Clinical Relevance of PD-1 Positive CD8 T-cells in Gastric Cancer

Authors:
Joan CHOO^1*, Ley Fang KUA^2*, Mu Yar SOE¹, Bernadette Reyna ASUNCION², Benjamin Kye Jyn TAN³, Chong Boon TEO³, Ryan Yong Kiat TAY³, Jimmy SO⁴, Asim SHABBIR⁴, Kim GUOWEI^4,6, Hon Lyn TAN^1,6, Gloria CHAN¹, Haoran MA⁵, Cheng Ean CHEE¹, Sriram SRIDHARAN², Patrick TAN^2,5,6,7,8,9, Raghav SUNDAR^1,3,5,6,10,+, Wei Peng YONG^1,2,+


Affliations:
¹Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore
²Cancer Science Institute, National University of Singapore, Singapore
³Yong Loo Lin School of Medicine, National University of Singapore, Singapore
⁴Department of Surgery, National University Cancer Institute, National University Health System, Singapore
⁵Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore
⁶Singapore Gastric Cancer Consortium, Singapore
⁷Department of Physiology, National University of Singapore, Singapore
⁸Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore
⁹SingHealth/Duke-NUS Institute of Precision Medicine, National Heart Centre Singapore, Singapore
¹⁰The N.1 Institute for Health, National University of Singapore, Singapore


Corresponding email address:
Raghav SUNDAR (mdcragh@nus.edu.sg)
Wei Peng YONG (wei_peng_yong@nuhs.edu.sg)

Background: We evaluated the relevance of PD-1⁺CD8⁺ T-cells in gastric cancer (GC) including prognostic significance, association with chemotherapy and immunotherapy sensitivity and correlations with the tumor microenvironment (TME).

Methods: Discovery cohort: GC samples were evaluated for AE1/3, CD8, PD-1, Ki-67 and Granzyme-B expression with fluorescence-based multiplex immunohistochemistry (mIHC). Validation cohorts: we analysed bulk RNAseq GC datasets from TCGA, the “3G” chemotherapy trial and an immunotherapy phase 2 trial. The cox proportional hazards model was used to identify factors that influenced overall survival (OS). To study the TME, we analysed single-cell RNAseq performed on GCs.

Results: In the discovery cohort of 350 GC samples, increased PD-1 expression of CD8 T-cells was prognostic for OS (HR 0.678, p=0.039). PD-1 expression in CD8 T-cells highly correlated with cytolytic [Granzyme-B+] (r= 0.714, p<0.001) and proliferative [Ki-67+] (r= 0.783, p=0.012) activity. Analysis of bulk RNAseq datasets showed tumors with high PD-1 and CD8Aexpression levels had improved OS when treated with immunotherapy (HR 0.117, p=0.036) and chemotherapy (HR 0.475, p=0.017). Analysis of an scRNAseq dataset of 152,423 cells from 40 GCs revealed that T-cell and NK-cell proportions were higher (24% vs 18% and 19% vs 15%, p<0.0001), while macrophage proportions were lower (7% vs 11%, p<0.0001) in CD8PD-1_highcompared to CD8PD-1_low tumors.

Conclusion: This is one of the largest GC cohorts of mIHC combined with analysis of multiple datasets providing orthogonal validation of the clinical relevance of PD-1⁺CD8⁺ T-cells being associated with improved OS. CD8PD-1_high tumors have distinct features of an immunologically active, T-cell inflamed TME.