Nuclear characteristics of the tumor cells such as nuclear pleomorphism, nuclearmegaly and nuclear hyperchromatism have long been used in the determination of the benign or malignant potential of the tumor and its biological behavior. These nuclear characteristics are still included in the grading systems for most solid tumors and are crucial for clinical and therapeutic management of patients. However, the molecular events underlying these nuclear changes, now recognized as a morphological feature of aneuploidy, are still unknown. Recent advances in our understanding of mitosis are linking dysregulation of mitotic kinases with aneuploidy and transformation in cultured mammalian cells.

In this Work Package, we are analyzing the expression dynamics of a selected set of key mitotic kinases in human tumors and are linking the pathological data to clinical end points. Such systematic studies will provide crucial insight on the clinical utility of mitotic kinases in cancer prevention, diagnosis and treatment.

- Characterization of existing antibodies against mitotic kinases

- Generation and characterization of antibodies against newly identified mitotic regulators

- DNA methylation analysis and expression profiling of mitotic regulators in human tumor samples

Kai Stoeber and Gareth Williams (University College London, UK)

1) Examining existing antibodies against mitotic kinases

We have determined the specificities of antibodies against Aurora A, Aurora B, Plk1 and Cdk1 by immunoblotting and immunofluorescence on cultured human cells. We have also optimized the procedures for using selected antibodies in enzyme-linked immunohistochemistry on standardized paraffin-waxed embedded tissue sections.

2) Generating antibodies against mitotic proteins

We are shortlisting mitotic regulators identified in the genome-wide RNAi screen for antibody production.

3) Expression profiling and analysis of mitotic regulators at protein level in various tumor samples

In the past two years, we have prioritized ovarian cancer in our study. 90% of ovarian tumors arise from the epithelium (Epithelial Ovarian Carcinoma). EOC is one of the most common cancers in woman and has the worst prognosis of all gynaecological malignancies.

143 ovarian cancers have been identified for which clinico-pathological parameters are available such as tumor cell types, invasiveness, tumor grade, chemotherapy response and survival. Expression profiling of Aurora A, Aurora B and Plk1 in combination with the standard makers has been performed on these samples by immunohistochemistry. Preliminary results are encouraging and raise the prospect of clinical usage of mitotic kinases as markers for cancer staging and personalization of therapy at the time of diagnosis.

In the future, we plan to extend our studies to breast cancer and male genito-urinary tract cancer.

- DNA replication licensing factors and Aurora kinases are linked to aneuploidy and clinical outcome in epithelial ovarian carcinoma.
Kulkarni AA, Loddo M, Leo E, Rashid M, Eward KL, Fanshawe TR, Butcher J, Frost A, Ledermann JA, Williams GH, Stoeber K. Clinical Cancer Research, in press. (PMID: )

- Mitogenic growth signalling, DNA replication licensing, and survival are linked in prostate cancer.
Dudderidge TJ, McCracken SR, Loddo M, Fanshawe TR, Kelly JD, Neal DE, Leung HY, Williams GH, Stoeber K. British Journal of Cancer. 2007;96(9):1384-93. (PMID: 17406359)

- DNA replication licensing and cell cycle kinetics of normal and neoplastic breast.
Shetty A, Loddo M, Fanshawe T, Prevost AT, Sainsbury R, Williams GH, Stoeber K. British Journal of Cancer. 2005;93(11):1295-300. (PMID: 16278669)

- Mcm2, geminin, and KI67 define proliferative state and are prognostic markers in renal cell carcinoma.
Dudderidge TJ, Stoeber K, Loddo M, Atkinson G, Fanshawe T, Griffiths DF, Williams GH. Clinical Cancer Research. 2005;11(7):2510-7. (PMID: 15814627)

- DNA replication licensing in peripheral B-cell lymphoma..
Obermann EC, Eward KL, Dogan A, Paul EA, Loddo M, Munson P, Williams GH, Stoeber K. Journal of Pathology. 2005;205(3):318-28. (PMID: 15682442)