In the contemporary era of cancer immunotherapy, an abundance of clinical and translational studies have reported radiotherapy (RT) and immunotherapies as a viable option for immunomodulation of many cancer subtypes, with many related clinical trials ongoing. In locally advanced disease, chemotherapy or chemoradiotherapy followed by surgical excision of the tumour remain the principal treatment strategy in oesophageal adenocarcinoma (OAC), however, the use of the host immune system to improve anti-tumour immunity is rapidly garnering increased support in the curative setting.

To immunophenotype OAC patients’ immune checkpoint (IC) expression with and without radiation and evaluate the effects of checkpoint blockade on cell viability.

In the contemporary era of cancer immunotherapy, an abundance of studies have demonstrated that combination RT and IC inhibitors (ICIs) are effective in the immunomodulation of many cancer subtypes, with many related clinical trials ongoing. Although surgical excision and elimination of tumour cells by chemotherapy or chemoradiotherapy remains the gold standard approach in OAC, the propagation of anti-tumour immune responses is rapidly garnering increased support in the curative setting. The aim of this body of work was to immunophenotype OAC patients’ IC expression with and without radiation and to establish the impact of checkpoint blockade on cell viability. This study was a hybrid combination of in vitro and ex vivo models. Quantification of serum immune proteins was performed by enzyme-linked immunosorbent assay. Flow cytometry staining was performed to evaluate IC expression for in vitro OAC cell lines and ex vivo OAC biopsies. Cell viability in the presence of radiation with and without IC blockade was assessed by a cell counting kit-8 assay.

We identified that conventional dosing and hypofractionated approaches resulted in increased IC expression (PD-1, PD-L1, TIM3, TIGIT) in vitro and ex vivo in OAC. There were two distinct subcohorts with one demonstrating significant upregulation of ICs and the contrary in the other cohort. Increasing IC expression post RT was associated with a more aggressive tumour phenotype and adverse features of tumour biology. The use of anti-PD-1 and anti-PD-L1 immunotherapies in combination with radiation resulted in a significant and synergistic reduction in viability of both radiosensitive and radioresistant OAC cells in vitro. Interleukin-21 (IL-21) and IL-31 significantly increased, with a concomitant reduction in IL-23 as a consequence of 4 Gray radiation. Similarly, radiation induced an anti-angiogenic tumour milieu with reduced expression of vascular endothelial growth factor-A, basic fibroblast growth factor, Flt-1 and placental growth factor.

The findings of the current study demonstrate synergistic potential for the use of ICIs and ionising radiation to potentiate established anti-tumour responses in the neoadjuvant setting and is of particular interest in those with advanced disease, adverse features of tumour biology and poor treatment responses to conventional therapies.

Oesophageal adenocarcinoma (OAC) is rapidly increasing in incidence in the western world, and five year survival rates rarely exceed 40%[1]. Multimodal therapy alongside surgical resection has become standard of care for locally advanced cancer of the oesophagus or the oesophagogastric junction[2]. One option is the CROSS regimen, which includes preoperative administration of carboplatin and paclitaxel with concomitant radiotherapy (RT)[3]. In Europe, radiation is delivered in 23 fractions of 1.8 Gray (Gy), giving a total dose of 41.4 Gy but this varies worldwide, with North American centres delivering up to 50.0-51.4 Gy[4], while Asian regimens can feature cumulative doses of 60 Gy[5].

Hypofractionated RT is where radiation is delivered in fewer fractions of 2.4 Gy to 5.0 Gy, but often the same cumulative dose[6]. This has the potential to reduce costs, increase patient comfort, and could be more effective compared to conventional treatment[7]. Randomised trials in breast and prostate cancer have found that both high- (≥ 5 Gy per fraction) and moderately (2.4-3.4 Gy per fraction) hypo-fractionated RT is non-inferior to traditional regimens[7-10]. As RT is a mainstay of treatment, and oesophageal malignancies are associated with considerable morbidity, there is interest in evaluating whether this paradigm can be applied in the upper gastrointestinal context.

Disappointingly, a pathologic complete response to treatment is observed in less than 30% of patients with oesophageal cancer undergoing chemoradiotherapy[11], and it is this small subgroup that benefits most in terms of survival[12]. More effective strategies are therefore required. One emerging approach is combining chemoradiotherapy with immune checkpoint blockade (ICB). The most widely used ICB involves blocking the interaction of PD-1 expressed on T cells and it’s ligand, PD-L1 expressed on tumour cells[13], and seeks to re-invigorate anti-tumour cytotoxic T cells[14]. Phase III trials of single agent ICB have delivered mixed results in chemorefractory advanced oesophagogastric cancer[15], but some recent encouraging results have been reported in earlier stage disease[16,17].

Radiation can sensitise tumours to immunotherapy through three main mechanisms[18]. First, radiation can increase neoantigen expression and induce immunogenic cell death, whereby release of damage associated molecular patterns (DAMPs) results in more efficient tumour antigen presentation and immune stimulation[19]. Second, radiation induced DNA damage can activate the GMP-AMP stimulator of interferon genes (cGAS-STING) cytosolic DNA sensor, resulting in type I interferon production[20,21]. Finally, RT can result in remodeling of the tumour microenvironment (TME), promoting infiltration of immune cells[22]. The latter effect is particularly affected by radiation dosage and some limited preclinical evidence suggests that hypofractionated RT can have more immunostimulatory effects than conventional fractionation[18]. However, the majority of studies in the literature to date have focused on more inherently immunogenic tumour models like melanoma or non-small cell lung cancer, or common malignancies like breast or colon cancer. There are a number of clinical studies evaluating hypofractionation in the context of squamous cell cancer of the oesophagus, however, data is lacking for OAC. In addition, there are no translational studies characterising immune response in OAC in the context of immunotherapy and thus was the premise for this study.

This study assessed the effects of hypofractionated RT on IC expression in oesophageal cancer cells in vitro and ex vivo and correlated this with clinical outcomes. We also assessed the synergistic effects of ICB and radiation on OAC cell lines. Through this, we aimed to enhance our understanding of the interplay between immunotherapy, radiation and the TME in oesophageal cancer, with the goal of identifying the most effective radiation dosing strategy to combine with immunotherapy.

The visceral appeal of modulating the host immune system is one of simplicity in an effort to harness a profound anti-tumour response and is a principle that has existed since the development of the field of cellular immunology as an entity. Quintessentially, skewing the hosts innate immune system to boost anti-tumour immunity consists of two processes compliant to exploitation: These being the stimulant as well as the reaction. The most logical way to perturb the tumour and its microenvironment is through promoting tumouricidal effects, through systemic chemotherapy or the radiation therapy delivered. Increasing and propagating the anti-tumour responses thereby facilitating immune activation with optimal kinetics may achieve a synergistic anti-tumour response, producing a more profoundly durable effect on the immune system than chemo (radio) therapy alone. In this context, the landmark Checkmate-577 trial has demonstrated significantly improved disease free survival in the adjuvant setting of resectable gastroesophageal cancer[16]. The findings of increased IC expression in vitro and ex vivo through the use of radiation in the current body of work provides promising translational therapeutic rationale for their use in the multimodal paradigm. RT propagates the priming and effector phases of the anti-tumour immune response rendering it an appropriate combination with IC inhibitors (ICIs)[24]. However, inherently radioresistant tumours may pose a particular therapeutic dilemma, as they may not have a similar synergism with ICB as radiosensitive tumours. Ionising radiation is currently under investigation in metastatic oesophageal cancer with pembrolizumab (NCT02642809) and is currently under investigation in the curative setting with neoadjuvant trimodal therapy of Pembrolizumab and chemoradiotherapy in oesophageal squamous cell carcinoma (NCT03792347), with a similar trial investigating durvalumab and chemoradiotherapy in squamous cell carcinoma (SCC) and OAC (NCT02735239). The SKY-SKRAPER-07 trial is currently evaluating anti-PD-L1 Atezolizumab with anti-TIGIT therapy following chemoradiotherapy in advanced oesophageal cancer (NCT04543617). Of note, in a study by Zhao et al[25], they reported that with PD-1 positivity correlated with TIM-3 expression, and CD8⁺ tumour infiltrating lymphocyte density as a risk factor for recurrence free and overall survival (OS) in oesophageal SCC. The increasing expression in OAC cells and a cohort of patients following RT in this study represents promising therapeutic targets in OAC.

The clinically important observation in this study that half of the patients assessed displayed a reduction in IC expression post RT is an interesting caveat, one which suggests very different susceptibility to ICB in combination with RT and therefore, the stratification of patients into potential responders and non-responders should be addressed. In the same vein, the activation of cGAS-STING signaling, which has been recognized to potentiate systemic anti-tumour immunity and subsequent tumour rejection by dual RT and checkpoint blockade administration is promising even in those with checkpoint downregulation. A study by Vanpouille-Box et al[26], highlighting the importance of the cGAS-STING pathway in response to combination RT and immunotherapy, reported the knockdown of cGAS in murine cancer cells abrogated the priming of CD8⁺ T cells in tumour-draining lymph nodes and spleen, and prevented the infiltration of abscopal tumours by CD8⁺ T cells. Importantly, the synergistic and significant reduction in viability of radioresistant OAC cancer cells which we observed in this study following the dual administration of ICIs and ionising radiation is very promising.

With respect to the use of hypofractionation in the curative setting for oesophageal cancer, there is an increasing volume of evidence demonstrating the safety and efficacy of this approach[27]. There are studies demonstrating a survival benefit of this approach particularly in the context of metastatic nodal disease. In one such study, hypofractionated radiotherapy (HFR) administered with taxane based chemotherapy in the management of post-surgery tracheoesophageal groove lymph node (TGLN) metastasis demonstrated improved OS in the HFR group compared with that of the conventional dosing treatment arm [24.1 mo (95%CI, 16.2-32.1 mo) vs 11.9 mo (95%CI, 9.2-14.4 mo), P = 0.024)[28]. Importantly, the study did not find a significant difference in pulmonary complications such as radiation pneumonitis (grades 3-4, 16.0% vs 7.1%; P = 0.314)[28].

Radiation induced lymphopenia is a frequent complication of multimodal cancer therapy and poorer outcomes are directly linked to the severity of lymphopenia[29]. Furthermore, it has been demonstrated that circulating lymphocyte count during neoadjuvant chemoradiation (CRT) in oesophageal cancer patients can predict pathological complete response (pCR) rates and low absolute circulating lymphocytes are associated with poorer outcomes[30]. The widespread adoption of immunotherapy has garnered new support and focus on the preservation of a pool of lymphocytes that are functional in enhancing immune function in the circulation and a study in pancreatic adenocarcinoma demonstrated hypofractionated CRT of 10 Gy in 3 doses over one week resulted in a decrease in the loss of T cells systemically compared to 28 daily doses of 1.8 Gy equating to 50.4 Gy[31,32].

The literature to date is concentrated primarily on evaluation of adverse events and of RT or immunotherapy in isolation. With improvements in targeted radiation delivery modalities, and technological advances, hypofractionated RT is now utilised without evidence of increased toxicities in a number of malignancies[33,34]. The Hypofractionated RT used in this instance was safe, well tolerated and provided robust survival results in those who could not receive chemoradiotherapy[35]. Furthermore, there is data in lung that conventional radiation dosing and immunotherapy is safe and feasible with no increases in adverse events[36-38].

The ATTRACTION 3 trial demonstrated a 50% reduction in serious adverse events in those treated with nivolumab vs conventional chemotherapy in Esophageal Squamous cell carcinoma[39]. In a study evaluating and immunotherapy in renal cell cancer, melanoma and lung, fatigue and pneumonitis were the most common adverse event. They found that toxicity did not correlate with hypofractionation or tumour type. Hypofractionated RT of pulmonary lesions was found to induce a complete response more consistently than in other sites. This study found that combining body Hypofractionated RT with immunotherapy is safe and viable, however, level I evidence is needed[40].

VEGF is a mitogen essential for angiogenesis and Ramucirumab is approved for use in advanced gastroesophageal cancer patients. The use of anti-VEGF agents have shown promise in promoting improved survival when used in combination with chemoradiotherapy in colon cancer, however treatment resistance is a common problem[41]. This can be due compensatory mechanisms resulting in resistance, namely hypoxia- induced increases of other angiogenic promoters such as PIGF[42]. VEGF, bFGF and PIGF are crucial angiogenic promoters linked with tumourigenesis and Flt-1, is involved in tumour growth and metastatic dissemination, most likely via stimulation of macrophage-lineage cells[43]. PIGF/Flt-1 signaling can contribute to colorectal cancer progression through increasing the phosphorylation of p38 mitogen-activated protein kinase (MAPK), thereby upregulating MMP9 expression; resulting in increasing cellular migration/invasion. Therefore inhibition of PIGF/Flt-1 signalling will have therapeutic potential in lower gastrointestinal cancers[44]. In the current study radiation therapy was demonstrated to reduce the expression of these promoters of angiogenesis, which is crucial in the mitigating the risk of metastatic disease for upper gastrointestinal cancers.

The subset of cytokines expressed post radiation and immunotherapy treatment play a key role in determining the subsequent immune response elicited. In this study the OAC tumour tissue released significantly more anti-tumour IL-21 and IL-31 in response to radiation. IL-21 is produced by numerous T helper cells, such as Th1 and Th17 cells, activated Natural Killer T cells[45]. It promotes B cell differentiation into plasma cells, regulates immunoglobulin production, reshaping the tumour microenvironment and influencing the proliferation and/or effector function of both CD4⁺ and CD8⁺ T cells, while limiting the differentiation of Tregs[46]. IL-21 has distinct anti-tumour properties as a consequence of its ability to increase the availability of CD8⁺ T cells through the induction of an early differentiation phenotype and Natural Killer cells[33]. IL-31 has immunoregulatory properties, with a study demonstrating that mice infused with IL-31 had tumour growth disruption and a decreased metastatic burden, supporting the use of IL-31 to offset the risk for metastatic disease development[47]. Similarly, in a breast cancer murine model, the tumouricidal effects of T cells are increased, and myeloid derived suppressor cells and tumour-associated macrophages are reduced in tumours with high expression of IL-31, with an immunophenotype supporting antitumour immunity[48]. While both IL-21 and IL-31 were significantly increased in the TCM, the expression of IL-23, which has been documented to promote tumour metastases was decreased. IL-23 has metastases promoting properties via suppressing the anti-tumour properties of T cells and the anti-metastatic function of NK cells[49]. In addition to this, IL-23 was found to be overexpressed in many human cancers including colorectal and gastric cancer, and was found to be a negative prognostic indicator[50]. Of note CRP, an acute phase protein and marker of vascular injury was found to increase with 2 Gy radiation in our study. There have been epidemiologic studies to suggest that elevated CRP levels in circulation are linked with poorer outcomes in those with solid cancers, whereas elevated levels in apparently healthy subjects, is a potential independent risk factor for future risk of developing cancer of any type including lung, colorectal and gastric cancers due to chronic low inflammatory states, which is of particular relevance in OAC[51]. Therefore, the exact role of CRP in response to RT and immunotherapy requires further study.

Co-stimulatory and IC molecules can have both immunostimulatory and immunosuppressive effects. In this study a range of soluble ICIs and ligands were significantly downregulated following 4 Gy radiation treatment of OAC tumour explants. The role of soluble receptors however, and its effects on immune function remain yet to be elucidated and therefore its potential use as an oncological treatment remain unclear. Through this body of work, we observed a significant down regulation of PD-1, PD-L1, TIM-3 and TIGIT and this was paralleled by a concomitant increase in OAC cell line surface expression and a cohort of OAC tumour explants, which may go some way to explain the decrease in the soluble forms of these IC proteins post irradiation. B7-H3 (also known as CD276) is an IC molecule, with many cancers exhibiting aberrant overexpression and such upregulation is associated with aggressiveness and a poor clinical prognosis[52]. Furthermore, there are studies demonstrating a vital role for B7-H3 in promoting tumourigenesis and metastatic dissemination, proliferation, invasion and migration[52-54]. CD276 promotes tumour proliferation and invasion[55]. In addition, soluble CD276 was found to stimulate the invasion and metastatic dissemination of pancreatic adenocarcinoma cells via the Toll like Receptor 4/nuclear factor kappa-light-chain-enhancer of activated B cells pathway (50). Overall, additional studies are required in gastroesophageal cancers to determine the true function of soluble IC proteins and how they pertain to treatment response and immune regulation.

CD28 which is a co-stimulatory molecule is essential in the augmentation of T cell activation and metabolism, driving tumour-infiltrating T cell glycolysis. It is antagonized by CTLA-4 and PD-1[56]. In the current study, soluble CD28 is reduced with radiation, which may be immunosuppressive. Soluble CD80-Fc has been found to maintain IFN-γ release by PD-1⁺ specific activated T cells even with PD-L1⁺ tumour cells[57]. Soluble GITR, which was reduced in this study, represents a potential immunotherapeutic target and is found to be expressed at high levels on Tregs[58,59]. A number of phase 1 trials have identified anti-GITR antibodies to have safe pharmacological profiles, with phase II trials ongoing evaluating its combination with RT and anti-PD-1 therapy (NCT04225039). New promising approaches are focusing on the activation of co-stimulatory pathways to enhance antitumour immune responses. GITR activation can result in the inhibition of T-cell (Treg) function and promote effector T-cell function[58], and may also provide theoretical basis for the clinical application of combinations with monoclonal antibody therapy such as bFGF in molecular targeted therapies[60].

Lastly, OX40 has been demonstrated to have a crucial part to play in maintaining the immune responses in the immediate term and ongoing responses through enhancing T cell expansion, differentiation, and survival[61]. OX40 activation can have a significant impact T cell receptor (TCR) signaling through the PI3-K/PKB pathway, influencing T cell division, survival and cytokine production. This can directly increase calcium influx, and lead to IL-2, IL-4, IL-5, and IFN-γ secretion[62]. OX40 triggering regressed Treg cells, thus this allows Dendritic cells to reach the lymph nodes draining the tumour and in doing so prime the tumour-specific CD8 lymphocytes response [63]. However, in the current study radiation induces a downregulation of OX40 in TCM which may indeed be an immunosuppressive consequence of radiation therapy and one which requires further investigation. Again, more robust studies will be helpful to determine the functions of soluble co-stimulatory molecules as they may have alternate functions compared with their cell membrane bound counterparts in the tumour microenvironment.

The introduction of ICIs has resulted in enhanced survival in melanoma and non-small cell lung cancer treatment and has evolved to involve the spectrum of solid gastrointestinal malignancies with positive results of the landmark Checkmate 577 trial in the adjuvant setting most notable to date. However, there remains many issues to be interrogated including an appropriate RT regimen in conjunction with immunotherapies. There is considerable translational, preclinical and clinical data in favour of fractionated RT, and timing of RT delivery and target delineation, there remains disparity and no universal approach applicable to the clinical setting. In the current study, IC blockade in combination with radiation synergistically reduces viability in radioresistant cells and Nivolumab appears most efficacious. There remains a need to delineate the effects of RT on host anti-tumour immunity. Additionally, lymphopenia induced by RT delivery may negate the effects of immunotherapy on offsetting T cell exhaustion, thus protocols that can minimize lymphopenia need careful design for maximal therapeutic potential. Finally, more concentrated and robust studies to determine and validate potential biomarkers to predict those who will be suitable for these treatment modalities are urgently required with profiling next-generation sequencing of tumour mutation burden based profiling, immune signatures, gene profiling signatures and the repertoire of T-cell receptors potential avenues to elucidate this.