•EIN can modify the TME in favour of cytotoxic ICs.•CD3+ and CD8+ T-cell tumour densities can be converted into IS.•High IS links with better patient outcome.•I-SUPPLY evaluates the EIN effect on TME IC infiltration through IS analysis.•EIN is supposed to favour an increase in GC immunogenic pattern. The 'immune pattern' of the esophagogastric junction and gastric cancer (GC) has been related to tumour progression and/or response to therapies. GC can be classified as immunogenic or immune-resistant based on the infiltration of the tumour microenvironment (TME) from different immune cells (ICs), the most significant of which are activated lymphocytes (a-Ly) CD8+ and CD4+. From the amount of a-Ly infiltration in TME, we can identify tumours with high Immunoscore (IS) which correlates with better prognosis in terms of disease-free survival and overall survival (OS). IC activation and consequent immunogenic TME requires high nutrient absorption and synthesis and accumulation of protein, lipid, and nucleotides. However, tumour cells (TCs) promote their own proliferation by stealing micronutrients to ICs, therefore leading to an immunosuppressive TME phenotype. This proof-of-concept protocol aims to assess whether a controlled enteral immune nutrition (EIN) supplementation can revert the TME in favour of ICs over TCs, in both auxotrophic and non-auxotrophic malignancies, witnessed by an increase in IS in surgical specimen over biopsy controls. Secondary endpoints are: (i) tumour regression grade assessment compared to historical data from FLOT4/AIO; (ii) combined proportion score ratio; (iii) relapse-free survival at 3 years and OS; and (iv) quality of life by the European Organisation for Research and Treatment of Cancer quality-of-life questionnaire (EORTC QLQ)-30. The 'immune pattern' of the esophagogastric junction and gastric cancer (GC) has been related to tumour progression and/or response to therapies. GC can be classified as immunogenic or immune-resistant based on the infiltration of the tumour microenvironment (TME) from different immune cells (ICs), the most significant of which are activated lymphocytes (a-Ly) CD8+ and CD4+. From the amount of a-Ly infiltration in TME, we can identify tumours with high Immunoscore (IS) which correlates with better prognosis in terms of disease-free survival and overall survival (OS). IC activation and consequent immunogenic TME requires high nutrient absorption and synthesis and accumulation of protein, lipid, and nucleotides. However, tumour cells (TCs) promote their own proliferation by stealing micronutrients to ICs, therefore leading to an immunosuppressive TME phenotype. This proof-of-concept protocol aims to assess whether a controlled enteral immune nutrition (EIN) supplementation can revert the TME in favour of ICs over TCs, in both auxotrophic and non-auxotrophic malignancies, witnessed by an increase in IS in surgical specimen over biopsy controls. Secondary endpoints are: (i) tumour regression grade assessment compared to historical data from FLOT4/AIO; (ii) combined proportion score ratio; (iii) relapse-free survival at 3 years and OS; and (iv) quality of life by the European Organisation for Research and Treatment of Cancer quality-of-life questionnaire (EORTC QLQ)-30. A more detailed explanation on background and rationale is provided on the Supplementary Material, available at https://doi.org/10.1016/j.esmogo.2023.100036. The background of this project lies in three main concepts: (i) the role of an immune-efficient tumour microenvironment (TME) [indirectly witnessed by the amount of activated lymphocytes (a-Ly) CD8+/CD4+], ranked as Immunoscore (IS), in keeping under control cancer development and tumour progression1Ren F. Zhao Q. Zhao M. et al.Immune infiltration profiling in gastric cancer and their clinical implications.Cancer Sci. 2021; 112: 3569-3584Crossref PubMed Scopus (34) Google Scholar, 2Pagès F. Mlecnik B. Marliot F. et al.International validation of the consensus immunoscore for the classification of colon cancer: a prognostic and accuracy study.Lancet. 2018; 391: 2128-2139Abstract Full Text Full Text PDF PubMed Scopus (1329) Google Scholar, 3Yun S. Koh J. Nam S.K. et al.Immunoscore is a strong predictor of survival in the prognosis of stage II/III gastric cancer patients following 5FU based adjuvant chemotherapy.Cancer Immunol Immunother. 2020; 70: 431-441Crossref PubMed Scopus (10) Google Scholar; (ii) the competition between immune cells (ICs) and tumour cells (TCs) in TME for micronutrients that affects lymphocyte differentiation as cytotoxic4Ricciardi S. Manfrini N. Alfieri R. et al.The translational machinery of human CD4(+) T cells is poised for activation and controls the switch from quiescence to metabolic remodeling.Cell Metab. 2018; 28: 895-906Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,5Pearce E.L. Poffenberger M.C. Chang C.H. Jones R.G. Fueling immunity: insights into metabolism and lymphocyte function.Science. 2013; 3421242454Crossref PubMed Scopus (944) Google Scholar; and (iii) the role of the enteral immune nutrition (EIN) in influencing the TME in favour of the cell-mediated immune response.6D'Ignazio A. Kabata P. Ambrosio M.R. et al.Preoperative oral immunonutrition in gastrointestinal surgical patients: how the tumour microenvironment can be modified.Clin Nutr ESPEN. 2020; 38: 153-159Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar,7Aida T. Furukawa K. Suzuki D. et al.Preoperative immunonutrition decreases postoperative complications by modulating prostaglandin E2 production and T-cell differentiation in patients undergoing pancreatoduodenectomy.Surgery. 2014; 155: 124e33Abstract Full Text Full Text PDF Scopus (85) Google Scholar Because of previous experiences showing that EIN can modify the TME IC infiltration, the I-SUPPLY trial aims to clarify its role in inducing an immune-efficient TME through an increase in IS, which eventually should lead to a better prognosis in terms of overall survival (OS)8Taniguchi K. Ota M. Yamada T. et al.Staging of gastric cancer with the Clinical Stage Prediction score.World J Surg Oncol. 2019; 17: 47Crossref PubMed Scopus (6) Google Scholar and disease-free survival (DFS).1Ren F. Zhao Q. Zhao M. et al.Immune infiltration profiling in gastric cancer and their clinical implications.Cancer Sci. 2021; 112: 3569-3584Crossref PubMed Scopus (34) Google Scholar This is a proof-of-concept, translational, prospective, non-randomized trial the primary endpoint (PE) of which is to assess whether a controlled EIN supplementation during the acme of the activity of cytotoxic neoadjuvant FLOT in the esophagogastric junction (EGJ) and gastric cancer (GC) can revert the TME in favour of ICs over TCs, in both auxotrophic and non-auxotrophic malignancies, witnessed by an increase in IS in surgical specimen over biopsy controls. We hypothesize that a supplementation of immune nutrients (IN) at the nadir of chemotherapy efficacy (maximum destruction of TCs) might leave IN more available for ICs so that, in a TME with less lactate, increased pH, and reduced hypoxia, they can migrate, activate, differentiate, and act at their best against TCs (Figure 1). As proof-of-concept, we expect an increase in IS in surgical specimens over biopsy controls. Secondary endpoints (SE) are (i) tumour regression grade assessment (using Becker regression criteria) compared to historical data from FLOT4/AIO9Al-Batran S.E. Hofh einz R.D. Pauligk C. et al.Histopathological regression after neoadjuvant docetaxel, oxaliplatin, fluorouracil, and leucovorin versus epirubicin, cisplatin, and fluorouracil or capecitabine in patients with resectable gastric or gastro-oesophageal junction adenocarcinoma (FLOT4-AIO): results from the phase 2 part of a multicentre, open-label, randomised phase 2/3 trial.Lancet Oncol. 2016; 17: 1697-1708Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar; (ii) combined proportion score ratio of the overall amount of programmed death-ligand 1-positive tumoral and non-tumoral cells (lymphocytes and macrophages) to the total number of viable TCs multiplied per 100,10Janjigian Y.Y. Shitara K. Moehler M. et al.First-line nivolumab plus chemotherapy versus chemotherapy alone for advanced gastric, gastroesophageal junction, and oesophageal adenocarcinoma (CheckMate 649): a randomised, open-label, phase 3 trial.Lancet. 2021; 398: 27-40Abstract Full Text Full Text PDF PubMed Scopus (1087) Google Scholar before and after EIN; (iii) relapse-free survival (RFS) at 3 years and OS. RFS will be calculated as the time from surgery to recurrence or death from any cause, and OS will be calculated as the time from surgery to death from any cause; 4) quality of life by the European Organisation for Research and Treatment of Cancer quality-of-life questionnaire (EORTC QLQ)-30. Patients with stage II-III GCs and EGJ (T2-T4 N0, any T N+, HER2 score 0), fit for standard neoadjuvant chemotherapy (CHT) of FLOT (each 2-week cycle of FLOT consisted of docetaxel 50 mg/m2 on day 1, oxaliplatin 85 mg/m2 on day 1, leucovorin 200 mg/m2 on day 1, and 5-fluorouracil 2600 mg/m2 as 24-h infusion on day 1), will start EIN from day 3 to day 8 of each cycle, twice a day, assuming such period of time as the main in which TCs are affected by cytotoxic effects of CHT. Exclusion criteria are stage IV disease, deficient mismatch repair (dMMR) tumours, immune nutrition before entering the study, chronic use of immunosuppressive drugs (corticosteroids, immunosuppressors like methotrexate or similar), and patients on total parenteral nutrition (NPT) (Table 1). After completing CHT and disease reassessment, in the absence of distant metastases, patients will undergo curative surgery (generally within 4 weeks after ending chemotherapy). A blood sample will be collected at baseline, after 4 weeks, and at the end of treatment to detect plasma CD4+/CD8+ ratio (CD4/CD8 ratio may provide information about immune activation, T-cell exhaustion, and T-cell replicative senescence) and interleukin (IL)-2, IL-4, and IL-6 circulating levels (Table 2). Body mass index (BMI) and nutritional risk score (NRS 2002), along with the presence of malnutrition, according to the Global Leadership Initiative on Malnutrition (GLIM) criteria, will be assessed at baseline and after ending treatment.Table 1Inclusion and exclusion criteriaInclusion criteria•Signed written informed consent obtained before any study-specific procedures•Histologically confirmed AJCC/UICC EGJ or gastric adenocarcinoma stage II and III, HER2 score 0 pMMR•No distant metastasis•Patients suitable for neoadjuvant standard chemotherapy FLOT (docetaxel 50 mg/m2 on day 1, oxaliplatin 85 mg/m2 on day 1, leucovorin 200 mg/m2 on day 1, and 5-FU 2600 mg/m2 as a 24-h infusion on day 1) with adequate bone marrow, liver, cardiac, and renal function (as for GCP)•Availability of biopsy specimen obtained before any oncologic treatment•Able to swallow or with enterostomy•Women of child-bearing potential and men must agree before study entry to use adequate contraception (hormonal or barrier method of birth control; abstinence) for the duration of study participation. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately. Men treated or enrolled in this protocol must also agree to use adequate contraception before the study enrolment and for the duration of study participation.•ECOG performance status ≤2•Age ≥18 yearsExclusion criteria•Distant metastasis at any site (stage IV disease)•Immune nutrition before entering the study•Chronic use of immunosuppressive drugs (corticosteroids, immunosuppressors like methotrexate or similar)•Patients on total parenteral nutrition (NPT)•Patients with any other condition or concurrent medical or psychiatric disease who, in the opinion of the investigator, are not eligible to enter the study or are unable to correctly follow the prescribed therapy according to the study protocol•Unavailability of biopsy specimen or patients who do not intend to provide specimen for the study•Pregnant or breastfeeding women•Patients ineligible for neoadjuvant chemotherapy of FLOT5-FU, 5-fluorouracil; AJCC, American Joint Committee on Cancer; ECOG, Eastern Cooperative Oncology Group; EGJ, esophagogastric junction; HER2, human epidermal growth factor receptor 2; GCP, good clinical practice; pMMR, proficient mismatch repair; UICC, Union for International Cancer Control. Open table in a new tab Table 2Secondary endpointsSecondary endpointsTRG assessment to be compared to historical data from FLOT4/AIO (using Becker regression criteria)CPS ratio of the overall amount of PD-L1-positive tumoral and non-tumoral cells (lymphocytes and macrophages) to the total number of viable tumour cells multiplied per 100, before and after EINRFS at 3 yearsOSQoL by EORTC QLQ-30Ancillary studyPlasma CD4+/CD8+ ratio collected at baseline, after 4 weeks, and at the end of treatmentCPS, combined proportion score; EIN, enteral immune nutrition; EORTC, European Organisation for Research and Treatment of Cancer; OS, overall survival; PD-L1, programmed death-ligand 1; QLQ, quality-of-life questionnaire; QoL, quality of life; RFS, relapse-free survival; TRG, tumour regression grade. Open table in a new tab 5-FU, 5-fluorouracil; AJCC, American Joint Committee on Cancer; ECOG, Eastern Cooperative Oncology Group; EGJ, esophagogastric junction; HER2, human epidermal growth factor receptor 2; GCP, good clinical practice; pMMR, proficient mismatch repair; UICC, Union for International Cancer Control. CPS, combined proportion score; EIN, enteral immune nutrition; EORTC, European Organisation for Research and Treatment of Cancer; OS, overall survival; PD-L1, programmed death-ligand 1; QLQ, quality-of-life questionnaire; QoL, quality of life; RFS, relapse-free survival; TRG, tumour regression grade. 4 mm-thick adjacent formalin-fixed paraffin-embedded sections will be stained with antibodies (Abs) against CD3+ and CD8+ and revealed with the ultraView Universal DAB IHC kit from Ventana. Slides will be digitalized at 10× magnification and 0.45 μm/pixel resolution. We plan to use image analysis software for automatic tumour detection, generation of the invasive margin (IM; 360 μm thick), and quantification of CD3+ and CD8+ stained cells. The IM, defined as a region of 360 μm width on each side of the frontier between malignant cells and peri-tumoral stroma, will be generated automatically. CD3+ and CD8+ T-cell densities will be converted into IS with pre-defined cut-offs. IS utilizes standardized percentile values (0%-100%). IS is categorized into two groups corresponding to IS Low and IS High (which gathers IS Intermediate [Int] and High) with a mean percentile of 0%-25%, >25%-70%, and >70%-100%, respectively. Moreover, to define the kind of tumour-infiltrating cells (TILs) and assess the amount of immune suppressive cells and programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) pathway, more IHC scoring will be evaluated: helper T cells CD4+, CD8 T memory (CD 103+), T regulatory cells (both CD4+ and Foxp3+/CD105+), and macrophages M1 (CD 68+) and M2 (CD 163+) (Table 3).Table 3Definition of primary endpoint: increase of IS in surgical specimen over biopsy controlsEventIS assessment in biopsy specimenIS Low (mean percentile of 0%-25%)IS High (Int >25%-70%, + High >70%-100%, respectively)IS assessment in surgical specimenIS Low (mean percentile of 0%-25%)IS High (Int >25%-70%, + High >70%-100%, respectively)Assessment of the amount of immune suppressive cells and PD-1-PD-L1 pathwayIHC scoring of helper T cells CD4+, CD8 T memory (CD 103+), T regulatory cells (both CD4+ and Foxp3+/CD105+), macrophages M1 (CD 68+) and M2 (CD 163+)Sample sizeThe required sample size, calculated by single proportion test, will be of 21 patients, assuming a probability alpha of 0.2 and beta of 0.2Expected IS increase20% increase in Intermediate/High IS gastric cancer over biopsy control (internal validation cohort), P value <0.05 considered statistically significantAssessment of prognostic value of IS (Low versus High)Univariate and multivariate analyses based on the Cox proportional hazard regression model. HRs and 95% CIs will be estimated for each valueCI, confidence interval; HR, hazard ratio; IHC, immunohistochemistry; IS, Immunoscore; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1. Open table in a new tab CI, confidence interval; HR, hazard ratio; IHC, immunohistochemistry; IS, Immunoscore; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1. We expect to find an IS increase in surgical specimens over biopsy. According to the historical controls, the rate of Intermediate/High IS is about 64%.11Jiang Y. Zhang Q. Hu Y. et al.Immunoscore signature. A prognostic tool in gastric cancer.Ann Surg. 2018; 267: 504-513Crossref PubMed Scopus (315) Google Scholar Considering an expected 20% increase in Intermediate/High IS GC over biopsy control (internal validation cohort), assuming a probability alpha of 0.2 and beta of 0.2, the required sample size will be 21 patients. For PE, a 'comparison of proportion test' will be carried out with a P value of <0.05 considered statistically significant, in order to detect an increase in the percentage of High IS among patients treated with EIN (estimated around 85%) compared to biopsy controls (estimated around 64%), assuming a probability alpha of 0.2 and beta of 0.2. OS and DFS curves will be described using the Kaplan–Meier method and the log-rank test will be utilized for survival analysis. The prognostic value of IS (Low versus High) will be assessed using univariate and multivariate analyses based on the Cox proportional hazard regression model. Hazard ratios (HRs) and 95% confidence intervals (CIs) will be estimated for each value. Statistical analysis will be carried out with the MedCalc statistical software package (MedCalc® v20.015). All P values will be two-sided and P < 0.05 will be considered as statistically significant. A blood sample will be collected at baseline, after 4 weeks, and at the end of treatment to detect plasma CD4+/CD8+ ratio (CD4/CD8 ratio may provide information about immune activation, T-cell exhaustion, and T-cell replicative senescence) and IL-2, IL-4, and IL-6 circulating levels. BMI and NRS 2002, along with the presence of malnutrition, according to the GLIM criteria, will be assessed at baseline and after ending treatment. Cancer development is associated with a tumour immune escape that can be resumed in two main mechanisms—firstly by the establishment of an immune suppressive TME with the prevalence of immune suppressive cells (Treg, M2 Macrophages, MDSCs), and secondly by the induction of immunosuppressive proteins like PD-L1 (in both TCs and some ICs), which can inhibit the effector function of cytotoxic T lymphocytes (CD8+/CD4 Th1). More importantly, emerging evidence indicates that TCs can suppress antitumour immune response by competing for and depleting essential nutrients or otherwise reducing the metabolic fitness of tumour-infiltrating ICs by creating an unfavourable TME through the 'Warburg effect'.4Ricciardi S. Manfrini N. Alfieri R. et al.The translational machinery of human CD4(+) T cells is poised for activation and controls the switch from quiescence to metabolic remodeling.Cell Metab. 2018; 28: 895-906Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar With EIN we assume to favour the induction of an immune-efficient TME by inducing a clonal selection of immune-efficient cells (CD8+, CD4Th1, M1 macrophages, B memory) taking advantage of the cytotoxic effect on TCs at the nadir of chemotherapy, when it is supposed that those will be less able to use micronutrients present in TME. It should eventually lead to a reduction in the number of ICs with an immunosuppressive phenotype (PD-L1+). EIN is strongly recommended in upper gastrointestinal cancer patients undergoing surgical resection,12Muscaritoli M. Arends J. Bachmann P. et al.ESPEN practical guideline: clinical nutrition in cancer.Clin Nutr. 2021; 40: 2898-2913Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar and previous studies demonstrated that it is not harmful in favouring cancer cell proliferation, being even associated with a better outcome in terms of OS and progression-free survival in an adjuvant setting.13Boisselier P. Kaminsky M.C. Thézenas S. et al.A double-blind phase III trial of immunomodulating nutritional formula during adjuvant chemoradiotherapy in head and neck cancer patients: IMPATOX.Am J Clin Nutr. 2020; 112: 1523-1531Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar The choice of administering EIN for a specific period comes from the assumption that there will be a synergic activity of cytotoxic effect of chemotherapy on TCs with a simultaneous gain for ICs in nutrient absorption, but also from previous experiences of low patient compliance when forced to introduce oral formulations for long. For what it concerns the PE, extensive literature has suggested that immune infiltrates in cancer are of clinical relevance. Most mature data come from the international consensus IS for the classification of colon cancer, while for GC the relationship between IS and outcome is still debated. In fact, it should be addressed whether the standard evaluation for IS is really representative of an immune-efficient TME or if we need to look for more markers of immune-efficient cells. Being a proof-of-concept trial, an issue of this trial can be the small sample size; however, we can get huge information on GC TME without asking patients to have more invasive procedures like biopsies (since that every step is framed in the standard of care of resectable GC). Moreover, if EIN proves to be effective in increasing IS in GC, it can lead to further investigation and a wider use of such strategy to improve patient's outcome, theoretically, for all malignancies. I-SUPPLY hypothesis is that a controlled EIN given during preoperative chemotherapy favours both a clonal selection of CD8+ cytotoxic lymphocytes and CD4+ T effectors that act as TH1h stimulating via ILs, interferon gamma, CD40L receptor interactions, and the establishment of an immune-efficient TME in both auxotrophic and non-auxotrophic malignancies, thus leading to an increase in GC immunogenic pattern.
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