1. Heat Shock Protein 90 (HSP90) is over-expressed in p16 negative oropharyngeal squamous cell carcinoma and its inhibition in vitro potentiates the effects of chemo-radiation Kirtesh Patel 1, Jing Wen 1, Kelly Magliocca 2, Susan Muller 2, Yuan Liu 3, Zhuo Georgia Chen 4, Nabil Saba 4, and Roberto Diaz 1 Departments of Radiation Oncology 1, Pathology 2, Statistics 3, Medical Oncology 4 ; Winship Cancer Institute of Emory University, Atlanta, GA BACKGROUND Fraction 4:  Despite treatment advances, 5-year overall survival for squamous cell carcinoma of the head and neck (SCCHN) is close to 50%.  Cisplatin and radiation therapy remain the current standard for treating locally advanced SCCHN.  Novel treatment approaches are urgently needed, especially in patients with human papilloma virus (HPV) negative disease who have worse outcomes despite multimodality therapy.  Ganetespib is an HSP90 inhibitor that has demonstrated activity in patients with NSCLC, but efficacy in SCCHN is unknown  Despite treatment advances, 5-year overall survival for squamous cell carcinoma of the head and neck (SCCHN) is close to 50%.  Cisplatin and radiation therapy remain the current standard for treating locally advanced SCCHN.  Novel treatment approaches are urgently needed, especially in patients with human papilloma virus (HPV) negative disease who have worse outcomes despite multimodality therapy.  Ganetespib is an HSP90 inhibitor that has demonstrated activity in patients with NSCLC, but efficacy in SCCHN is unknown METHODS  Using our IRB approved head and neck cancer database, we obtained twenty oropharyngeal squamous cell carcinoma (SCC) tissue samples: ten p16 positive, ten p16 negative. We subsequently analyzed, via immunohistochemistry, HSP90 protein levels.  Using HPV-positive and HPV- negative SCC cell lines, we compared the effect of the HSP90 inhibitor ganetespib on proliferation and apoptosis.  Clonogenic survival of HPV-negative and positive cells treated with ganetespib and radiation therapy was then investigated.  Using our IRB approved head and neck cancer database, we obtained twenty oropharyngeal squamous cell carcinoma (SCC) tissue samples: ten p16 positive, ten p16 negative. We subsequently analyzed, via immunohistochemistry, HSP90 protein levels.  Using HPV-positive and HPV- negative SCC cell lines, we compared the effect of the HSP90 inhibitor ganetespib on proliferation and apoptosis.  Clonogenic survival of HPV-negative and positive cells treated with ganetespib and radiation therapy was then investigated. RESULTS P16+ (n = n10) P16- (n = 10) % Male90%80% % Caucasian100%60% % Current Smoker10%60%p = 0.01 T stage T140%10% T260%70% T30%20% N N030%40% N110% N260%50% Mean % HSP90 expression 51.079.5P = 0.016 Median % HSP90 expression 87.687.5P = 0.012 CONCLUSIONS Ganetespib inhibited HPV-negative SCCHN proliferation and potentiated cell kill when combined with radiation therapy in vitro. With HSP90 expression higher in p16 negative patients, further exploration of the clinical activity of HSP90 inhibitors in SCCHN is warranted. Ganetespib inhibited HPV-negative SCCHN proliferation and potentiated cell kill when combined with radiation therapy in vitro. With HSP90 expression higher in p16 negative patients, further exploration of the clinical activity of HSP90 inhibitors in SCCHN is warranted. 1B – P16 POSITIVE 1A – P16 NEGATIVE CLINICAL & PATHOLOGIC CHARACTERISTICS OF 20 OROPHARYNGEAL SCCHN PATIENTS STAINED WITH HSP90 HPV+ cell ( n = 3) HPV- cells ( n = 4) Cellular Metabolic activity 72.1%30.6%p = 0.03 HSP90 INHIBITION WITH GANETESPIB (50NM FOR 24HRS) DEMONSTRATES INCREASED CYTOTOXICTY IN 4 HPV NEGATIVE CELL LINES RELATIVE TO 3 HPV POSITIVE CELL LINES HSP90 IS UPREGULATED IN P16 NEGATIVE SCCHN TISSUEHPV NEGATIVE CELLS DEMONSTRATE INCREASED APOPTOSIS TO HSP90 INHIBITION HSP90 INHIBITION SENSITIZES BOTH HPV- AND HPV+ CELLS TO IONIZING RADIATION

2. Heat Shock Protein 90 (HSP90) is over- expressed in p16 negative oropharyngeal squamous cell carcinoma and its inhibition in vitro potentiates the effects of chemo- radiation Discussant Slides Kirtesh Patel 1, Jing Wen 1, Kelly Magliocca 2, Susan Muller 2, Yuan Liu 3, Zhuo Georgia Chen 4, Nabil Saba 4, and Roberto Diaz 1 Departments of Radiation Oncology 1, Pathology 2, Statistics 3, Medical Oncology 4 ; Winship Cancer Institute of Emory University, Atlanta, GA

3. Heat Shock Protein 90 (HSP90) is over-expressed in p16 negative oropharyngeal squamous cell carcinoma and its inhibition in vitro potentiates the effects of chemo-radiation Results – HSP90 was over-expressed in p16- oropharynx squamous cell carcinoma of the head neck tissue samples compared to p16+ samples Patients’ samples were well balanced except for expected differences in smoking – HSP90 inhibition with ganetespib decreased cellular proliferation of HPV- cells relative to HPV+ cells – Ganetespib induced apoptosis in HPV- cells, but not HPV+ cells – HPV- cells were more resistant to radiation therapy than HPV+ cells. Ganetespib sensitized both to radiation therapy alone

4. Heat Shock Protein 90 (HSP90) is over-expressed in p16 negative oropharyngeal squamous cell carcinoma and its inhibition in vitro potentiates the effects of chemo-radiation Strength of this research – Used ganetespib, an HSP90 inhibitor which has demonstrated safety in numerous clinical trials Ganetespib does not have ocular toxicity of other HSP90 inhibitors – In Vitro Doses (50nM for 24hrs) of ganetespib reflect those achieved in humans from phase 1 clinical trials – Compared expression of HSP90 in human tissue samples and stratified for p16 status – Compared efficacy of ganetespib alone and in combination with radiation therapy in both HPV negative and HPV positive cells

5. Heat Shock Protein 90 (HSP90) is over-expressed in p16 negative oropharyngeal squamous cell carcinoma and its inhibition in vitro potentiates the effects of chemo-radiation Limitations Tissue samples focused on p16 expression, not HPV+ expression via in situ hybridization. P16 can be overexpressed in HPV- cells. Findings would be more definitive if had access to greater number of HPV negative and HPV positive cell lines. Ganetespib demonstrated greater cytotoxicity and apoptotic ability in HPV+ cells relative to HPV- cells. Further studies are necessary to characterize this mechanism.