Cancer Care

1. Cancer Care High Stromal Expression of CAIX is Predictive of Poorer Overall Survival in Patients with HPV-negative Head & Neck Cancer Treated with Concurrent Chemoradiotherapy Nigel Brockton1,2, Joseph Dort2,3,4, Harold Lau2, Desiree Hao2, Sony Brar1, Alex Klimowicz5, Corinne Doll2, Anthony Magliocco2,5 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 • Endogenous Markers of Hypoxia (EMH) such as carbonic anhydrase IX (CAIX)) are regulated by Hypoxia Inducible Factors (HIF) 1α and 2α • EMH have not demonstrated prognostic equivalence to direct oxygen measurement. However, expression of EMH may be affected by HPV infection or associated inflammatory responses • Previously, stratifying patients by p16 status (HPV surrogate) improved the predictive utility of CAIX expression in tumour tissue of HPV-ve tumours; non-statistically significant inverse association between epithelial CAIX expression and survival (Figure 4) • Both epithelial and stromal hypoxia or related gene expression may contribute to prognosis and therapy response • METHODS • Consecutive cases of biopsy-proven, locally advanced HNSCC were identified from the Multidisciplinary Head & Neck Clinic database • Tissue micro-arrays were constructed from triplicate 0.6mm cores from paraffin-embedded tumor tissue. • Semi-quantitative immunohistochemistry (IHC) staining for p16 status • Automated, quantitative IHC system (AQUA HistoRx™) was used to quantify the intensity and tissue localization for CAIX (Figures 1 & 2) • We compared the expression (“low” ≤ median; “high” > median) of epithelial (eCAIX) and stromal (sCAIX) expression in groups defined by p16 status, as a surrogate for active HPV involvement in tumor formation • We compared Kaplan-Meier survival curves for overall survival associated with each group. Overall survival was compared by the exact Wilcoxon-Gehan test. • 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 eCAIX (n=46) and sCAIX (n=49) • Overall survival was significantly better in patients with tumours with low sCAIX expression (median survival 1.7years vs 4.4 years in high vs low sCAIX respectively) • 2 Year Survival (p16-ve): sCAIX low: 91%, sCAIX high: 36% Figure 2: HistoRx™ PM-2000 image analysis platform Figure 1: AQUA HistoRXImmunofluorescent staining and tissue localized quantitative analysis of CAIX expression Figure 3: Overall survival in all patients, stratified by stromal CAIX expression Figure 4: Overall survival in p16-negative patients stratified by epithelial CAIX expression Figure 5: Overall survival in p16-negative patients stratified by stromal CAIX expression • DISCUSSION • Patients with HPV-negative HNSCC have a generally poor prognosis • Low stromal CAIX expression is predictive of better overall survival in patients with HPV-negative LA-HNSCC • The increased predictive performance of stromal CAIX when stratified by p16 status (HPV surrogate) supports the hypothesis that EHM may be affected by HPV infection (Figures 3 & 5) • High sCAIX may confer a more invasive tumour phenotype by inducing necrosis of normal cells, selecting for acid-resistant tumor cells, inducing extracellular matrix degradation and angiogenesis RELEVANT IMPACT • HPV testing will likely become routine in the clinical management of HNSCC • Combined p16 status and stromal CAIX expression could provide a more accurate prognosis than p16 or HPV status alone • Currently available carbonic anhydrase inhibitors may improve outcomes in HPV-negative HNSCC patients. ACKNOWLEDGEMENTS This work was funded by the Alberta Cancer Research Institute Bridge & Pilot funding and establishment funds from the Alberta Cancer Foundation.