Novel blood and tissue Biomarkers for Breast and Prostate Cancers Elena Klenova (email: klenovae@essex.ac.uk) NHS Consultants Chandra Sekharan John Corr Phil Murray Ian Seddon University of Essex team Metodi Metodiev Yukti Gupta Viv D’Arcy Dawn Farrar Jay Mani Naresh Pore Anu Apte Adhip Mandal Zubair Cheema Research Nurses Kathy Rooke Andrea Appleby Louise Robb Devi Basu Essex Biomedical Sciences Institute (EBSI)

PLAN OF THE TALK Protein BORIS -a potential breast and prostate blood and tissue cancer biomarker. BORIS belongs to the Cancer-Testis antigen family which is normally present only in the testis but aberrantly activated in various malignancies. (Loukinov et al 2002, PNAS; Klenova et al 2002, Sem Cancer Biol) 2. Identification and characterization of new blood biomarkers for early diagnosis, prognosis and treatment of breast and prostate cancer

BORIS – a novel breast tumour tissue biomarker Immunohistochemistry analysis IRS = Immuno Reactivity Score (calculated to assess the levels of a protein) Western blot analysis

BORIS levels are higher in breast tumour tissues with different phenotypes than in normal breast tissues BR – breast reduction PP – paired peripheral All – all tumours High levels of BORIS correlate with high levels of the Oestrogen and Progesterone Receptors D’Arcy et al, British Journal of Cancer (2008).

BORIS – a novel prostate tumour tissue biomarker BORIS staining on Prostate Tumour Tissue Sections IRS= 9 IRS= 8 IRS= 3 Non Tumour BPH Prostate Tumour IRS=0 IRS=0 Secondary only Cheema, Hari et al in preparation (2013).

BORIS in Tissues: Clinical Correlations Gleason Score Tumour stage

Clinical Correlation: BORIS and Androgen Receptor in prostate tumors Mean BORIS IRS

Blood Leukocyte Biomarker BORIS – a novel blood biomarker detected in leukocytes of breast cancer patients Cancer Patient T Interaction between tumour and immune system Change in protein profiles Blood Leukocyte Biomarker

Immunohistochemistry analysis Western blot analysis IRS = Immuno Reactivity Score (calculated to assess the levels of a protein) D'Arcy V et al. Clin Cancer Res 2006;12:5978-5986

BORIS is detected in the leukocytes of breast cancer patients (a) phenotypes of matching tumours (b) BORIS levels increase with the tumour size D'Arcy V et al. Clin Cancer Res 2006;12:5978-5986

BORIS is detected in leukocytes of prostate cancer patients Healthy Donor IRS= 8 IRS= 6 IRS= 4 IRS= 0 Secondary Only

BORIS in Leukocytes: Clinical Correlations

Assays for BORIS detection from whole blood (“point of care device”)   ELISA (Enzyme linked immunosorbent assay) Immunohistological methods. Real-time RT-PCR Immunofluorometric methods (flow cytometry)

Identification of novel blood leukocyte-based biomarkers Cancer Patient T Interaction between tumour and immune system Change in protein profiles Blood Leukocyte Biomarker

Identification and characterization of new blood biomarkers for (i) early diagnosis and prognosis or breast and prostate cancers, and (ii) treatment monitoring of breast cancer Patient cohorts Breast and prostate cancer patients and healthy donors. Metastatic breast cancer patients undergoing chemo - and endocrine therapies.

The Search for Novel Blood Biomarkers for Breast and Prostate Cancers Blood collection Plasma White Blood Cells Cell pellets X RBC 2 D gel electrophoresis Cell pellets Protein identification by Mass Spectrometry Protein extraction Candidate Validation Massive parallel protein profiling using the Orbitrap Velos instrument

Protein Identification/ Candidate Blood Biomarkers for metastatic cancer treatment 78 Candidates 11 No Ids ( unknown? Novel biomarkers?) 7 Bacteria proteins Several biomarkers are currently being validated.

Conclusions Protein BORIS has been identified and evaluated as a novel potential breast and prostate blood and tissue cancer biomarker. Work in progress to further evaluate BORIS for the use in the clinic. More new blood biomarkers for early diagnosis, prognosis and treatment of breast and prostate cancer are being identified using high throughput genomics and proteomics techniques. Work in progress to validate these biomarkers in relation to clinical outcomes. 3. In some cases biomarkers = DRUG TARGETS ( new dimensions)