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Abstract
Background Combining immune checkpoint inhibitors (ICIs) with immunogenic chemotherapies is a promising approach in oesophageal adenocarcinoma (OAC) to convert ‘cold’ tumours to ‘hot’ tumours expanding the efficacy of ICIs to a greater spectrum of patients.1 However, there is a vast array of immune checkpoints (ICs) expressed by T cells and the effect of ICIs in combination with chemotherapy regimens is largely unknown.2
Methods The expression profile of a range of ICs on circulating and tumour-infiltrating T cells was assessed using flow cytometry prior to and post-neoadjuvant treatment and correlated with clinical parameters (n=20). PBMCs isolated from OAC blood were treated with single agent ICIs alone (single agent anti-PD-1, anti-PD-L1, anti-A2aR and anti-TIM-3 inhibition) and in combination with FLOT (5-Fluorouracil, oxaliplatin and docetaxel) and CROSS (carboplatin and paclitaxel) chemotherapy regimens. The production of anti-tumour cytokines by T cells was assessed in vitro by flow cytometry (n=6).
Results In the treatment-naïve and post-treatment setting, a range of ICs were expressed by circulating T cells and were significantly increased on tumour-infiltrating T cells, which included PD-L1, PD-L2, CD160, PD-1, CTLA-4, TIGIT, TIM-3, LAG-3, A2aR and ICOS (p<0.05) (figure 1). Pre-treatment circulating PD-1+ T cells positively correlated with pathological nodal status (p<0.05), (figure 2). Whereas tumour-infiltrating CD3+CTLA-4+ cells positively correlated with nodal metastasis and lymphovascular invasion (p<0.05). The percentage of tumour-infiltrating CD3+CTLA-4+ and CD3+ICOS+ cells was significantly lower post-neoadjuvant treatment (p<0.05) (figure 3). However, post-neoadjuvant treatment circulating CD3+PD-1+ cells and CD3+CD4+TIGIT+ cells positively correlated with a better treatment response, determined by PET/CT (p<0.05), (figure 4). ICIs enhanced T cell production of anti-tumour cytokines IL-2 and IFN-y alone and in combination with chemotherapy in vitro from treatment-naïve OAC patients (p<0.05).
The percentage of T cells expressing PD-1 was significantly increased in tumour tissue compared with peripheral blood circulation in the treatment-naïve setting of OAC patients CD3+, CD3+ CD4+ and CD3+ CD8+ cells were screened for the surface expression of PD-1 in OAC patient blood pre-treatment blood and tumour tissue (n=20). Mann Whitney test **<0.001.
Pre-treatment circulating PD-1+ T cells positively correlated with pathological nodal status and tumour-infiltrating CD3+CTLA-4+ cells positively correlated with nodal metastasis and lymphovascular invasion. The percentage of CD3+, CD3+CD4+ and CD3+CD8+ cells expressing ICs in peripheral circulation and infiltrating OAC tissue in the treatment-naïve setting was correlated with each other, patient demographics and clinical features of the tumour. Patient demographics and clinical features included gender (female=0, male=1), age, tumour type (OAC=0 and OGJ=1), neo-adjuvant treatment received (CROSS=0 and FLOT=1), treatment response (determined by radiographic features using PET/CT), tumour regression grade (TRG), clinical tumour stage and nodal involvement, pathological tumour stage and nodal involvement, body mass index (BMI kg/m2), peri-neural invasion, serosal invasion and lymph-vascular invasion. BMI and weight measurement was recorded post-treatment. Spearman correlation. Only significant data shown.
The percentage of tumour-infiltrating T cells expressing CTLA-4 and ICOS were significantly lower post-neoadjuvant therapy in OAC tumour tissue. CD3+, CD3+ CD4+ and CD3+ CD8+ cells were screened for the surface expression of CTLA-4 and ICOS infiltrating OAC tissue pre-treatment and post-treatment (n=20). Mann Whitney test *<0.001.
The percentage of circulating CD3+PD-1+ cells and CD3+CD4+TIGIT+ cells positively correlated with a better treatment response in the post-treatment setting. The percentage of CD3+, CD3+CD4+ and CD3+CD8+ cells expressing ICs in peripheral circulation and infiltrating OAC tissue in the post-neoadjuvant treatment setting was correlated with each other, patient demographics and clinical features of the tumour. Patient demographics and clinical features included gender (female=0, male=1), age, tumour type (OAC=0 and OGJ=1), neo-adjuvant treatment received (CROSS=0 and FLOT=1), treatment response (determined by radiographic features using PET/CT), tumour regression grade (TRG), clinical tumour stage and nodal involvement, pathological tumour stage and nodal involvement, body mass index (BMI kg/m2), peri-neural invasion, serosal invasion and lymph-vascular invasion. BMI and weight measurement was recorded post-treatment. Spearman correlation. Only significant data shown.
Conclusions T cells expressing ICs in circulation and infiltrating OAC tissue were adverse prognostic markers in the pre-treatment setting, perhaps due to their role in enabling tumour immune evasion and subsequent tumour progression. In contrast, T cells expressing ICs post-chemotherapy treatment in peripheral circulation were favorable prognostic markers. ICs are typically expressed by ‘hot’ tumours2 therefore, the presence of ICs in the post-treatment setting may be as a result of an induced-anti-tumour immune response following immunogenic chemotherapy/chemoradiotherapy treatment and may be a useful strategy for stratifying patients into chemotherapy/chemoradiotherapy responders or non-responders. A therapeutic rationale is also highlighted for combining ICIs with chemotherapy regimens in OAC patients to enhance anti-tumour T cell-mediated responses and potentially boost response rates to chemotherapy treatment.
Acknowledgements We would like to thank all the patients who kindly donated their samples to our research
Ethics Approval The work was performed in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving human samples.
Consent Patients provided informed consent for sample and data acquisition, and the study received full ethical approval from the St. James’s Hospital/AMNCH Ethical Review Board.
References
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