RELEVANT IMPACT

1. Does the Utility of Endogenous Markers of Hypoxia Differ by Tumour HPV Status in Head & Neck Cancer? Nigel Brockton 1,2, Joseph Dort 2,3,4, Harold Lau 2, Desiree Hao 2, Sony Brar 1, Corinne Doll 2, Anthony Magliocco 2,5 1Division of Population Health, Alberta Cancer Board, Calgary, Alberta; 2Department of Oncology, University of Calgary, Calgary, Alberta; 3Department of Community Health Sciences, University of Calgary, Calgary, Alberta; 4Department of Surgery, University of Calgary, Calgary, Alberta; 5Department of Pathology, University of Calgary/ Alberta Cancer Board, Calgary, Alberta. • BACKGROUND • Head & Neck Squamous Cell Carcinoma (HNSCC): • 5th most common malignancy worldwide • Main risk factors: alcohol intake and tobacco smoking • Emerging major risk factor: Human Papilloma Virus (HPV) infection (high risk sub-types) • HPV+ve tumours: • later stage at diagnosis • more favorable prognosis • p16 up-regulation (surrogate marker for HPV infection) • Direct measurement of tumor oxygen status is prognostically useful but has practical limitations • Genes regulated by Hypoxia Inducible Factors (HIF) 1α and 2α (e.g. Glucose Transporter 1 (GLUT1) and Carbonic Anhydrase IX (CA9)) are candidate endogenous markers of hypoxia (EMH) • EMH have not demonstrated prognostic equivalence to direct oxygen measurement. However, expression of EMH may be affected by HPV infection or associated inflammatory responses • Stratifying patients by p16 status (HPV surrogate) may improve the prognostic utility of EMH • RESULTS • 91 cases of locally advanced HNSCC identified; 59 tumor blocks were available • Semi-quantitative IHC for p16 was successful for 55 tumours • Quantitative IHC data were successfully obtained for CAIX (n=46) and GLUT1 (n=45) • Previous analysis confirmed p16 positivity conferred prognostic advantage (Figure 1) • DISCUSSION • Our analyses suggest that high CAIX expression may be a prognostic marker in HPV-ve locally advanced HNSCC • GLUT1 does not appear to be an informative prognostic marker in our cohort (Figures 2 & 4) • The Carbonic Anhydrase 9 gene (CA9) is an exclusive target for HIF-1α; GLUT1 expression is mediated by both HIF-1α and HIF-2 α • The increased prognostic performance of CAIX when stratified by p16 status (HPV surrogate) supports our hypothesis that HPV infection may modify EMH utility (Figures 3 & 5) Figure 2: Overall survival stratified by GLUT1 expression Figure 3: Overall survival stratified by CAIX expression Figure 1: Overall survival stratified by p16 status • METHODS • Consecutive cases of biopsy-proven, locally advanced HNSCC were identified from the Multidisciplinary Head & Neck Clinic database • Triplicate 0.6mm cores from paraffin-embedded tumor tissue were assembled into tissue micro-arrays • Semi-quantitative immunohistochemistry (IHC) staining for p16 • Automated, quantitative IHC system (AQUA HistoRx™) was used to quantify the intensity and distribution of staining for candidate EMH proteins: CAIX and GLUT1 (plates 1 & 2) • We compared the expression (“low” ≤ median; “high” > median) of GLUT1 and CAIX in each group, defined by p16 staining, as a surrogate for active HPV involvement in tumor formation • We compared Kaplan-Meier survival curves for overall survival associated with each group Figure 4: Overall survival for p16-ve patients stratified by GLUT1 expression 1 Year Survival (p16-ve): CAIX low: 91%, CAIX high: 60% RELEVANT IMPACT • It is likely that testing for HPV status will become part of the clinical management for HNSCC • Combined p16 status and CAIX status could provide a more accurate prognosis than p16 status alone • Combined p16 and CAIX is a candidate short-term prognostic marker Plate 1: AQUA HistoRX™ false color image of GLUT1 expression ACKNOWLEDGEMENTS This work was funded by the Alberta Cancer Research Institute Bridge & Pilot funding and establishment funds from the Alberta Cancer Foundation. Plate 2: AQUA HistoRX™ false color image of CAIX expression Figure 5: Overall survival for p16-ve patients stratified by CAIX expression