Doxorubicin is considered to be the most effective agent in the treatment of breast cancer patients. Unfortunately, resistance to this agent is common, representing a major obstacle to successful treatment. The identification of novel biomarkers that are able to predict treatment response may allow therapy to be tailored to individual patients. Antibody microarrays provide a powerful new technique, enabling the global comparative analysis of many proteins simultaneously. This technology may identify a panel of proteins to discriminate between drug-resistant and drug-sensitive samples. The Panorama Cell Signaling Antibody Microarray was exploited to analyze the MDA-MB-231 breast cancer cell line and a novel derivative, which displays significant resistance to doxorubicin at clinically relevant concentrations. The microarray comprised 224 antibodies selected from a variety of pathways, including apoptotic and cell signaling pathways. A standard z2.0-fold cutoff value was used to determine differentially expressed proteins. A decrease in the expression of mitogen-activated protein kinaseactivated monophosphotyrosine (phosphorylated extracellular signal-regulated kinase; 2.8-fold decrease), cyclin D2 (2.5-fold decrease), cytokeratin 18 (2.5-fold decrease), cyclin B1 (2.4-fold decrease), and heterogeneous nuclear ribonucleoprotein m3-m4 (2.0-fold decrease) was associated with doxorubicin resistance. Western blotting was exploited to confirm results from the antibody microarray experiment. These results suggest that antibody microarrays can be used to identify novel biomarkers and further validation may reveal mechanisms of chemotherapy resistance and identify potential therapeutic targets.

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Background-Despite intensive research into the molecular abnormalities associated with colorectal cancer (CRC), no diagnostic tests have emerged which usefully complement standard histopathological assessments. Aims-To assess the feasibility of using immunohistochemistry to detect replication error (RER) positive CRCs and determine the incidence of RER positivity within distinct patient subgroups. Methods-502 CRCs were analysed for RER positivity (at least two markers aVected) and/or expression of hMSH2 and hMLH1. Results-There were 15/30 (50%) patients with metachronous CRCs, 16/51 (31%) with synchronous CRCs, 14/45 (31%) with a proximal colon carcinoma, and 4/23 (17%) who developed a CRC under the age of 50 showed RER positivity. However, 0/54 patients who developed a solitary carcinoma of the rectum/left colon over the age of 50 showed RER positivity. Immunohistochemical analysis revealed that 66/66 (100%) RER positive carcinomas were associated with complete lack of expression of either hMSH2 or hMLH1. This correlation was confirmed using a further 101 proximal colon carcinomas. Patients with a mismatch repair defective carcinoma showed improved survival but a 5.54 times relative risk of developing a metachronous CRC. A prospective immunohistochemical study revealed 13/117 (11%) patients had a mismatch repair defective carcinoma. A fivefold excess of hMLH1 defective cases was noted. Conclusions-All RER positive carcinomas were identified by the immunohistochemical test. This is the first simple laboratory test which can be performed routinely on all CRCs. It will provide a method for selecting patients who should be investigated for HNPCC, oVered long term follow up, and who may not respond to standard chemotherapy regimens. (Gut 1999;45:409-415)

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