Abstract 3142: Tumor-infiltrating lymphocytes and CD4/FOXP3 ratios reliably predict survival using digital image analysis
Authors: Craig Stephanie G. , Bankhead Pete , Bingham Victoria , McQuaid Stephen , Tumelty Maria , McDade Simon S. , Salto-Tellez Manuel , James Jacqueline A. 
Published: August 2018

Abstract
Background:Tumour infiltrating lymphocytes (TILs) have been associated with improved prognosis in cancer, including oropharyngeal squamous cell carcinoma (OPSCC); however, the survival benefit appears modest and favours HPV positive OPSCC. The current literature presents a mixed view on the best method to score TILs and if it would be beneficial to score all cancers of the head and neck. Use of immunotherapies to stimulate immune cell activation is increasing in the clinic and as such a robust method of assessment is needed if we want to apply this routinely to OPSCC. This study investigates the prognostic value of assessing TILs by H&E in HNSCC using digital pathology solutions against human eye visual assessment and by comparing TIL estimates in H&Es against markers of adaptive immunity (CD3, CD4,CD8 and FOXP3) using digital image analysis.
Methods:This study utilised 190 HNSCC arising from the oropharynx. TMAs constructed, in triplicate, from these samples were stained with either H&E or IHC for CD3, CD4, CD8 and FOXP3. Visual estimates of TIL counts by human eye from H&E stained slides were assessed on the whole core at x10 magnification based on published thresholds by two observers to form a consensus score. Image analysis was performed on whole cores using image analysis software QuPath on H&E and IHC stained slides. HPV status was determined by p16 IHC and high risk HPV RNAScope. Statistical analyses were performed using R.
Results:Scoring of TILs in H&Es by visual estimates was prone to misestimation and reduced survival estimates when using predetermined thresholds compared to machine scoring by image analysis. Comparison of digital pathology solutions assessing the TIL population in H&E and IHC stained slides found the percentage of TILs present to be significantly higher in the HPV positive's compared to the HPV negative's in the H&E, CD3 and CD8 immunostained slides but not CD4 or FOXP3. When dichotomised into high and low TILs only H&E based TIL counts and CD4/FOXP3 ratios were found to be independently predictive of survival when adjusted for age and smoking history.
Conclusion:Image analysis creates continuous variables from which patient TIL counts can be reliably assessed. The adaptive immune landscape of HPV negative OPSCC is significantly different to HPV positive OPSCC of which only total TIL counts (H&E) and CD4/FOXP3 ratios appears to be predictive of five-year overall survival.
Citation Format: Stephanie G. Craig, Pete Bankhead, Victoria Bingham, Stephen McQuaid, Maria Tumelty, Simon S. McDade, Manuel Salto-Tellez, Jacqueline A. James. Tumor-infiltrating lymphocytes and CD4/FOXP3 ratios reliably predict survival using digital image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3142.