Abstract
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.
Key words: immunonutrition supplementation, Immunoscore, immune nutrients, gastric cancer, tumour microenvironment, perioperative chemotherapy
Graphical abstract
Specifications table
| Subject area | Medicine and Dentistry |
|---|---|
| More specific subject area | Immunonutrition gastric cancer |
| Name of your trial in progress | The I-SUPPLY trial |
| Trial design | This is a proof-of-concept, translational, prospective, non-randomized trial PE of which is to assess whether a controlled EIN supplementation during the acme of the activity of cytotoxic neoadjuvant FLOT in EGJ and gastric cancer can revert the TME in favour of ICs over TCs, witnessed by an increase in IS in surgical specimen over biopsy controls. Patients with stage II-III GCs and EGJ (T2-T4 N0, any T N+, HER2 score 0, pMMR), fit for standard neoadjuvant 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-FU 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. Considering an expected 20% increase in Intermediate/High IS gastric cancer over biopsy control (internal validation cohort), assuming a probability alpha of 0.2 and beta of 0.2, the required sample size will be of 21 patients. |
| Investigator Initiated Study (IIS)Period | Recruitment period: 24 months First inclusion planned: December 2023 Last inclusion: December 2025 Treatment period: 2 months for each patient Follow-up period: 5 years |
| Ethics | The current study has been approved by the Sardinian Regional Ethic Committee (Italy), and permission to conduct the study has been obtained from the management of all participating facilities. Written informed consent for participation will be obtained from all participants. |
| Value of the trial in progress |
|
CHT, chemotherapy; DFS, disease-free survival; EGJ, esophagogastric junction; EIN, enteral immune nutrition; GC, gastric cancer; HER2, human epidermal growth factor receptor 2; IC, immune cell; IL, interleukin; IS, Immunoscore; OS, overall survival; PE, primary endpoint; pMMR, proficient mismatch repair; TC, tumour cell; TME, tumour microenvironment; TRG, tumour regression grade.
Highlights
-
•
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.
Description of protocol
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.
Background and rationale
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 progression1, 2, 3; (ii) the competition between immune cells (ICs) and tumour cells (TCs) in TME for micronutrients that affects lymphocyte differentiation as cytotoxic4,5; and (iii) the role of the enteral immune nutrition (EIN) in influencing the TME in favour of the cell-mediated immune response.6,7 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)8 and disease-free survival (DFS).1
Objectives
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.
Figure 1.
EIN given during chemotherapy should favour a clonal selection of CD8+ cytotoxic lymphocytes and CD4+ T effectors that act as Th1 helper stimulating via interleukins (IL2 and IL4), interferon gamma and CD40L receptor interactions, the establishment of an immune-efficient TME characterized by M1 polarized macrophages, selection of B memory lymphocytes, and reduction of immunosuppressor Treg. Dots in yellow, blue and green indicated as immune micronutrients. EIN, enteral immune nutrition; TME, tumour microenvironment; APC, antigen-presenting cells; MDSCs, myeloid-derived suppressor cells.
Secondary endpoints (SE) are (i) tumour regression grade assessment (using Becker regression criteria) compared to historical data from FLOT4/AIO9; (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 100,10 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.
Methods
Trial design
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 1.
Inclusion and exclusion criteria
| Inclusion criteria |
|---|
|
|
|
|
|
|
|
|
|
| Exclusion criteria |
|---|
|
|
|
|
|
|
|
|
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.
Table 2.
Secondary endpoints
| Secondary endpoints |
|---|
| TRG 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 EIN |
| RFS at 3 years |
| OS |
| QoL by EORTC QLQ-30 |
| Ancillary study |
|---|
| Plasma CD4+/CD8+ ratio collected at baseline, after 4 weeks, and at the end of treatment |
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.
Tumour sampling, IS, and immunohistochemical (IHC) testing
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 3.
Definition of primary endpoint: increase of IS in surgical specimen over biopsy controls
| Event | |
|---|---|
| IS assessment in biopsy specimen | IS Low (mean percentile of 0%-25%) IS High (Int >25%-70%, + High >70%-100%, respectively) |
| IS assessment in surgical specimen | IS 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 pathway | IHC 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 size | The required sample size, calculated by single proportion test, will be of 21 patients, assuming a probability alpha of 0.2 and beta of 0.2 |
| Expected IS increase | 20% increase in Intermediate/High IS gastric cancer over biopsy control (internal validation cohort), P value <0.05 considered statistically significant |
| Assessment 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 value |
CI, confidence interval; HR, hazard ratio; IHC, immunohistochemistry; IS, Immunoscore; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.
Statistical considerations
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%.11 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.
Translational research
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.
Protocol validation
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’.4 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,12 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.13 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.
Conclusion
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.
Acknowledgements
The authors would like to thank the patients, their families, investigators, and the study team. The authors would also like to thank the Société des Produits Nestlé SA for providing the formula for EIN free of charges.
Funding
This a monocentric, investigator-initiated clinical trial. Funds for immunohistochemical (IHC) tests and laboratory analysis will be provided by the Medicine Department of Cagliari University (Hybrid Hub H2UB: Cellular Models and COMputational micro and nanotEcnologies for InnovAtive therapies—COMETA). Oral Formula for EIN will be provided by Société des Produits Nestlé SA as a donation (Impact Oral®) free of charges.
This study is also partially funded by the Italian Ministry of University and Research (no grant number), PNRR, mission 4, component 2, investment 1.3, (partnership extended to Universities, research centres, companies funding for base research) title HEAL ITALIA, project number PE00000019, CUP F53C22000750006 (MS University of Cagliari).
Disclosure
The authors have declared no conflicts of interest.
Supplementary data
References
- 1.Ren F., Zhao Q., Zhao M., et al. Immune infiltration profiling in gastric cancer and their clinical implications. Cancer Sci. 2021;112:3569–3584. doi: 10.1111/cas.15057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Pagès 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(10135):2128–2139. doi: 10.1016/S0140-6736(18)30789-X. [DOI] [PubMed] [Google Scholar]
- 3.Yun 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;70:431–441. doi: 10.1007/s00262-020-02694-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Ricciardi 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(6):895–906. doi: 10.1016/j.cmet.2018.08.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Pearce E.L., Poffenberger M.C., Chang C.H., Jones R.G. Fueling immunity: insights into metabolism and lymphocyte function. Science. 2013;342(6155) doi: 10.1126/science.1242454. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.D’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;38:153–159. doi: 10.1016/j.clnesp.2020.05.012. [DOI] [PubMed] [Google Scholar]
- 7.Aida 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(1):124e33. doi: 10.1016/j.surg.2013.05.040. [DOI] [PubMed] [Google Scholar]
- 8.Taniguchi K., Ota M., Yamada T., et al. Staging of gastric cancer with the Clinical Stage Prediction score. World J Surg Oncol. 2019;17:47. doi: 10.1186/s12957-019-1589-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Al-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;17:1697–1708. doi: 10.1016/S1470-2045(16)30531-9. [DOI] [PubMed] [Google Scholar]
- 10.Janjigian 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;398:27–40. doi: 10.1016/S0140-6736(21)00797-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Jiang Y., Zhang Q., Hu Y., et al. Immunoscore signature. A prognostic tool in gastric cancer. Ann Surg. 2018;267:504–513. doi: 10.1097/SLA.0000000000002116. [DOI] [PubMed] [Google Scholar]
- 12.Muscaritoli M., Arends J., Bachmann P., et al. ESPEN practical guideline: clinical nutrition in cancer. Clin Nutr. 2021;40:2898–2913. doi: 10.1016/j.clnu.2021.02.005. [DOI] [PubMed] [Google Scholar]
- 13.Boisselier 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;112:1523–1531. doi: 10.1093/ajcn/nqaa227. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.