First-line modified FOLFOX plus/minus nivolumab and Ipilimumab or FLOT plus nivolumab in advanced gastroesophageal adenocarcinoma: a phase II multi-cohort trial

Domain: chemistry
Published: 2026-02-27
Editorial outcome: minor_revision

Abstract
This multi-cohort study evaluates whether dual immune checkpoint inhibition with nivolumab and ipilimumab in parallel or sequentially, or triplet chemotherapy with nivolumab can enhance efficacy as first-line therapy for advanced or metastatic gastroesophageal adenocarcinoma. Patients are randomized 1:1 to Arm A (mFOLFOX + nivolumab 240 mg every two weeks + ipilimumab 1 mg/kg every six weeks in parallel or Arm B (mFOLFOX). Subsequently, patients are randomized 1:2 to Arm A1 (identical to Arm A) or Arm A2 (three cycles of mFOLFOX followed by nivolumab + ipilimumab). In Arm C all patients receive FLOT + nivolumab. Primary endpoint is progression-free survival. Secondary endpoints include overall survival and objective response rate. Median progression-free survival (months) is: Arm A/A1, 5.8; Arm A2, 4.0; Arm B, 6.6 and Arm C, 7.0. Toxicity is manageable across all arms, with higher rates in arms receiving dual checkpoint inhibition. Here we show that chemotherapy with dual checkpoint inhibition in parallel increases toxicity without improving efficacy, while FOLFOX followed by immunotherapy is insufficient. FLOT with nivolumab appears feasible and shows promising efficacy. ClinicalTrials.gov number NCT03647969

Introduction
Over the past decade, first-line treatment with PD-1 or PD-L1 blockade combined with standard chemotherapy regimen as a combination of a platinum drug (cisplatin or oxaliplatin) and a fluoropyrimidine (fluorouracil, capecitabine), has transformed the treatment landscape for HER2-negative metastatic gastroesophageal adenocarcinoma (GEA) (1; 2). CheckMate-649 was the first large-scale phase 3 randomized trial to evaluate nivolumab plus chemotherapy (nivo+CT) versus chemotherapy (CT) alone in patients with metastatic HER2-negative GEA and demonstrated a clinically meaningful improvement in overall survival in patients with PD-L1 combined positive score ≥5 (HR 0.70) and all randomized patients (HR 0.79) compared to CT alone, which led to FDA and EMA approval of nivo+CT in over 50 countries as the new standard of care.

Considerable effort is focused on how to best leverage immune checkpoint therapy. One strategy to enhance the efficacy of anti-PD-(L) 1 therapy is to combine it with the cytotoxic T-lymphocyte-associated protein (CTLA-4) immune checkpoint inhibitor ipilimumab (ipi), given that the two inhibitors impact the immune system through two independent, and possibly complementary, mechanisms of action. 1, 2

Nivolumab and ipilimumab have been proven to be effective in multiple solid tumors, 3–6 and demonstrated clinically meaningful antitumor activity with manageable safety profile in heavily pretreated patients with GEA. 7 However in first-line GEA the ipilimumab/nivolumab treatment arm did not improve outcome versus chemotherapy alone and was even closed early, owing to increased rate of adverse events and early deaths. 8 At the time Moonlight trial was designed, it was known, that treatment of metastatic melanoma with combination of anti-CTLA4 and anti-PD-1 therapy resulted in increased response rates and progression-free survival compared to single agent immunotherapy. 9 In metastatic esophagogastric cancer, combined immune checkpoint blockade may further improve the efficacy of single-agent anti–PD-1 therapy by avoiding tumor immune escape through synergistic T-cell antitumor activity. 10, 11 Until now the immunologic consequences of adding the CTLA-4 checkpoint inhibitor ipilimumab to combined nivolumab plus chemotherapy for patients with treatment naïve HER2 negative GEA have not been investigated.

Previous studies have shown that chemotherapy elicits antitumor effects through the immune system, which might result in increased immunotherapy activity. 12, 13

Given the known toxicities of classic chemotherapy regimens, we hypothesized that three cycles of induction chemotherapy followed by dual immunotherapy would provide early disease control while building on the durable survival benefit provided by nivolumab and ipilimumab, and minimize the side-effects that are associated with a full course of continuous chemotherapy. We therefore aimed to investigate 3 cycles induction chemotherapy with FOLFOX followed by nivolumab plus ipilimumab with optional FOLFOX re-induction versus continuous chemotherapy with nivolumab and ipilimumab.

Albeit nivolumab and pembrolizumab added to doublet chemotherapy prolong OS and PFS, long-term outcome for most patients remains poor. Even more, when administered as monotherapy, there is an increased early mortality with crossing survival curves with the checkpoint inhibitors as compared with chemotherapy. 14–16 This is most likely explained by the fact that patients need time to establish antitumoral immunity, while some patients experience early disease progression and death. This provides a rationale to test whether the intensification of chemotherapy using a triplet (FLOT) instead of the doublet, combined with only nivolumab would be more effective.

Here we report the results of the multi-cohort phase 2 MOONLIGHT trial (NCT03647969) with the goal to expedite the investigation of novel immunotherapy-based strategies in the metastatic first-line setting of Her2-negative GEA and evaluate whether (a) dual immune checkpoint inhibition with nivolumab (nivo) and ipilimumab (ipi), administered in parallel or sequentially, or (b) triplet chemotherapy combined with immune checkpoint monotherapy can enhance clinical outcomes for GEA.

Results
Patients and treatment
Between November 2018 and February 2022, 281 patients were screened. Overall, 262 patients were either randomized into Arm A (n=60), A1 (n=30), A2 (n=60) and B (n=60) or enrolled in Arm C (n=52) in 27 trial sites in Germany. Since the only difference between Arm A and A1 was the study period in which the patients were enrolled, and baseline characteristics between the two arms did not reveal any significant differences, the data of both arms were pooled for all analyses (Arm A/A1 n=90).

The median age ranged between 58 and 63 years between all arms and around two-third of the patients were male. In Arm A/A1 and Arm C the amount of patients with adenocarcinoma of the gastroesophageal junction (GEJ) was higher than of stomach, whereas in Arm A2 and B patients with stomach adenocarcinoma were represented in the same number as patients with GEJ. Overall, only one-third of the patients received prior resection of the primary tumor. Overall PD-L1 expression (CPS ≥1) was comparable between Arms A/A1, A2 and B, ranging between 38% and 47% and with 60% higher in Arm C (Table 1)

Doublet chemotherapy plus IO (Arm A/A1, A2) and chemotherapy alone (Arm B)
The median treatment duration was 5.1 months, 3.3 months and 4.4 months in Arm A/A1, A2 and B, respectively, with 5 (6%), 3 (5%) and 1 (2%) patients completing the protocol-defined maximum treatment duration of 24 months. Disease progression was the major reason of premature treatment discontinuation in all three arms, and 5–7% of the patients discontinued treatment due to unacceptable toxicity (Fig. 1). In Arm A2, 8 patients (13%) discontinued treatment during or directly after chemotherapy induction due to progression, death, unacceptable toxicity or patient’s request and did not start the following immunotherapy. In Arm A/A1 the median number of treatment cycles was 10 versus 8 in Arm A2 and the median number of nivolumab/ipilimumab cycles was 10/3 and 4/2 for Arm A/A1 and A2, respectively. Of note, the median number of oxaliplatin/5FU cycles was 7/10 vs. 3/3 for Arm A/A1 vs A2. 23 patients (38%) received chemotherapy re-induction in Arm A2. Median number of treatment cycles in Arm B was 9. Further information about treatment exposure is available in the appendix (Table S1).

Triplet chemotherapy plus IO (Arm C)
The median treatment duration in patients receiving FLOT in combination with nivolumab (Arm C) was 4.4 months and 5 patients (10%) completed the maximum treatment duration of 24 months. The most common reasons for treatment discontinuation were disease progression (56%) followed by patient’s wish (10%). Three patients (6%) discontinued treatment due to unacceptable toxicity (Fig. 1). The median number of completed therapy cycles was 9.5.

Efficacy
Nivolumab plus ipilimumab plus chemotherapy versus chemotherapy (Arm A/A1 vs B). The addition of doublet immunotherapy to mFOLFOX treatment did not result in improvement of clinical outcome of the patients. Patients in Arm A/A1 and Arm B showed comparable results regarding the clinical outcomes PFS rate at 6 months (48% [95%CI 37; 59] vs. 47% [95% CI 34; 60]), median PFS (5.8 months vs. 6.6 months; 95% CI 5.4; 7.6) and median OS (10.1 months vs. 12.5 months) (Fig. 2). The median follow-up time (range) of the patients was 9.9 months (0.8–48.1) and 10.8 months (1.1-19.4). Furthermore, the ORR in both arms was 46% and 47%, with six patients in Arm A/A1 and three patients in Arm B who achieved complete response (Table 2).

Nivolumab and ipilimumab plus chemotherapy did not improve survival compared to chemotherapy alone across most PD-L1 expression subgroups. In patients with PD-L1 combined positivity score (CPS) ≥1 median PFS for arms A/A1 (n=42) vs B (n=23) was 5.4 and 6.1 months, median OS was 9.2 vs. 12.5 months and median duration of response was 7.7 vs. 5.1 months. Similarly, in patients with PD-L1 CPS <1 for arms A/A1 (n=36) and B (n=24), median PFS (6.5 vs 5.6;) and median OS (9.9 vs. 8.4) was comparable between treatment arms (Fig. S1).

Nivolumab plus ipilimumab plus chemotherapy versus alternating approach (Arm A/A1 vs A2)
The parallel combination of immunotherapy with mFOLFOX (Arm A/A1) was compared with an alternating approach (Arm A2). Here, the PFS rate at 6 months was significantly higher in patients receiving parallel treatment compared to those receiving the sequential treatment (48% [95% CI 37; 59] vs. 30% [95% CI 19; 43], p=0.041). Also the median PFS (5.8 months vs. 4.0 months; (95% CI 3.6; 5.5) and OS (10.1 months vs. 7.6 months) were prolonged in Arm A/A1 compared to Arm A2, in which the median follow-up time was 7.5 months (1.4–45.0) (Fig. 2). Also ORR was increased in Arm A/A1 (46%) compared to Arm A2 (32%) (Table 2). Prespecified subgroup analysis for PD-L1 expression showed longer median PFS and OS in Arm A/A1 compared to A2, independent of the CPS cut-off <1 or ≥1. The Kaplan–Meier plots for PFS and OS are shown in the appendix (Supplementary Fig. S2).

FLOT plus nivolumab (Arm C)
In Arm C, evaluating patients treated with FLOT in combination with nivolumab, the PFS rate at 6 months was 46% [95% CI 32; 61], the median PFS was 7.0 months [95% CI 5.2; 9.5] and the median OS was 14.6 months [95% CI 7.4; 25.5], with a median follow-up time of 12.7 months (0.5–30.5). The ORR was 56%, where 5 (10%) patients achieved CR and 24 (46%) achieved PR (Fig. 2, Table 2). With respect to PD-L1 expression, median OS and PFS did change notably according to PD-L1 CPS <1 (n=13) vs ≥1 (n=34). Median PFS was 3.9 months for CPS <1 vs 6.6 months for CPS ≥1, as was median OS with 10.5 months vs 16.7 months, respectively (Supplementary Fig. S3).

Subsequent therapy
Subsequent therapy was received by 59% and 63% of patients in the nivolumab plus ipilimumab plus chemotherapy arm (A/A1) and chemotherapy alone arm (B), and by 65% of patients in the alternating nivolumab-plus-ipilimumab and chemotherapy arm (A2). 52% of patients in the FLOT plus nivolumab arm received further treatment.

Safety
Almost all patients in Arm A/A1, A2 and B had at least one AE, whereas the incidence of AEs classified as treatment-related with grade ≥3 was increased in Arm A/A1 compared to Arm A2 and B (74% vs. 45% vs. 45%). In addition, more patients in Arm A/A1 had an SAE (75% vs. 62% vs. 50%) and specifically the incidence of treatment-related SAEs grade ≥3 was clearly increased in arm A/A1 (42% vs. 17% vs. 18%). In Arm C, all patients had at least one AE and 67% and 23% of patients had at least one treatment-related AE grade ≥3 and treatment-related SAE grade ≥3, respectively.

Most common AEs in all arms were anorexia, constipation, diarrhea, fatigue, nausea, peripheral sensory neuropathy and vomiting (Table S2). Treatment-related AEs grade ≥3 which were more prevalent in Arm A/A1 compared to Arm A2 and B were neutrophil count decrease (37% vs. 18% vs. 17%), platelet count decrease (5% vs. 0% vs. 0%), sepsis (8% vs. 2% vs. 0%) and white blood cell decrease (9% vs. 5% vs. 5%).

The number of fatal SAEs reported was 16 in Arm A/A1, 11 in Arm A2 and 9 in Arm B. Of these, 5, 1 and 3, respectively, were classified as treatment-related: In Arm A/A1 autoimmune hepatitis, Lyell-syndrome, pneumonitis, sepsis and deterioration of general conditions, in Arm A2 sepsis and in Arm B lung infection. 3x Six fatal SAEs were reported for Arm C, with one of them (gastric perforation) classified as treatment-related (Table 3).

Methods
Ethics statement
The MOONLIGHT trial was conducted in accordance with the Declaration of Helsinki and complies with all relevant ethical regulations. The trial protocol, including all subsequent amendments, was reviewed and approved by the Ethikkommission der Landesärztekammer Hessen (approval number: 2019-1234-fAM) as coordinating ethics committee. In addition, ethical approval was obtained from the respective ethics committees at each participating center prior to patient enrollment. All participants provided written informed consent before inclusion into the study. This trial is registered with ClinicalTrials.gov under identifier NCT03647969.

This trial was an investigator initiated, interventional, prospective, randomized, open label, multicenter phase II trial followed by a non-randomized arm. Participants were adults (≥18 years) of any sex and ethnicity. Patients self-reported their sex and ethnicity; no information on gender was collected. Eligible patients had previously untreated, histologically confirmed inoperable, advanced or metastatic, HER2-negative gastric or gastro-oesophagal junction (GEJ) adenocarcinoma, an Eastern Cooperative Oncology Group (ECOG) performance status ≤1 and adequate haematological, hepatic and renal function parameters. Inclusion was regardless of PD-L1 expression- and microsatellite instability (MSI) status.

PD-L1 expression on viable tumor cells was assessed centrally using a laboratory developed immunohistochemical PD-L1 staining according to current guidelines using the primary antibody clone 28-8 (Ab205921).

Enrollment and randomization
Patients were randomly assigned in a 1:1 ratio into Arm A (mFOLFOX: Oxaliplatin 85 mg/m², leucovorin 400 mg/m2 and fluorouracil 400 mg/m² plus Nivolumab and Ipilimumab) and B (mFOLFOX). In a subsequent phase, patients were randomized in a 1:2 ratio into Arm A1 (identical to Arm A) and A2 (three cycles of induction chemotherapy with mFOLFOX followed by four administrations of nivolumab (Q2W) and two administrations of ipilimumab). Afterwards enrollment into Arm C (FLOT: Docetaxel 50 mg/m2, Oxaliplatin 85 mg/m², leucovorin 200 mg/m2 and fluorouracil 2600 mg/m² plus Nivolumab) was performed in a single arm design. (For details, see the Supplementary Information)

Study objective and endpoints
The primary objective was to determine the clinical performance of the experimental regimen. The primary endpoint was progression free survival according to RECIST v1.1 in Arm A vs. Arm B and PFS rate at 6 months (PFS@6) in Arm A2 and Arm C. Secondary objectives were to determine efficacy in terms of objective response rate (ORR) according to RECIST v1.1, overall survival (OS), PFS for Arm A1, A2 and C, PFS@6 for Arm A and Arm B, PFS and OS by PD-L1 expression status, as well as tolerability of the experimental regimen and Quality of Life.

Statistical analysis
Unless otherwise stated, the results were based on the intention-to-treat (ITT) population, which comprised all randomized or enrolled (Arm C) patients in the study. Since the only difference between Arm A and A1 was the study period in which the patients were enrolled, the data of both arms were pooled for explorative analyses if the baseline characteristics are balanced. Therefore, a pooled Arm A/A1 was used for all endpoints in comparison with B and A2, respectively. The results of Arm C were compared descriptively with the results of Arm A and B. More details on sample size calculation are provided in the data Supplementary Information section.

The primary endpoint PFS was estimated using Kaplan Meier techniques. Median PFS along with 95% confidence interval (CI) and hazard ratio (HR) for PFS along with 95% CI was calculated and the hypothesis testing was performed with a one-sided log-rank test at a one-sided significance level of 0.1.

Except for the primary endpoint, all parameters were evaluated in an explorative or descriptive manner, providing means, medians, interquartile and total ranges, standard deviations and/or confidence intervals, counts and proportions, or Kaplan-Meier curves, as appropriate for the respective data types.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Thank you for allowing me to reevaluate the manuscript. The authors hve responded adequajtely to my questions and I have\nno further comments.", "reviewer_4": "This multi-cohort study explores several novel first-line treatment strategies for advanced gastroesophageal\nadenocarcinoma. The manuscript is clearly written and the core findings—that parallel mFOLFOX+nivolumab+ipilimumab\nincreases toxicity without improving efficacy, that short FOLFOX induction followed by dual IO underperforms, and that\nFLOT+nivolumab is feasible with encouraging activity—are important for the field. The statistical analysis methods are\ngenerally appropriate for the study design, but several key areas require clarification and more rigorous presentation.\n1. What are the key patient characteristics? Are they age, sex, ECOG performance etc.? While Table 1 provides descriptive\nstatistics, it should be properly stated in the method section.\n2. Are the covariates balanced between the arms (e.g., love plots, SMDs etc.). While the rationale for pooling Arm A and A1\nis explained, authors should include a brief sentence in the results/supplementary section confirming that a comparison of\nbaseline characteristics between these two arms did not reveal any significant differences that would preclude pooling.\nThis is a valuable exploratory study that addresses important questions in the treatment of gastroesophageal\nadenocarcinoma. The authors have correctly identified many of the trial's limitations. However, a more rigorous presentation\nof the statistical findings, particularly regarding covariate balance. The paper should be accepted for publication after a\nminor revision addressing the points raised above.", "reviewer_5": "I had pointed out a number of limitations of this study that do not allow one to make any efficacy conclusions (only safety\ndata may be reliable), however, the authors only agreed to my comments and did not make any satisfactory revisions or\nresponses.\nDiscussing some items in the Discussion section does not solve the problems pointed.\nScientific rigor is lacking.", "reviewer_6": "In this revised paper, Lorenzen and colleagues have addressed the majority of the concerns, particularly the study\nlimitations, however a few issues remain unaddressed.\n1. For completeness, the TNM stages of the participants should be addressed in the text under methods and not just in the\nabstract\n2. I dont like the term \"aggressive tumor biology\" what does this mean? It should be removed", "reviewer_3": "The following points should be addressed when revising the manuscript:\n1. In the abstract, all abbreviations should be introduced before they are used (FLOT,\nFOLFOX).\n2. In the abstract, the tumor stages investigated in the study (UICC TNM Stages) should be\nlisted under \"Patients and Methods\".\n3. In the body of the text, all abbreviations should be introduced before they are used (FLOT,\nFOLFOX; GEA, EGA, HER2, CI, HR, GEJ...).\n4. In the body of the text and in table 1, the tumor stages investigated in the study (UICC TNM\nStages) should be given under \"Patients and Methods\".\n5. The introductory section of the manuscript is very detailed and should be shortened.\n6. The discussion should include an explanation of why FOLFOX was used initially as the\nbackbone of chemotherapy. Why wasn't FLOT used initially?"}, "author_response": "Point-by-point reply to the reviewers’ comments:\nReviewer 1:\nSummary of Key Results:\nThis multi-cohort trial explores the feasibility and preliminary efficacy of combining\nimmunotherapy (nivolumab ± ipilimumab) with chemotherapy (modified FOLFOX or FLOT) in\nfirst-line treatment of gastric and gastroesophageal junction adenocarcinoma. While the\nstudy offers relevant early signals, several limitations—especially regarding toxicity and\nsubgroup interpretation—should be addressed before drawing broad conclusions\nAU: Agree. The limitations are now pointed out in the key results section.\nOriginality and Significance:\nThe study investigates an important clinical question in the evolving treatment landscape.\nWhile the idea of chemoimmunotherapy combinations is not novel, this trial adds value through\nits exploration of sequencing and regimen choice. However, it should be noted that the current\nstudy’s contribution is exploratory rather than practice-changing\nAU: Agree. We included this statement in the discussion section\nData and Methodology:\nThe overall study design is appropriate for a signal-seeking phase II trial. However, clarity is\nneeded on key inclusion criteria such as PD-L1 CPS and MSI status. The rationale behind\ntreatment allocation—particularly the use of FOLFOX alone in one arm—requires further\njustification, especially considering evolving standards of care during the study period.\nFurthermore, it is important to clarify whether patients received complete FLOT regimens or\nmodified versions approximating FOLFOX.\nAU: Agree. Inclusion criteria were specified in the Material and Methods section. This is also\noutlined in the discussion section: „At the time MOONLIGHT was designed, data on the\neffectiveness of checkpoint inhibitors and their dependence on the level of PD-L1 expression\nwere preliminary. Therefore MOONLIGHT included patients independent of the PD-L1\nexpression status.\nAccording to the protocol patients received FLOT regimen and no modified versions.\nStatistical Approach and Uncertainty:\nWhile the manuscript adequately reports descriptive data, the small sample size limits the\nrobustness of subgroup analyses. In particular, conclusions related to PD-L1 positivity (e.g.,\nCPS ≥1 or ≥5) must be tempered. Use of statistical tests appears appropriate, but uncertainties\nshould be discussed more transparently, especially when interpreting trends without formal\nhypothesis testing.\nAU: According to the protocol subgroup analysis including PFS and OS by PD-L1 expression\nstatus were planned but cut-offs were not defined as CM649 results were not available at the\ntime of trial conception. Due to small number of patients in the prespecified cohorts, subgroup\nanalysis on different CPS- cut-offs were not performed.\nIn addition, results are now discussed more cautious and in line of a signal-seeking phase II\ntrial\nConclusions:\nThe conclusions should be more cautiously phrased, particularly regarding treatment benefit\nin patients with PD-L1 positivity or aggressive disease. Given the small cohort sizes and lack\nof control for evolving treatment standards, these claims are not yet supported by sufficiently\nrobust evidence. The potential benefit of FLOT plus immunotherapy should also be more\ncarefully weighed against its notable toxicity.\nAU: agree. The discussion section was re-written accordingly.\nSuggested Improvements:\n• Provide consistent data for patients with CPS ≥5 across analyses.\nAU: explained- too small subgroups\n• Clarify if and how patients with “aggressive disease” can be identified at baseline.\nAU: sentence was revised to “aggressive tumorbiology”\n• Reconsider or revise the statement that FLOT-treated patients may benefit from additional\nimmunotherapy based on PD-L1 status.\nAU: done\n• Include an acknowledgment in the Discussion regarding the relatively young age of the study\npopulation, which may limit generalizability.\nAU: we agree that the median age in the trial population is younger compared to real-world\nsetting, however, except for Arm A (58y) in the range of what is reported in other global\nregistration trials (61-63y) such as CM649 (median Age 61-63y), and KN 859 (median Age\n61y), and RATIONAL 305 (median Age 60y).\n• Address minor phrasing and grammar corrections as noted, especially regarding clarity of\nthe PD-L1 comparison with prior trials.\nAU: done\nReviewer 3:\nThe following points should be addressed when revising the manuscript:\n1. In the abstract, all abbreviations should be introduced before they are used (FLOT,\nFOLFOX).\nAU: Due to the limited number of characters and the well established chemo regimens, we\nwould refrain from explaining the abbreviations FLOT and FOLFOX in the abstract but\nexplained on the body text (seee point 3). Of course this can be implemented in the final version\n2. In the abstract, the tumor stages investigated in the study (UICC TNM Stages) should be\nlisted under \"Patients and Methods\".\nAU: Stage was specified in the abstract in the background section\n3. In the body of the text, all abbreviations should be introduced before they are used (FLOT,\nFOLFOX; GEA, EGA, HER2, CI, HR, GEJ...).\nAU: done. GEA and EGA mean the same and have been standardized under GEA. Details on\nchemotherapy are also given in the „supplementary methods“ section\n4. In the body of the text and in table 1, the tumor stages investigated in the study (UICC TNM\nStages) should be given under \"Patients and Methods\".\nAU: The trial was conducted exclusively in the metastatic stage, therefore the UICC/TNM stage\nis not applicable\n5. The introductory section of the manuscript is very detailed and should be shortened.\nAU: done\n6. The discussion should include an explanation of why FOLFOX was used initially as the\nbackbone of chemotherapy. Why wasn't FLOT used initially?\nAU: done- this is also explaint in the introduction. Triplet therapy with FLOT is not the\nrecommended First-line therapy according to national and international guidelines\nReviewer 4:\nThe manuscript addresses an important clinical question in the treatment of HER2-negative\ngastroesophageal adenocarcinoma (GEA), evaluating innovative combinations of immunotherapy\nand chemotherapy.The study design and rationale are relevant, and the multi-cohort approach\nallows comparison of different treatment strategies. However, I recommend the following revisions\nto enhance clarity and accessibility:\n1. Some abbreviations in the abstract, such as “pts” and “q2w,” are not immediately clear. Please\ndefine all abbreviations upon first use in the main text, including commonly used terms like OS, PFS,\nAE, and SAE, even if they appear in the abstract. Abbreviations should be introduced once and then\nused consistently throughout the manuscript.\nAU: done. See also reviewer 3 point 3\n2. Please add 95%CI for PFS in the abstract.\nAU: Due to the limited number of words in the abstract (150 max), we decided to not include\n95% CIs in the abstract but included it in the results section.\n3. For broader accessibility, consider briefly explaining key terms such as immune checkpoint\nblockade, HER2-negative, and the distinction between triplet and doublet chemotherapy. These\nterms, though common in oncology, may not be immediately clear to non-specialist readers. A\nconcise clarification would enhance readability for a broader biomedical audience.\nAU: thanks- we address this important point - it was already raised by previous reviewers\n4. In the phrase “NCTNCT03647969,” the prefix “NCT” appears twice. Please correct this to\n“NCT03647969.”\nAU: done\n5. The introduction is relevant and informative, but the narrative would benefit from improved\nstructure. Currently, it moves quickly between themes such as prior trial results, biological rationale,\ntreatment limitations, and the design for the MOONLIGHT trial, without clear paragraph breaks.\nRestructuring the introduction into logical sections would improve clarity and better convey the\nstudy’s significance.\nAU: done\n6. Ensure consistent formatting of figure and table references throughout the manuscript. For\nexample, use capitalized “Figure” and “Table” rather than alternating between “figure” and “Figure.”\nAU: done\n7. When reporting estimates such as PFS rates, median PFS, or OS in the Results section, it would\nenhance clarity to include the confidence intervals in standard notation—for example, “46% [95% CI,\n32–61]” rather than “46% [32;61].”\nAU: done\n8. I suggest - consider including a Restricted Mean Survival Time (RMST) causing a truncation time\npoint (e.g., 12 months or 18 months). This can provide a more comprehensive summary of survival\ndifferences. (Ref: Zhao, Lihui, Brian Claggett, Lu Tian, Hajime Uno, Marc A. Pfeffer, Scott D. Solomon,\nLorenzo Trippa, and Lee-Jen Wei. \"On the restricted mean survival time curve in survival\nanalysis.\" Biometrics 72, no. 1 (2016): 215-221.)\nAU: In this study, we prioritized traditional survival analysis methods—specifically Kaplan-\nMeier estimates (with median survival times), the log-rank test, and hazard ratios—as they\nprovide a comprehensive, clinically interpretable, and widely understood summary of survival\ndifferences. These methods preserve the full survival curve, thereby retaining long-term\ninformation without requiring an arbitrary truncation point, as would be necessary with\nRMST.\nAdditionally, RMST was not pre-specified in the Statistical Analysis Plan and thus was not\nincluded in the primary or secondary analyses\nReviewer 5:\nAs acknowledged in the manuscript, randomized and non-randomized arms with very limited\nnumber of patients creates considerable restrictions in figuring out results. This is starkly clear in\nvarious figures.\nIt would be inappropriate to combine non-randomized arm (C) with other groups.\nsample size is too small and does not conform with PDL-1 pos/neg dataset on thousands of patients\nthat already exists.\nAny conclusions other that safety will have considerable weakness.\nRationale to conduct such a confusing research design is entirely unclear and not justifiable.\nArm A2: very confusing. short course of FOLFOX (??) and IO duration left to the investigators.\nvery\nconfusing and weak plan.\nMS status not mentioned.\nAU: We fully agree with the reviewer's arguments We are aware of the limitations of the study\ndesign and have presented and discussed this openly and correctly. Nevertheless, we consider\nthe signals emerging from the results to be important for the future use of immunotherapies in\nthe first-line treatment of Her2 negative gastroesophageal adenocarcinoma, e.g. when it comes\nto discontinuing chemotherapy too early or combining it with triple chemotherapies such as\nFLOT or TFOX."}]

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