Role of Serine Protease Inhibitor (SERPINB3) in radiation resistant invasive Squamous Cell Carcinoma of the cervix Heena Singh, BHSc, Health Sciences TBCC Translational Lab, University of Calgary, 2500 University Drive NW, Calgary, AB Introduction What is SERPINB3? (3) Results Results Cervical cancer is the second most common cancer in women that results in approximately 274 000 deaths worldwide(1). An estimated 75% of sexually active individuals will receive an HPV infection and women in their early twenties generally demonstrate the highest rate of HPV infection (2). Furthermore, 93% of invasive cervical cancer causes contain high risk types of HPV(2). Aside from the pronounced correlation with HPV, early stages of cervical cancer are predominantly asymptomatic. Advanced cases of cervical cancer may present symptoms such as leg or back pain, fatigue, heavy vaginal bleeding, and loss of appetite, however, such symptoms can easily be mistaken or disregarded as non life threatening by most individuals (2) .Therefore, there is a significant need to identify reliable prognostic and predictive factors for better selection of patients for treatment with chemoradiation. Initially found as an independent prognostic marker in cervical cancer serum; prognostic marker in lung, esophageal, head & neck & cancers. SERPINB3 has been found to prevent TNF-α induced cell death by inhibiting Cytochrome C release from the mitochondria. Molecular target location of SERPINB3 is suggested to be upstream of caspase 3 thus suggesting that they are protected from apoptotic death by immune killer cells In 293T cells, pro-apoptotic effect of p38 MAPK was reduced by SERPINB3 induced cells at the step of phosphorylation both BEFORE and AFTER radiation. Figure 3: SERPINB3 probe set 3 is strongly correlated with the response group in this study (p=0.024, N=23) Modified from Vidalino et. al (2009) Conclusion Higher levels of SERPINB3 is correlated significantly with OS and PFS, however SERPINB3 should protect against radiation. Further analysis is needed to determine possibilities of mutations and further assessment of localization. Why Study Cervical Cancer? Early stages of cervical cancer are mainly asymptomatic Advantage of cervical cancer as model system- Its ease in observable direct biopsies during the course of therapy. Majority of cancer patients receive radiation therapy (RT)- Yet only a subset are sensitive to radiation treatment. 40% of cervical cancer patients fail to respond to radiation / chemotherapy in advanced cervical cancer cases. In such instances, tumors are resistant to RT Significant overuse of RT- Resulting in health system usage and risk of toxicity in the subset of patients with radio resistant tumors. Problem- There are no reliable markers of radiation responsiveness that can be used in the routine clinical setting. Future Directions Methods Run real time PCR on remaining samples Run IHC analysis of all samples (using AQUA score analysis) Interpret both qPCR and IHC results to recent research and published data Increase patient dataset Look at possible mutations and localization of SERPINB3 near the mitochondria Microarray Data (11NR, 12R) Data analysis Genes of Interest Statistical Analysis (SPSS & X-tile software) Figure 1: Average SERPINB3 scores of 3 probe sets demonstrate high levels of SERPINB3 correlate significantly with both overall survival (p=0.004, where total N=22; Low group=11, and High group=11) and progression free survival (p=0.006, N=21; Low=10 , High=11 ). Formalin embedded tumor block RNA extraction Real Time PCR Acknowledgments The O’ Brien Centre has been instrumental for funding this research project. Furthermore, this project could not have been possible without the help of everyone at the Tom Baker Translation Lab as well as my supervisors, Dr. JB McIntyre, Dr. Corrine Doll and Dr. Tony Magliocco. References Formalin embedded DNA blocks IHC Interpretation of results 1) Parkin, D. M., Bray, F., Ferlay, J., & Pisani, P. (2005). Global cancer statistics, 2002. CA: A Cancer Journal for Clinicians, 55(2), 74-108. 2) Walboomers, J. M. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J. A., Shah, K. V., Snijders, P. J. F., Peto, J., Meijer, C. J. L. M. & Muñoz, N. (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. The Journal of Pathology, 189: 12–19. 3) Vidalino, L., Doria, A., Quarta, S., Zen, M., Gatta, A., & Pontisso, P. (2009). SERPINB3, apoptosis and autoimmunity. Autoimmunity Reviews, 9(2), 108-112. Goal NR R R R NR R NR NR R NR The aim of this project is to identify and validate candidate radiation response biomarkers (such as SERPINB3) using immunohistochemistry and qPCR to validate current gene expression data which suggests host immune factors have an important role in tumor response to treatment. This research will be analyzing patients that have only received RTX and have had squamous cell carcinoma. Figure 2: Malignant cervix is standardized with normal cervix (SF08-22232-B1) while using housekeeping genes such as 18S, huB2M, huBA, and huTFR. In general, all responders demonstrated high levels of SERPINB3 while all 5 nonresponders consistently had low levels of SERPINB3. Analysis of sample 86-1584-2 did not show any results due to a low levels of samples.