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Development of a Robust Nanoimmunoassay and Immunohistochemical Assay for ASNS (MD Anderson, AACR 2012)

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The enzyme-drug L-asparaginase (L-ASP) has been used for four decades to treat acute lymphoblastic leukemia. However, its unique mechanism of action is still poorly understood, and its clinical efficacy has proven unpredictable. Those problems have prompted a continuing search for biomarkers that predict L-ASP response. We previously found that the expression of asparagine synθse (ASNS) is strongly negatively correlated with L-ASP anticancer activity in ovarian cancer cell lines, suggesting that L-ASP might be effective against a low-ASNS subset of ovarian cancers if salient characteristics of the cell lines reflect clinical ovarian tumors. However, quantitatively robust, single-antibody assays for ASNS expression have been absent from the literature. We therefore used a capillary-based isoelectric focusing (IEF) platform (the NanoPro 1000) to screen twelve ASNS antibodies for their specificity and sensitivity. Only two antibodies exhibited completely on-target activity (as shown by signal ablation by ASNS siRNA) and sufficient sensitivity. The on-target activity corresponded to a single band on Western blot and a single peak on the NanoPro 1000, suggesting the existence of just one ASNS protein isoform. Optimized, final NanoPro assay conditions yielded less than 8% CV, a 160-fold dynamic range, and Z′-factor of 0.82, indicating a robust assay that is amenable to high-throughput screening. We next used the best ASNS antibody to develop an immunohistochemistry (IHC) assay for ASNS. As with the NanoPro assay, optimized IHC conditions yielded a large dynamic range of staining intensity, and staining was completely ablated by ASNS siRNA. To test the hypothesis that subsets of various cancer types express very low levels of ASNS, we have initiated ASNS IHC of more than 20 tissue arrays representing a wide variety of cancer types. Using a 3-point scoring system (0 = negative, 1 = low, 2 = high), among the tumor samples assayed, 90/136 (66%) of bladder cancer, 63/133 (47%) of bone cancer, 32/149 (22%) of breast cancer, 29/115 (25%) of brain cancer, 51/168 (30%) of colon cancer, 2/85 (2%) of endocrine system cancer, 23/99 (23%) of liver cancer, 7/64 (11%) of head and neck cancer, 7/136 (5%) of lung cancer, 13/53 (25%) of lymphoma, 1/25 (4%) of bone marrow lymphoma, 2/35 (6%) of lymphoma from spleen, 9/109 (8%) of melanoma, 81/396 (21%) of ovarian cancer, 3/29 (10%) of uterine cancer, 27/73 (37%) of pancreatic cancer, 5/119 (4%) of prostate cancer, 10/125 (8%) of renal cancer, 25/138 (18%) of testicular cancer, and 8/39 (21%) of thyroid cancer were ASNS-negative (score = 0), suggesting that a subset of each cancer type may be sensitive to the drug L-asparaginase. Efforts are underway to apply the NanoPro assay to the NCI-60 cell line panel and to continue performing ASNS IHC to survey tissue arrays for ASNS expression.

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