Abstract #1582: Monoclonal antibody based biomarkers for colorectal cancer (CRC) detection in serum

CRC is the third most common cancer in both men and women. There are several recommended CRC screening methods: colonoscopy is the gold standard, yet is invasive and expensive, and FOBT has low sensitivity, yet is cost-effective. Only 44% of US adults over 50 undergo screening. Our aim is to develop easy-to-use and cost-effective serum-based immunoassays for CRC detection. We have developed proprietary approaches to the discovery of biomarkers (Bm) using an in-house generated library of monoclonal antibodies (mAb), a large collection of clinical samples from patients with major cancers, and a proprietary multiplex screening platform, known as Matrix Protein Array Technology (MPAT). The MPAT enables us to screen a large number of clinical samples with a large number of antibodies. Briefly, a protein matrix comprising samples from cancer patients, benign and normal controls, is replica printed on the MPAT solid support. Each sample matrix is simultaneously interrogated with a different mAb. mAb-Bm reaction is detected using fluorescence-based Odyssey imaging system (Li-Cor), followed by spot quantification and statistical analysis to select mAb-based Bm that are significantly and differentially expressed in cases versus controls. We first examined by MPAT mAb in tissue protein extracts derived from patients with CRC (173), benign and inflammatory conditions of the colon (23), and normal controls (240), in addition to breast, lung, and ovary cases and controls, amounting to 1329 samples. Several mAb-based Bm show excellent discriminatory power between cancer and control, with up to 90% sensitivity at 90% specificity, as determined by ROC curves using statistical package GB-STAT (Dynamic Microsystems). Immunohistochemistry on in-house and commercial tissue microarrays (75 cases) confirmed cancer specificity, and mAb specifically stained CRC tissues, with membrane, nuclear and cytoplasmic localization. Some candidate Bm appeared to be secreted and overexpressed in CRC patient serum, when tested by Western blot. Candidate serum Bm were further tested by antibody capture immunoassay in 288 serum samples comprising 32 CRC (12 stage I/II and 20 stage III/IV) and 264 non-CRC (50 normal, 50 cardiovascular diseases, 64 lung cancer and 100 prostate cancer). mAb-Bm reactivity was detected by cheluminescence using a CCD camera, and quantified with Scanalyze software. Based on ROC curve analysis, most mAb sensitivity ranged from 60 to 70% at 80% specificity when comparing CRC to non-CRC group. Also most mAb showed low sensitivity for lung or prostate cancer, when compared to non cancer samples, further confirming their CRC specificity. We are also developing two-antibody ELISA sandwich assays using optimal capture and detection mAb pairs compared to CEA standard. These serum-based immunodiagnostic assays would have tremendous applications in CRC detection, whether alone or in combination with recommended screening methods.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1582.