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    Research Grants Awarded

    Use of a Panel of Mitotic Kinase Antibodies to Predict Pre-malignancy Outcomes in Human Breast Cancer

    Study Section:
    Detection, Diagnosis and Prognosis

    Scientific Abstract:
    Background. Aneuploidy is the most defining characteristic of human ductal carcinoma in-situ (DCIS) and primary invasive ductal breast cancer (IDBC). There is growing evidence that mitotic kinases (MK) over-expression precipitates the cascade of centrosome amplification, chromosomal instability, and aneuploidy, thus driving the malignant phenotype of BC. Objectives/Hypothesis . We propose that unique signatures of MK over-expression in pre-malignant lesions [dysplasia, atypical ductal hyperplasia (ADH) and DCIS] will have predictive value in determining their malignancy outcomes. On this basis, we intend to develop a panel of potent and highly specific monoclonal antibodies (MCA) for MKs [Aurora, Polo-like, and Nek (NIMA), including their phosphorylated-(active) forms]. Specific Aims . 1. To develop a panel of specific and sensitive MCA for the MKs Aurora, B, C, Polo-like 1-3, Nek (NIMA) 2 and 8, and to each of their phosphorylated -(active) forms. 2 . To systematic evaluate these MCAs in paraffin-embedded sections of human breast tissue specimens obtained from normal tissue derived from breast reductions and from breast tissue containing focal dyplasias, DCISs, and IDBCs. Study Design . Two 20 amino acid sequences will be selected representing the NH 4 - and COOH-terminus of each MK selected for MCA production from the NCBI protein sequence database ( http://www.ncbi.nlm.nih.gov/ ), and assessed for suitable peptide sequences for antigen production. The peptides will be synthesized and screened by ELISA. Female Balb/c mice will be immunized with each peptide. Spleens will be removed after immunization for hybridoma production. The presence of the desired MCA will be done by ELISA, for subsequent cloning, and characterization. Paraffin serial sections from patients diagnosed with DCIS and IDBC will be stained for H&E to verify morphology, and by immunohistochemistry to assess their MK expression, both intensity and distribution. Relevance . MKs play a crucial role in the oncogenesis of BC. Of the few MK antibodies that are commercially available, some are not functional in paraffin sections of human breast tissues. Therefore, our goal is to develop a uniformly reliable panel of specific and sensitive MK MCAs for evaluating early pre-malignant stages of human IDBC. It is anticipated that the MK expression pattern in pre-malignant stages will discriminate which lesions would progress to invasive disease.

    Lay Abstract:
    Background . A conspicuous failure in breast cancer (BC) detection is the inability to predict which atypical hyperplasias (ADH) and carcinoma in-situ (DCISs) will progress to invasive disease. Of the ~50,000 new DCISs detected yearly in American women, only 35-40% evolve into invasive BCs. Also, there remains an inability to discriminate which DCIS patients would best benefit from certain adjuvant therapies. Objectives/Hypothesis . While current human BC biomarkers are useful, their expression has been largely unreliable for assessing the ultimate fate of DCIS and the progression of even earlier pre-malignant lesions. Therefore, new molecular markers with greater predictive value are needed to improve the prognosis, and therefore, the prevention, and treatment of BC pre-malignancy. Aneuploidy is the most distinctive feature in human DCISs and invasive ductal BCs. Our strategy is to develop a panel of mitotic kinase (MK) antibodies to determine which pre-malignant lesions will evolve into invasive BC by distinguishing unique patterns and intensities of expression in breast tissue biopsy sections. MKs play a major role in cell division and, when over-expressed, in the generation of aneuploidy in BC pre-malignancy. Specific Aims . We propose: 1. To develop a panel of specific and sensitive MK [Aurora, Polo-like, and Nek (NIMA)] antibodies, both native and phosphorylated (active) forms. 2 . To systematically evaluate the MK expression in paraffin-embedded tissue sections of normal human breast reductions, and breast containing focal dyplasias including ADH, and different histopathologic classified DCISs, that will be compared to invasive ductal BCs. Study design . Highly specific and reactive antibodies will be prepared against all of the MKs [Aurora, Polo-like, and Nek (NIMA)]. Initially, a total of 16 MK antibodies will be prepared. A further goal is to use these antibodies for Western blot analyses to quantitate MK expression in micro-dissected pre-malignancies. Potential Outcomes and Benefits . This novel approach will establish a relationship between MK over-expression and early human BC pre-malignant stages. We anticipate that the combined emergent MK expression patterns will have predictive power for BC pre-malignancy outcomes. An obvious benefit of these studies is the reduction of unnecessary surgical interventions in women when DCISs are initially diagnosed.