of patients who benefit from treatment and thus avoid ineffective treatment and associated side effects. In this study, several commercially available cell lines as well as primary epithelial mixed cultures and clonal cell lines established from colorectal cancer patients were treated with different drugs, such as 5-fluorouracil, oxaliplatin, and drug combinations, e.g., FOLFOX. The determination of chemosensitivity of these cell lines to drug treatment was performed using two different assays, the ATPlite™-(Perkin Elmer) and FMC-assay (according to Larsson et al.). Both assays showed time- and dose-dependent drug responses which differed significantly among the 18 colorectal cell cultures. The analysis of obtained in vitro chemosensitivity data allowed us to cluster sensitive versus resistant cell lines by determining IC50-values. For the discovery of differentially expressed proteins which might be linked to a specific phenotypes of intrinsic chemoresistance we generated protein expression patterns from all 18 cell lines using a combination of reversed phase highperformance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (RP-HPLC MALDI MS). Up to 3500 m/z ion signals in the molecular mass range between 2500-35000 m/z were detected reflecting a complex pattern of small proteins and peptides. For protein identification, RP-HPLC fractionated samples were purified and digested by trypsin. Fragments of interest were subjected to MS/MS analysis in the TOF/TOF mode. The corresponding MS/MS spectra were used to search the NCBInr database using MASCOT software. Based on these data, we hope to gain more understanding of the biological processes of chemoresistance concerning important cellular drug responses (e.g., signal transduction pathways) and thereby obtain insight into new drug targets for future therapeutic intervention. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B43.
Colorectal cancer (CRC) ranks worldwide at third place for tumor diseases and account for an annual mortality rate of 492.000 cases. Despite the advances in treatment of CRC, patients mostly benefit from an early detection of malignancy. Although several molecular events are known to be associated with tumor development and progression the accuracy of currently practiced early detection of CRC has to be optimized. Therefore, the discovery of new and more reliable biomarker for diagnosis of early stages of cancer is still a challenge. Towards this end, our study was designed to identify protein biomarkers which are closely associated with the molecular events leading from epithelial adenoma to the early stages of carcinoma. Tissues from patients with epithelial adenoma (high dysplasia), carcinoma (stage I) and matched normal tissue (n = 10 for each group) were selected and analysed using our novel biomarker discovery platform. The workflow combines the cell specificity and the selectivity of laser capture microdissection (LCM) with the resolution power and sensitivity of liquid-chromatography (LC)-matrix-assisted-laser-desorption/ionization mass spectrometry (LC-MALDI-MS). According to this LCM-LC-MALDI-MS strategy microdissected cells were lysed and extracted proteins were digested with trypsin. Obtained peptides were separated by capillary reversed phase HPLC. Tryptic fragments were subsequently detected by reflector MALDI-MS measurements. Differential peptide analysis was performed to discover robust and significant expression differences between patient groups. Selected peptides were subsequently fragmented by MS/MS experiments to reveal protein identity. Up to 7000 ion signals ranging from m/z 800 to 4000 per sample were generated and used for statistical analysis. Our targeted biomarker discovery approach resulted in the identification of more than 30 biomarker candidates. The identified early detection biomarker candidates are involved in diverse cellular functions and can be linked to early or late events of tumor progression. Among these hNRPU, HSP90 and SFPQ were so far further analysed. All three biomarker candidates were significantly regulated between groups, showing increasing expression from normal across adenoma to carcinoma tissue. Currently these biomarkers are being validated using antibody based assays. In this study we were able to show that the newly established high resolution proteomic workflow is applicable for the detection and identification of regulated peptides in microdissected tissue compartments. In summary the established comparative proteomic workflow is suitable for the discovery of biomarker for miscellaneous applications (e.g. early detection, prognosis and prediction of response to therapy) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3177. doi:10.1158/1538-7445.AM2011-3177
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