EXPRESSION OF PD-L1 IN TRIPLE NEGATIVE BREAST CANCER (SP142) AND IMPLEMENTATION OF VENTANA HER2 Dual ISH DNA ASSAY Alexander Makanga Senior Biomedical Scientist  (Immunohistochemistry) Gwyddonydd Biomeddygol Arbenigol   (Imiwnohistocemeg)

Introduction Breast cancer remains one of the leading causes of death in women with over 400,000 estimated deaths every year (Worldwide Breast Cancer. 2017). Triple-negative breast cancer (TNBC), accounts for about 15% of all breast cancer cases (Ribeiro et al., 2013). Phenotypically TNBC are very aggressive tumours and usually have poor outcomes compared to other subtypes of breast cancers. TNBC are characterised by lack of expression of hormone receptors such as oestrogen (ER), progesterone (PR) and human epidermal growth factor receptor 2 (HER-2).

TNBC patients are unable to benefit from targeted therapy i. e TNBC patients are unable to benefit from targeted therapy i.e. Tamoxifen for ER-positive cancers and Trastuzumab (Herceptin). Surgery, chemotherapy and radiotherapy seem to be the only options for these patients. Chemotherapy and radiotherapy resistance is common in this group of patients, equating to relapse and poor survival rates.

Therefore there is urgent clinical need to identify and develop new therapeutic targets to improve prognosis and disease outcome in these patients. Immunotherapy i.e. Tecentriq (atezolizumab), Pembruzamab (Keytruda), Nivolumab (Opdivo) are an emerging class of drugs which might be beneficial to TNBC patients if the tumour cells express PDL-1.

DIAGNOSIS FOR BREAST CANCER Haematoxylin and eosin Immunohistochemistry (ER,PR and HER-2) In-situ hybridization (For equivocal cases)

INTRODUCTION OF HER2 Dual ISH DNA HER2 Dual ISH DNA  is equivalent to Fluorescence in-situ hybridisation HER2 Dual ISH DNA  probe Cocktail assay is fully automated on the BenchMark IHC/ISH instruments. New improved HER2 Dual ISH DNA  probe Cocktail assay provide quick result

WHAT IS PD-L1? PD-L1 is a death ligand receptor which is involved in the cross- talk between normal cells and T lymphocyte to prevent autoimmunity. However, tumour cells use the same mechanisms to evade immune surveillance. In this project, we wanted to investigate if lymphocyte infiltration has a role in the expression of PD-L1 in TNBC.

Importance of PDL-1 IN CANCER PDL-1 and PD1 in normal cells inactivates T cells thereby preventing autoimmunity Tumour cell using PDL-1 and PD1 in the same way as normal cells inactivates T cells thereby preventing evading Immune surveillance Anti -PD-1 preventing cross talk between tumour cell and T Cell. The helps the T cell to remain active and destroy tumour cell. https://www.agilent.com/cs/library/usermanuals/public/29171_22C3-ihc-pharmdx-interpretation-manual-eu.pdf [Accessed 6 Oct. 2018].

RESEARCH QUESTIONS, AIMS AND OBJECTIVES Can TNBC patients respond to Immune modulating drugs This study aim to assess expression of PDL-1 in TNBC, which might provide other options for these patients. Immunotherapy i.e. Keytruda and Opdivo are an emerging class of drugs which might benefit TNBC patients if the tumour cells express PDL-1.

Hypothesis Expression of PDL-1 is closely related to lymphocyte infiltration. Therefore TN tumour cells infiltrated by lymphocytes are likely to express PD-L1

Materials & Methods Approximately 50 TNBC tissue blocks will be included in this study. Ethical approval was sought and approved. Patients are being recruited across BCUHB Tissue blocks of all cases have been retrieved from the various storage sites within the health board.

PRELIMINARY RESULTS Immunohistochemical stain with confirmed Ventana SP142 clone of PD-L1 showed positive staining in 11 cases (31%) and these were all confirmed severe lymphocyte infiltrated cases. These results suggest that TNBC patients with positive PD-L1 result might benefit from anti-PD-L1 blockade immune therapy drugs such as Tecentriq.

PRELIMINARY RESULTS Our results have coincided with FDA approval of Ventana PD-L1 (SP142) assay as a companion diagnostic for Roche cancer immunotherapy drug Tecentriq. This assay is aimed at identifying TNBC patients who might benefit from Tecentriq. This is exciting news for TNBC patients as this provides another option in the management of this subtype of breast cancer.

  Conclusion This research seeks to investigate a novel approach to the management of TNBC and will provide an insight into alternative treatment regimes. Significant findings from this study will provide an opportunity for larger multi-centre collaborations employing larger sample cohorts.

Reference Alberts, B., Hunt, T., Johnson, A., Lewis, J., Morgan, D., Raff, M., Roberts, K., Walter, P. and Wilson, J. (2015). Molecular biology of the cell. 6th ed. New York: Garland Science, pp.353-379. Hartl, D. (2014). Essential genetics. Burlington, MA: Jones & Bartlett Learning. https://www.agilent.com/cs/library/usermanuals/public/29171_22C3-ihc- pharmdx-interpretation-manual-eu.pdf [Accessed 6 Oct. 2018]. Ribeiro, E., Ganzinelli, M., Andreis, D., Bertoni, R., Giardini, R., Fox, S., Broggini, M., Bottini, A., Zanoni, V., Bazzola, L., Foroni, C., Generali, D. and Damia, G. (2013). Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes. PLoS ONE, 8(6), p.e66243. Worldwide Breast Cancer. (2017). Breast Cancer Statistics Worldwide. [online] Available at: http://www.worldwidebreastcancer.com/breast-cancer- statistics-worldwide/ [Accessed 25 Nov. 2017].

QUESTIONS