Neoadjuvant serplulimab combined with chemotherapy for resectable oesophageal squamous cell carcinoma: a single-arm, phase 2 trial

Domain: neuroscience
Published: 2025-12-21
Editorial outcome: major_revision

Abstract
In this phase 2 trial, patients diagnosed with locally advanced oesophageal squamous cell carcinoma underwent three cycles of neoadjuvant therapy comprising serplulimab, carboplatin, and albumin paclitaxel. The primary endpoint was the pathological complete response assessed by two independent pathologists and was defined as no residual viable tumours at both the primary tumour site and all the lymph nodes. The secondary endpoints included adverse events and overall survival time. In total, 85.4% of patients experienced adverse events of any grade, and 4.2% reported Grade 3 adverse events; 45 patients underwent pathological evaluation, 14 (31.1%, 95% confidence interval, 18.2–46.6%) achieved pathological complete response, and the two-year survival rate was 88.3%. Imaging mass cytometry revealed that patients without pathological complete response presented immunosuppressive molecule expression, and expanded immunosuppressive fibroblast neighbourhoods. Here we show, serplulimab and chemotherapy are associated with modest antitumour activity and a tolerable safety profile in patients with locally advanced oesophageal squamous cell carcinoma. A phase 3 randomised trial featuring continued follow-up is essential for a formal evaluation. Clinical Trials.gov identifier: NCT05659251.

Introduction
The most prevalent histological form of oesophageal cancer is oesophageal squamous cell carcinoma (ESCC), which accounts for more than 80% of cases1,2. Because early-stage ESCC has few clinical symptoms, the majority of patients are often diagnosed at a more advanced stage, leading to relatively poor survival rates despite the application of multimodal treatment strategies3,4. Neoadjuvant chemoradiotherapy followed by surgery is the principal therapeutic approach used for potentially resectable ESCC5. However, recent multicentre evidence indicates that neoadjuvant chemoradiotherapy and neoadjuvant chemotherapy, followed by surgery, yield comparable 3-year survival rates of 64.1% and 54.9%, respectively6. In alignment with findings from the JCOG1109 NExT trial, the addition of radiotherapy to doublet chemotherapy did not lead to a significant increase in survival rates compared with doublet chemotherapy alone, and the 3-year survival rate was significantly greater for patients receiving triplet chemotherapy (72.1%) than for those receiving doublet chemotherapy (62.6%), with no significant difference observed in patients who underwent neoadjuvant chemoradiotherapy (68.3%)7. Therefore, there is a pressing need to explore additional multimodal neoadjuvant therapeutic strategies for the treatment of locally advanced ESCC. In recent years, immunotherapy for resectable ESCC patients has evolved from adjuvant treatment to neoadjuvant therapy8,9. This change offers a new treatment approach for patients with locally advanced ESCC. Theoretically, neoadjuvant immunotherapy, which functions by inhibiting the immune evasion of tumour cells and enhancing the capacity of the immune system to eradicate these cells through activation of the immune response10, has the potential to reduce the risk of postoperative recurrence and improve survival rates. Nevertheless, the considerable variability in treatment response presents a significant challenge, with the spatial heterogeneity of the tumour microenvironment (TME) and dynamic immunosuppressive mechanisms being critical determinants of immunotherapy efficacy11. Therefore, the integration of high-dimensional spatial omics is imperative for elucidating the mechanisms underlying the diverse responses to immunotherapy.

In patients with advanced ESCC, the combination of serplulimab (HLX10, a fully human anti-PD-1 antibody) and chemotherapy is associated with favourable antitumour activity and manageable safety as an initial treatment, as evidenced by ASTRUM-0072. Serplulimab combined with chemotherapy has been approved as a first-line treatment for advanced ESCC in China. In addition, the combination of immunotherapy with chemotherapy has been shown to have synergistic antitumour effects on ESCC in several studies12,13. Imaging mass cytometry (IMC) integrates mass spectrometry with cytometry to visualise and quantify multiple proteins at the single-cell level within tissue sections, offering valuable insights into the TME in cancer research14,15. Although the IMC technique has been utilised in neoadjuvant immunotherapy for locally advanced ESCC16, there remains a paucity of research employing this method to compare pathological complete response (PCR) and non-PCR patients. Furthermore, studies are needed to investigate the functional relationships between spatial multicellular interaction patterns and the potential identification of cell phenotypes that may predict the efficacy of immunotherapy in locally advanced ESCC.

Here, we show this phase 2 trial investigates the efficacy and safety of a combination of serplulimab with chemotherapy alongside subsequent minimally invasive oesophagectomy for locally advanced ESCC.

Results
Patient and surgery summary
At a total of 55 patients were evaluated between December 1, 2022, and October 31, 2024 (Fig. 1). Seven patients were excluded for having either unresectable tumours or a history of other tumours; 48 patients were successfully enroled in this study and received treatment. Three patients completed the neoadjuvant therapy but subsequently refused to receive surgery and declined to continue participating in the study. Thus, a total of 45 patients who underwent oesophagostomy were evaluated to determine pathological efficacy and survival outcomes.

As detailed in Table 1, the average age of the enroled patients was 64.5 years, with more than half (52.1%) of the patients being older than 65 years. Prior to neoadjuvant treatment, 27 patients (56.3%), 19 patients (39.6%), and 2 patients (4.2%) were diagnosed with clinical stages II, III, and IVA disease, respectively.

As shown in Table 2, the mean duration between the last neoadjuvant therapy and surgery was 33.7 days, and there were no surgical delays related to neoadjuvant treatment. A total of 28 patients (62.2%) underwent the minimally invasive Ivor‒Lewis procedure, whereas 17 patients (37.8%) underwent the minimally invasive McKeown procedure. The median operative duration was 240 minutes, with a median blood loss of 50 mL, and the median number of resected lymph nodes was 33. In addition, no patients required conversion to open surgery, and all patients successfully achieved R0 resection. The major postoperative complications included anastomotic leakage and vocal cord paralysis, each of which occurred in two patients (4.4%). One patient suffered from chylothorax and underwent reoperation, followed by unexpected admission to the intensive care unit. No mortality was observed within the 90-day postoperative period in this study. Following surgery, 46.7% of patients opted for maintenance therapy with serplulimab, 37.8% of patients selected a combination of serplulimab and chemotherapy, and 4.4% of patients selected a combination of serplulimab and radiotherapy as adjuvant treatment. In contrast, 11.1% of patients did not receive any further adjuvant therapy.

Efficacy
No patients experienced disease progression during neoadjuvant treatment. Among these patients, three patients (6.2%; 95% confidence interval [CI], 1.3–17.2%) achieved a complete response, 30 patients (62.5%; 95% CI, 47.4–76.0%) achieved a partial response, and 15 patients (31.2%, 18.7–46.3%) had stable disease. Therefore, the disease control rate was 100.0% (95% CI, 92.6–100.0%), and objective responses were observed in 33 patients (68.8%; 95% CI, 53.7–81.3%) (Table 2).

Among the 45 patients who underwent pathological evaluation (Fig. 2A), 19 (42.2%) patients had lymph node metastasis, and 22 (48.9%) patients had a lower stage than the clinical stage. Fourteen patients (31.1%; 95% CI, 18.2–46.6%) exhibited a PCR (ypT0N0), whereas the other 31 patients (68.9%; 95% CI, 53.4–81.8%) did not exhibit a PCR (Table 2). Among these patients, 4 patients exhibited PCR for the primary tumour but also had positive lymph nodes (ypT0N1-3). With respect to the data cut-off on April 1, 2025, the median follow-up duration was 18.1 months, during which 45 patients completed the follow-up. The median survival, event-free survival time and disease-free survival times were not reached. A total of four patients died, and three fatalities were attributed to distant metastasis (specifically, one due to liver metastasis and two due to multiple metastases). The overall survival rate at two years was 88.3% (Fig. 2B). Notably, no patients in the PCR group died, whereas the two-year survival rate in the non-PCR group was 84.3%, with no significant difference between the two groups (p = 0.219) (Fig. 2C). During follow-up, 2 patients experienced local recurrence, and 3 patients experienced distant metastasis. The event-free survival rate at two years was 82.4% (Fig. S1A), and the disease-free survival rate at two years was 82.7% (Fig. S1B).

Treatment-related AEs
Table 3 presents a summary of treatment-related adverse events (AEs) observed during neoadjuvant therapy. Overall, 85.4% (41 out of 48) of patients experienced AEs of any grade, whereas only 2 patients (4.2%) reported treatment-related AEs classified as Grade 3. The most frequently observed treatment-related AEs included anaemia (72.9%), decreased white blood cell count (47.9%), decreased neutrophil count (39.6%), nausea (37.5%), decreased appetite (25.0%), decreased platelet count (20.8%) and increased alanine aminotransferase (20.8%). The Grade 3 treatment-related AEs included one case each of anaemia and increased alanine aminotransferase. In this study, no immune-related AEs, including myositis, pneumonia, hypothyroidism, or pituitary dysfunction, were observed. No Grade 4 or 5 AEs were reported, and the AEs did not cause dose reductions or treatment discontinuation.

Immune landscape and cellular distribution characteristics
No significant differences were observed in the baseline demographic or clinical baseline data between the 10 patients in the PCR and non-PCR groups used for IMC (Supplementary Table 1). Automated cell segmentation of stained images using the DeepCell algorithm indicated that the non-PCR group presented a significantly greater cell density per unit area than the PCR group did (p = 0.032) (Fig. 3A–C). Both groups demonstrated substantial immune cell infiltration, which was predominantly localised in stromal regions (Fig. 3D, E, Fig. S2B, C). Despite the abundance of immune cells had no siginifcant difference between the two groups (Fig. S2D), the dense spatial distribution of immune cells was associated with elevated expression levels of immunosuppressive molecules in the non-PCR group when compared with PCR group, including HIF1A, CD163, and FoxP3 (Fig. 3F, G, Fig. S2E).

Multicellular composition and functional assessment
We used canonical immune markers to identify 25 distinct cell populations (Fig. 4A, Fig. S2A), which consisted of 13 non immune subsets (Fig. S3A) and 12 immune subsets (Fig. S3B). Proportional analysis indicated that regulatory T cells (Tregs) constituted the most abundant immune infiltrate in both groups. However, the non-PCR group was predominantly characterised by Ki67+ Tregs (Fig. 4B, C). Additionally, cellular infiltration frequency analysis revealed significantly greater prevalences of HIF1Ahi/Hsp90hi tumour cells, PDL1+Ki67+ tumour cells, FAP+ fibroblasts, Tregs, M2 macrophages, and MPO+ neutrophils in the non-PCR group than in the PCR group. In contrast, the numbers of αSMA+ fibroblasts, collagen I+ fibroblasts, and CD8+ Trm cells were significantly greater in the PCR group than in the non-PCR group (Fig. 4D).

Cell‒cell interactions and spatial neighbourhood characteristics
Figure 5A presents a quantitative analysis of the cell‒cell interaction intensities, identifying four distinct interaction modules between the non-PCR and PCR groups. In BOX1, epithelial cells characterised by high expression levels of HIF1A and HSP90 in the non-PCR group presented strong positive correlations with other epithelial cells. BOX2 shows that MPO+ CXCL10+ macrophages in non-PCR tumours engaged in robust interactions with tumour cells. BOX3 highlights a tightly interconnected network centred on CD44⁻FAP+ fibroblast subsets, demonstrating strong interactions with Ki67+ Tregs, Ki67- Tregs, M2 macrophages, and CD8+ TRMs in non-PCR patients. These interactions were attenuated or absent in PCR patients. BOX4 reveals that Tregs engaged in significant interactions with multiple immune cell populations, including neutrophils, B cells, and CD8+ TRMs and M2 macrophages. This type of cellular crosstalk, particularly through the Tregs–M2 macrophage axis, may sustain the immunosuppressive microenvironment in non-PCR patients.

We performed cellular neighbourhood analysis to explore functional synergies among nearby cell populations in the tissue microenvironment. By analysing the 20 nearest neighbours of each cell, we identified the structural characteristics of 10 distinct cellular neighbourhoods (CNs) (Fig. S4A). Both groups exhibited significant infiltration of CD8+ T cells (CN9); however, the PCR group exhibited a diffuse pattern of CD8+ T cell infiltration, whereas the non-PCR group presented CD8+ T cells spatially confined by immunosuppressive cell aggregates surrounding tumour nests (Fig. 5B, Fig. S5). The immunosuppressive cellular neighbourhood CN6, centred on FAP+ fibroblasts as spatial organisers. This domain was coenriched Ki67+ Tregs, TCF1hi Tregs, and PD-L1hi M2 macrophages (Fig. S4A), collectively forming a functional immunosuppressive hub. No significant difference in the quantity of tertiary lymphoid structures (TLSs) (CN4) was detected between the PCR and non-PCR groups (Fig. 5C). Nevertheless, functional profiling indicated that TLSs in the non-PCR group presented elevated expression levels of immunosuppressive molecules, including FOXP3, HIF1A, CD38, PD1, and HSP90 (Fig. S4B).

Methods
Patients
The protocol was reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University (Approval No. 2022–1022); this approval is available in the supplementary information. This study was registered with ClinicalTrials.gov (NCT05659251) on December 13, 2022. We screened patients aged 18–75 years with histologically confirmed ESCC. Prior to enrolment, standard examinations included magnetic resonance imaging of the brain, enhanced computed tomography scans of the chest and abdomen, neck ultrasound, and single-photon emission computed tomography bone scans. Attending physicians in the thoracic and oncology departments were responsible for staging these patients and evaluating their resectability. The eligibility criteria were as follows: stage II-IVA ESCC as per the 8th edition of the American Joint Committee on Cancer staging manual; provision of informed consent; Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; and no prior history of anticancer treatment. Patients who exhibited distant metastasis; who were receiving antiviral therapy (i.e., chronic hepatitis B virus carriers who needed antiviral therapy) at the time of this study; who had a history of radiotherapy, chemotherapy, or molecular targeted therapy; who had a history of other malignancies within the past five years; who had either active autoimmune diseases or immune deficiencies or a history of such issues; or who were intolerant to oesophagectomy were excluded from the study. Individuals of both sex who met the defined inclusion and exclusion criteria were allowed to participate in the trial.

Study design and treatment
This prospective, single-arm, phase 2 trial was initiated by an investigator and conducted at the Second Affiliated Hospital of Zhejiang University. After the participants provided informed consent, all patients underwent three cycles of neoadjuvant therapy, with each cycle administered at 21-day intervals. The treatment regimens included intravenous administration of serplulimab at a dosage of 4.5 mg/kg on Day 1, albumin-bound paclitaxel at a dosage of 260 mg/m² on Day 1, and carboplatin with an area under the drug concentration–time curve of 5 on Day 1. Following the completion of the final cycles of neoadjuvant therapy, minimally invasive oesophagectomy (Ivor–Lewis or McKeown) was performed 4–6 weeks later. The resection should extend at least 5 cm from the tumour’s origin, as determined by pre-neoadjuvant endoscopy. The initial follow-up was conducted one month post treatment, followed by subsequent assessments at three-month intervals for a duration of two years and, thereafter, at six-month intervals. Base on the final pathological assessment, the investigator and the participant could collaboratively determine the appropriate subsequent treatment. Patients were then eligible to receive serplulimab for a period of up to one year. For patients who achieved PCR, a single administration of serplulimab was recommended. In contrast, patients who did not achieve PCR were advised to undergo an additional cycle of chemotherapy along with continued immunotherapy. Furthermore, for patients with positive lymph nodes, radiotherapy was recommended.

Endpoints and assessments
The principal endpoint of this study was PCR, which was evaluated within one month after the surgery by two independent senior pathologists and defined as the absence of residual viable tumours at both the primary tumour site and all harvested lymph nodes.

The secondary endpoints included the R0 resection rate, AEs, objective response rate, event-free survival time, disease-free survival time and overall survival time. The R0 resection rate refers to the absence of cancer cells at the margin of the incision according to the pathological results. AEs in all treated patients were assessed and classified by the investigators according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0. Three weeks after the last neoadjuvant, enhanced thoracic computed tomography was performed, and the radiologist assessed the objective response rate according to the Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1. The surgical outcomes, including the operative duration, postoperative hospital length of stay, incidence of major complications, and 90-day mortality rate, were recorded. In addition, the overall survival time was calculated from the date of enrolment to either the last follow-up on April 1, 2025, or the date of death from any cause. The event-free survival time was calculated from the date of enrolment to the time of the first date of disease recurrence or date of death due to any cause, whichever occurred first. Disease-free survival time was calculated from the date of surgery to the time of the first date of disease recurrence or date of death due to any cause, whichever occurred first.

IMC preprocessing and clustering
Patients who achieved PCR at both the primary tumour site and the lymph nodes were assigned to the PCR group, whereas the other patients were assigned to the non-PCR group. Haematoxylin and eosin (HE) staining was performed to identify regions of interest with dense immune cell infiltration. After IMC staging, imaging was conducted using a Hyperion system (Fluidigm, USA) at 1 μm resolution. The raw data were segmented with DeepCell to extract spatial, morphological, and marker features. The IMC staining procedures, all the markers used in this study and data preprocessing are detailed in the supplementary methods.

Statistical analysis
The sample size calculation was performed with PASS version 21.0. Previous research has revealed a 9% (95% CI, 6–14%) PCR rate for neoadjuvant chemotherapy in patients with ESCC21. Thus, we hypothesised that the addition of serplulimab to chemotherapy may increase the PCR rate to 29%. At a significance level (α) of 0.025 (one-sided), a sample size of 45 participants was considered sufficient to achieve a statistical power of at least 94% to detect such a difference. To accommodate an anticipated dropout rate of 10% in this study, we planned to enrol a total of 50 participants.

Data analyses for clinical data were originally planned in the statistical analysis plane and were performed with the assistance of SPSS version 23.0 (IBM, Armonk, NY). The data are expressed as medians (ranges), means (standard deviations) or proportions (percentages) where suitable. The PCR rate was summarised for the efficacy-evaluable population as the number of patients (percentage), and 95% CI was employed to estimate the potential range for the PCR. The 95% CI was calculated using the Clopper–Pearson method. To evaluate the cumulative survival rate, the Kaplan–Meier method was used, and the log-rank test was used to compare differences between the PCR and non-PCR groups.

The IMC analysis in this study was a post hoc analysis that was not included in the initial research design. The imcRtools package (version 1.14.0) was used to create a spatial experiment object, integrating segmentation results with raw images. The multichannel images, nuclear masks and cytoplasmic masks were imported into a CytoImageList using Cytomapper (version 1.20.0). Single-cell expression data were associated with spatial coordinates and image metadata. FastMNN was used to correct batch effects. PhenoGraph was used to identify the subpopulations, and the cluster markers were determined using Wilcoxon tests.

The test interaction function in the imcRtools package was used to calculate empirical p values by permuting the cell labels 1,000 times. Colocalization was categorised as follows: interaction, in which the observed counts exceeded the 95th percentile of the null distribution; avoidance, in which the observed counts were below the 5th percentile; and cross-sample aggregation, in which the significance scores (−1, 0, and +1) were summed across images and visualised as a heatmap. To construct the spatial graph, a k-nearest neighbour graph (k=20, validated through spatial autocorrelation) was constructed based on cell centroids. In neighbourhood profiling, the aggregate neighbour function was used to compute phenotype composition vectors for the local neighbourhood of each cell. CN clustering was performed via k-means clustering (k=8, determined by the elbow method) to partition cells into CNs, which were subsequently functionally annotated. Data analysis for IMC was performed with the assistance of R version 4.4.2. A t test was used to evaluate the differences in cell proportions and functional marker expression between the PCR and non-PCR groups, and statistical significance was defined by a two-tailed p value less than 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors report a single arm phase 2 trial combining the immune checkpoint inhibitor serplulimab and chemotherapy in a\nhighly select group of patients with operable squamous cell cancer of the esophagus. The authors report the treatment as\nsafe and feasible and encouraging rates of pathologic complete response, R0 resection, surgical tolerance and short term\nfollow up of disease-free survival are reported. Increasingly pathologic complete response has not been shown to be a\nuseful surrogate endpoint in the evaluation of neoadjuvant treatment strategies, although it is appropriate for signal finding to\njustify a phase 3 trial. Problematic is the allowance for unclear reasons, in addition to planned postoperative immunotherapy,\nfor the arbitrary application of additional chemotherapy and radiotherapy. What was the justification for this variance which\nwill confound any interpretation of future outcomes of disease free and overall survival? Which patients received additional\nchemotherapy and why, and which patients (albeit a small minority) received radiotherapy? Was this determined by nodal\npositive count, pathologic response, or other issues? As with any phase 2 trial the authors admit phase 3 validation is\nrequired, is this planned? Why was this trial pursued, given prior trials looking at Camrelizumab added to preoperative\nchemotherapy? It is unclear how propensity score matched analyses can realistically and meaningfully be applied to a small\n48 patient phase 2 trial. The literature citation is inadequate and ignores one of the most salient phase 3 trials, NEXT from\nJapan. This trial used a chemotherapy triplet regimen of DCF prior to surgery, which was shown to be superior to doublet\nchemotherapy and superior to doublet chemotherapy plus radiotherapy. The path CR rate on this trial for DCF was 19%.\nThis key omitted study citation needs to be included and put in context. The correlative science, although of interest, is\nconfusing and complex. The conclusions drawn about the key questions raised by these observations, and potential targets\nof future drug development, are highly speculative. How can these targets be exploited as they are identified after\nchemoimmunotherapy treatment and surgery, arguably too late to act.\nSpecific comments are outlined below:\nIntroduction: Currently the only approved indication of immunotherapy added to the management of locally advanced\nesophageal squamous cancer is after patients receive chemoradiotherapy and surgery. This indication needs to be more\nteased out. The introduction ignores the pivotal NEXT trial which suggests three drug chemotherapy should be the preferred\npreoperative chemotherapy regimen in esophageal squamous cancer when the use of radiotherapy is not considered. The\nauthors cite the positive phase 3 trial in metastatic esophageal squamous cancer adding serplulimab to palliative\nchemotherapy, there are multiple other such studies published, and it is unclear what advantage this agent offers over\nsimilar agents.\nPatients: How were they staged? EUS, CT scan, PET scan, bronchoscopy? This needs to be stated.\nStudy design: Why was anything goes permitted after surgery including immunotherapy alone, immunotherapy plus\nchemotherapy, or immunotherapy, chemotherapy, and radiation therapy? This complicates any interpretation of disease\nfree and overall survival outcomes.\nResults: Patients: There is no mention of testing for PDL-1 status. This biomarker may predict response to immunotherapy\nagents. The failure to test or report this biomarker is a deficiency of the report.\nDiscussion: The mention of a study adding an EGFR targeted agent to immunotherapy and chemotherapy in metastatic\ndisease is irrelevant and should be deleted. The NEXT trial, as well as the CHECKMATE 577 study, require in depth\ndiscussion and reviews, the NEXT trial is completely ignored by these authors. It is not permissible to make comparisons of\nthis limited phase 2 trial to larger controlled phase 3 trials, and it is not permissible to claim results from this trial are\nsomehow superior. Some discussion about why additional chemotherapy and radiotherapy were permitted to administer, in\naddition to immunotherapy, is required. The “strategies” reported from the correlative science are overstated and with too\nmuch confidence, and the realistic pursuit of studying the proposed subsets with specific druggable targets is unlikely.\nWould similar alterations in the microenvironment be seen in patients getting chemotherapy alone without immunotherapy?\nThis is always the problem in a single arm, uncontrolled study without a control group for reference.", "reviewer_2": "This is a very interesting manuscript reporting several results for a phase 2 single-arm trial. I have some comments on the\nanalytical approaches and results presentation, which can hopefully help improving the quality of this manuscript.\n- Makor comment. It is a bit unclear where the different analyses originally included in the SAP are located, and which of the\nanalysis presented in the manuscript are post-hoc analyses. The Authors should clearly discuss all analyses originally\nincluded in SAP and their results, even if null. If some analyses from the SAP are not included in the manuscript, please\nmotivate why.\n- Even though included in the original SAP, it would be useful to mention why the Authors report a CI for a descriptive\nmeasure (the observed # of achieved PCR out the overall sample, which is a descriptive measure, reported both in abstract\nand text) and how this CI is calculated.\n- Lines 36-38. If available, given that these are the only strong positive results of the trial, it would be useful to include some\nstatistical testing or quantitative measure in the abstract to substantiate these claims.\n- Line 40, the abstract does not discuss safety in methods or results so I would not include in main abstracts conclusion\n- Line 103. Could you better explain what the mention of AUC in this section means?\n- Lines 108 to 149 include a lot of description of the statistical analyses conducted. I'd recommend clearly including analyses\ndescription under the \"Statistical Analysis\" section, which is currently located after line 149. Related to my first comment I'd\nalso recommend clearly distinguishing within this section what analysis were originally planned in the SAP (primary) and\nwhat were post-hoc (or secondary).\n- Line 110. When was PCR assessed? Over time? What was the follow-up, planned and observed?\n- Line 144. I'd recommend including more details (maybe in supplement) of these analyses. For example, how was the k=20\nselected? Through cross validation? The number of groups seem pretty high given the number of individuals.\n- Line 159. SPSS is included in software description but several R packages are also mentioned throughout the text. Please\nclarify what software was used for each analysis.\n- Line 178-181. Given that the Authors stress in the Methods section that both men and women were included, the result that\nall participants were actually men needs some further explanation. How does this affect generalizability?\n- Line 199 (and other instances). The heading of table 1 says \"baseline\", but it looks like this information on who achieved\ncomplete response is not a baseline characteristics. Please clarify in table and text what variables represent baseline\ninformation and what refer to later assessment.\n- Line 200. I do not see this result in Table 2 as indicated in the text.\n- Line 210. Figure 2C also include a p-value that is not described nor discussed in the text. Is this a logrank test? What is its\ninterpretation?\n- Lines 222-223. This result is expected since they are matching by propensity score. What would be useful to include,\ninstead, is some information on what variables were included in the propensity score model and how these were selected.\nThis can be added in a supplement.\n- The caption of Figure 1 could be expanded a bit.", "reviewer_3": "The paper presents the results a single-arm phase 2 trial assessing serplulimab, a fully human anti-PD1 antibody, combined\nto chemotherapy for the neoadjuvant treatment of locally- advanced resectable esophageal SCC (ESCC). They further\nexplored functional relationships between spatial multicellular interaction patterns and pathological response (pCR or not),\nusing imaging mass cytometry.\nSerplulimab has already shown favorable anti-tumor activity combined to chemotherapy in the treatment of advanced PD-L1\npositive (CPS>1) ESCC in a Chinese randomized, double-blind phase 3 study (ASTRUM007), showing improvement of OS\nas compared to placebo plus chemotherapy arm, and was approved in China following this study in first line setting of\nadvanced ESCC.\nThis study was a single-arm, phase 2 trial aiming at assessing the pathological complete response rate of patients with\nlocally advanced ESCC undergoing three cycles of neoadjuvant therapy comprising serplulimab, carboplatin, and albumin\npaclitaxel. Among the 45 patients who underwent oesophagostomy, all patients achieved R0\nresection, and 14 patients\n(31.1%, 95% CI, 18.2–46.6%) achieved PCR.\nUsing Imaging mass cytometry (IMC), authors further compared the heterogeneity of the tumour microenvironment (TME)\nbetween non-PCR and PCR patients. Compared with PCR patients, authors claimed that non-PCR patients presented\nincreased TME cell density, immunosuppressive molecule expression, enriched regulatory T cells/M2 macrophages,\nspatially confined CD8⁺ T cells, and expanded immunosuppressive fibroblast neighborhoods.\nThis paper show interesting anti-tumor activity and efficacy with the neo-adjuvant treatment assessed in this phase II\nincluding serplulimab.\nSome caveats need to be underlined :\nIn general, language needs to be verified by native speaker English; the manuscript needs to be rephrased for better\nintelligibility.\nThe results should be interpreted with caution, as some statements made by the author appear to overstate the findings,\nespecially regarding the results with IMC analyses.\nFor each part of the results in the manuscript, there is no conclusion made. Especially for IMC results, the results are mainly\ndescriptive results, presented as descriptive data without any bibliography, interpretation or conclusion on these results,\nmaking it difficult to find the value of these results.\nOther team have assessed IMC to elucidate the dynamics within the tumor microenvironment and correlation with\npathological response to neo-adjuvant chemotherapy plus immunotherapy :\nSpatial proteomic profiling elucidates immune determinants of neoadjuvant chemo-immunotherapy in esophageal\nsquamous cell carcinoma. Oncogene . 2024 Sep;43(37):2751-2767. doi: 10.1038/s41388-024-03123-z.\nManuscript would need major revisions at least before new revision, and could not be published as it is in Nature\nCommunication.\nIn more details:\n-Abstract needs to be formatted including secondary endpoints.\n-Authors need to cite paper describing other data with IMC analyses describing the TME in SCC of the esophagus, like this\npaper : Spatial proteomic profiling elucidates immune determinants of neoadjuvant chemo-immunotherapy in esophageal\nsquamous cell carcinoma. Oncogene . 2024 Sep;43(37):2751-2767. doi: 10.1038/s41388-024-03123-z.\n- Table 1 and 2 should be structured with one table resuming baseline characteristics, and one table resuming efficacy\nresults after neo-adjuvant treatment\nSome precision needs to be added:\n- At what timepoint CT-scan are done to evaluate objective response rate before surgery ?\n- Delay of the planned surgery need to be added as a surgical outcome\n- pCR rate according to clinical stage needs to be detailed\n- line 90 “who were receiving antiviral therapy » : to which antiviral therapy this sentence refer ?\n- line 93 : the sentence needs to be removed, it should not be stated like that in the methods : The sex of the participants was\ndetermined on the basis of the identity information provided by the patients. The study did not specifically assess or report\ngender, which is influenced by social and cultural contexts. Both male and female patients were included in the study.\n- line 156 : this is not a randomized study, authors cannot claim that they are planning to demonstrate the superiority of\nserplulimab in combination with chemotherapy over chemotherapy alone\n-line 191 : « Following surgery, 46.7% of patients opted for maintenance therapy with serplulimab, 37.8% of patients\nselected a combination of serplulimab and chemotherapy, and 4.4% of patients selected a combination of serplulimab and\nradiotherapy as adjuvant treatment » : the choice was leaved to the patient ? there were no standard of care following\nguidelines ? On what was based the decision of adjuvant treatment ? the pCR rate versus not ? the clinical stage ? (some\npatients with pCR still received adjuvant treatment). This needs to be detailed.\nThe type of adjuvant treatment needs to be put in perspective with the type of pathological response, and the initial clinical\nstage.\n- line 208, results should be presented with statistics, this result being non-significant.\n- in the section “Treatment-related AEs\", authors need to precise if there were any immune-related AEs.\n-line 222: it seems in Sup table 1 that stage II were more frequent among pCR patient than non pCR patients.\n- line 225: when authors state that results are significant, numerical values plus statistics needs to be detailed.\n- Figure S1A: a precision could be made to categorize the canonical markers : like myeloid, lymphocytes, epithelial, stroma\ncells\n- part showing IMC results should globally be enriched with bibliography.\n- figure 3: results should be presented with quantification on the total of patients analyzed (10 in pCR versus 10 in non-pCR),\nfor example for figure 3 D to 3G : results should be presented with quantifications; authors do not show sufficient data to\nclaim the results they describe for figure 3D to 3G concern all the pCR vers non pCR groups, because what is shown is only\nthe spatial distribution for 2 pCR patients and 2 non pCR patients.\n- Figure 5 A : legend should be more explicit, what are the statistical analyzes ?\n- line 247 for figure 5A: BOX3 do not highlight interaction with CD8 T cells as it is stated in the text.\n- line 249 : for BOX4, it is difficult to interpret that Tregs particularly interact with M2 macrophages, Tregs in BOX4 also\ninteract with Neutrophils, B cells, CD8 TRM cells.\n- Figure 5A: it is difficult from this figure to understand what the differences of cell-cell interactions between the two groups of\npCR are versus non pCR patients.\n- line 254, FIGURE 5B: results refer to CD8 T cell infiltration between group of patients with pCR versus non pCR?\nHowever, figure 5B show an example of one FFPE tissue from one patient of each group, so how can author conclude on\nthe whole group of pCR versus non pCR patients ?\n- line 257 : CN6 do not reflect only the FAP+ fibroblast cells, it is difficult to understand why authors are emphasizing this\npopulation, from figure S3A CN6 represents a group of CD8 TRM cells, Tregs, M2 cells.\n- line 262: results also show an increase in FOXP3 molecule reflecting Tregs in the non-PCR group.\n- line 267: patients were treated with immunotherapy plus chemotherapy, authors cannot state that the results they observe\nin the TME are due to immunotherapy treatment only, there is no comparison arm with patients treated with chemotherapy\nalone.\n- in the first part of discussion, authors should also discuss other publications assessing anti-PD1 molecules combined to\nchemotherapy as neoadjuvant treatment to describe pCR rates (like this paper : Spatial proteomic profiling elucidates\nimmune determinants of neoadjuvant chemo-immunotherapy in esophageal squamous cell carcinoma, and the papers cited\nafter describing survival rates ref 24, ref 25: what are the pCR rates in the publications ?).\n-line 290 : “ In addition, previous research has suggested that a decrease in viable tumour cells following neoadjuvant\ntherapy is associated with improved survival outcomes in cancer patients (20)”. Authors are stating that, but without putting\nthese findings in perspective with their results.\n- line 358 : results stated in the sentence are not clearly evidenced in the results presented in the paper. Figure 5B show\nsequestered spatial distribution of cD8 T cells in one sample of FPFPE slide from one non-pCR patient, no results show the\nsame tendency across all the non-pCR patients.\n-for the reproductivity of the results, authors should detailed the antibodies used for IMC analyses in the method"}, "author_response": "Point-by-point responses to the reviewers’ comments:\nFirst, we thank all the reviewers for their kind advice. Your comments are very\nimportant to our work. We have studied the comments carefully and have tried our best\nto revise our manuscript. The revised portions are marked in red (Track changes on).\nThe main corrections in the paper and the responses to the reviewers’ comments are as\nfollows and are marked in blue.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nReviewer #1: We would like to express my sincere thanks to you for your kind\ncomments. We agree with your suggestions, which have been of great help in improving\nthe quality of our manuscript. We have changed the manuscript based on your advice.\nThe authors report a single arm phase 2 trial combining the immune checkpoint\ninhibitor serplulimab and chemotherapy in a highly select group of patients with\noperable squamous cell cancer of the esophagus. The authors report the treatment as\nsafe and feasible and encouraging rates of pathologic complete response, R0 resection,\nsurgical tolerance and short term follow up of disease-free survival are reported.\nIncreasingly pathologic complete response has not been shown to be a useful surrogate\nendpoint in the evaluation of neoadjuvant treatment strategies, although it is appropriate\nfor signal finding to justify a phase 3 trial.\nProblematic is the allowance for unclear reasons, in addition to planned postoperative\nimmunotherapy, for the arbitrary application of additional chemotherapy and\nradiotherapy. What was the justification for this variance which will confound any\ninterpretation of future outcomes of disease free and overall survival? Which patients\nreceived additional chemotherapy and why, and which patients (albeit a small minority)\nreceived radiotherapy? Was this determined by nodal positive count, pathologic\nresponse, or other issues?\nResponse: Thank you for raising this important issue. For oesophageal squamous cell\n1\ncarcinoma, postoperative chemotherapy, radiotherapy, and chemoradiotherapy are\ncommon. Although adjuvant therapy remains a critical determinant of long-term\nsurvival, the completion of adjuvant chemotherapy presents substantial challenges for\npatients who often experience compromised health following radical oesophagectomy,\nas described in the JCOG1109 NEXT study. Given the exploratory nature of this study,\nthe primary objective was to investigate neoadjuvant treatment, and adjuvant therapy\nwas not restricted. Like other neoadjuvant immunotherapy approaches, immunotherapy\nmay be administered for one year to all patients to improve survival outcomes. This\ntext has been added to the Discussion section.\nBased on the final pathological assessment, the investigator and the participant could\ncollaboratively determine the appropriate subsequent treatment. Patients were then\neligible to receive serplulimab for a period of up to one year. For patients who achieved\nPCR, a single administration of serplulimab was recommended. In contrast, patients\nwho did not achieve PCR were advised to undergo an additional cycle of chemotherapy\nalong with continued immunotherapy. Furthermore, for patients with positive lymph\nnodes, radiotherapy was recommended. This text has been added to the Methods section.\nAs with any phase 2 trial the authors admit phase 3 validation is required, is this planned?\nResponse: We agree that randomized validation is essential. We are planning a\nmulticentre, randomized, phase III trial to formally evaluate the efficacy of neoadjuvant\nserplulimab plus chemotherapy compared with standard regimens in patients with\nresectable ESCC. This has been mentioned at the end of the Discussion section.\nWhy was this trial pursued, given prior trials looking at Camrelizumab added to\npreoperative chemotherapy?\nResponse: Although previous studies have evaluated the use of neoadjuvant\ncamrelizumab in ESCC, our trial is pioneering in its exploration of serplulimab, a\nstructurally distinct anti-PD-1 antibody, within this context. Serplulimab has\ndemonstrated superior efficacy in advanced ESCC, as evidenced by the ASTRUM-007\n2\ntrial, and is extensively utilized in China. This investigator-initiated exploratory study\naims to assess the efficacy and safety of combining serplulimab with chemotherapy,\nfollowed by minimally invasive oesophagectomy, for the treatment of locally advanced\nESCC. Our objective is to contribute to the development of multimodal neoadjuvant\ntherapeutic strategies for this condition.\nIt is unclear how propensity score matched analyses can realistically and meaningfully\nbe applied to a small 48 patient phase 2 trial.\nResponse: Thank you for your valuable suggestion. We apologize for the previously\nemployed methodology, which was neither meaningful nor appropriate. As the number\nof patients in clinical stage III was significantly higher in the non-PCR group compared\nto the PCR group, our intention was to minimize selection bias to the greatest extent\npossible. As you correctly noted, selection bias is an unavoidable challenge in small-\nsample clinical studies. We have removed this methodology and have provided a\ndetailed discussion of selection bias in the limitations section.\nThe literature citation is inadequate and ignores one of the most salient phase 3 trials,\nNEXT from Japan. This trial used a chemotherapy triplet regimen of DCF prior to\nsurgery, which was shown to be superior to doublet chemotherapy and superior to\ndoublet chemotherapy plus radiotherapy. The path CR rate on this trial for DCF was\n19%. This key omitted study citation needs to be included and put in context.\nResponse: In our previous version, the JCOG1109 NEXT trial was cited as reference\n21. We acknowledge that we did not provide a detailed elaboration of this significant\nstudy. In the current revision, we have cited this study and provided more\ncomprehensive information in both the Introduction and Discussion sections.\nThe correlative science, although of interest, is confusing and complex. The\nconclusions drawn about the key questions raised by these observations, and potential\ntargets of future drug development, are highly speculative. How can these targets be\n3\nexploited as they are identified after chemoimmunotherapy treatment and surgery,\narguably too late to act.\nResponse: Thank you for your valuable suggestion. We apologize for these highly\nspeculative conclusions raised by the IMC observed. This text has been deleted.\nSpecific comments are outlined below:\nIntroduction: Currently the only approved indication of immunotherapy added to the\nmanagement of locally advanced esophageal squamous cancer is after patients receive\nchemoradiotherapy and surgery. This indication needs to be more\nteased out. The introduction ignores the pivotal NEXT trial which suggests three drug\nchemotherapy should be the preferred preoperative chemotherapy regimen in esophageal\nsquamous cancer when the use of radiotherapy is not considered. The authors cite the\npositive phase 3 trial in metastatic esophageal squamous cancer adding serplulimab to\npalliative chemotherapy, there are multiple other such studies published, and it is\nunclear what advantage this agent offers over similar agents.\nResponse: Thank you for this suggestion. The Introduction has been revised, and the\nJCOG1109 NEXT trial has been cited. In addition, a more comprehensive discussion\nhas been included in the Introduction section.\nPatients: How were they staged? EUS, CT scan, PET scan, bronchoscopy? This needs\nto be stated.\nResponse: Prior to enrolment, standard examinations included magnetic resonance\nimaging of the brain, enhanced computed tomography scans of the chest and abdomen,\nneck ultrasound, and single-photon emission computed tomography bone scans.\nAttending physicians in the thoracic and oncology departments were responsible for\nstaging these patients and evaluating their resectability. This information has been\nadded to the Methods section.\nStudy design: Why was anything goes permitted after surgery including\n4\nimmunotherapy alone, immunotherapy plus chemotherapy, or immunotherapy,\nchemotherapy, and radiation therapy? This complicates any interpretation of disease\nfree and overall survival outcomes.\nResponse: Although adjuvant therapy remains a critical determinant of long-term\nsurvival, the completion of adjuvant chemotherapy presents substantial challenges for\npatients who often experience compromised health following radical oesophagectomy,\nas described in the JCOG1109 NEXT study. The CheckMate577 study demonstrated\nthat a 12-month regimen of nivolumab is efficacious for patients with residual disease\nafter neoadjuvant chemoradiotherapy and oesophagectomy. As an adjuvant treatment,\nnivolumab significantly enhanced disease-free survival (hazard ratio 0.69, 95% CI\n0.56–0.86, p<0.001) while maintaining manageable toxicity and exerting minimal\nimpact on health-related quality of life compared with placebo. Consequently, its use is\nnow recommended, as single-agent immune checkpoint inhibitors, in contrast to\ncytotoxic chemotherapy, present an appealing option in the adjuvant setting. Given the\nexploratory nature of this study, the primary objective was to investigate neoadjuvant\ntreatment. Like other neoadjuvant immunotherapy approaches, adjuvant therapy is not\nrestricted. Immunotherapy may be administered for one year to all patients without\nrestrictions to improve survival outcomes. This text has been added to the Discussion\nsection.\nResults: Patients: There is no mention of testing for PDL-1 status. This biomarker may\npredict response to immunotherapy agents. The failure to test or report this biomarker\nis a deficiency of the report.\nResponse: At the beginning of the experimental design, the expression level of PD-L1\nin tumours was not considered. The expression level of PD-L1 can serve as an initial\npredictor of the immunotherapy response in patients with ESCC. However, low or even\nnegative expression does not necessarily preclude the efficacy of immunotherapy. Post\nhoc analysis of the JUPITER-06 trial demonstrated that, despite low PD-L1 expression,\nthe combination of PD-1 antibodies and chemotherapy was superior to chemotherapy\n5\nalone in these patients. This deficiency was added to the limitations.\nDiscussion: The mention of a study adding an EGFR targeted agent to immunotherapy\nand chemotherapy for metastatic disease has been deleted. The JCOG 1109 NEXT\ntrial and CHECKMATE 577 study have been added to the Discussion section. A\ndiscussion of adjuvant therapy has been added. The “strategies” reported from the\ncorrelative science have been deleted. The limitations of single-arm and uncontrolled\nstudies have been described.\nReviewer #2: Thank you for your careful review and valuable suggestions. I have\nchanged my manuscript based on your advice.\nThis is a very interesting manuscript reporting several results for a phase 2 single-arm\ntrial. I have some comments on the analytical approaches and results presentation,\nwhich can hopefully help improving the quality of this manuscript.\n- Makor comment. It is a bit unclear where the different analyses originally included in\nthe SAP are located, and which of the analysis presented in the manuscript are post-hoc\nanalyses. The Authors should clearly discuss all analyses originally included in SAP\nand their results, even if null. If some analyses from the SAP are not included in the\nmanuscript, please motivate why.\nResponse: Thank you for this valuable suggestion. All the clinical data included in the\nSAP have been reported. The disease control, objective response, event-free survival\nand disease-free survival rates have been added to the Results section. A clear\ndistinction within this section regarding what analysis was originally planned in the\nSAP and what was post hoc has been added to the Statistical Analysis section.\n- Even though included in the original SAP, it would be useful to mention why the\nAuthors report a CI for a descriptive measure (the observed # of achieved PCR out the\noverall sample, which is a descriptive measure, reported both in abstract and text) and\nhow this CI is calculated.\nResponse: The PCR rate was summarized for the efficacy-evaluable population as the\nnumber of patients (percentage), and 95% CI was employed to estimate the potential\nrange for the PCR. The 95% CI was calculated using the Clopper‒Pearson method. This\ntext has been added to the Statistical Analysis section.\n- Lines 36-38. If available, given that these are the only strong positive results of the\ntrial, it would be useful to include some statistical testing or quantitative measure in the\n7\nabstract to substantiate these claims.\nResponse: The quantitative measure used was the pathological complete response (PCR)\nassessed by two independent pathologists and was defined as the absence of residual\nviable tumours at both the primary tumour site and all the lymph nodes; this information\nhas been added to the Abstract.\n- Line 40, the abstract does not discuss safety in methods or results so I would not\ninclude in main abstracts conclusion\nResponse: Thank you for this suggestion. As suggested by reviewer 3, the secondary\nend points, including some safety results, have been added to the Abstract.\n- Line 103. Could you better explain what the mention of AUC in this section means?\nResponse: We apologize for this misleading abbreviation. This term represents the area\nunder the drug concentration‒time curve. This abbreviation has been deleted.\n- Lines 108 to 149 include a lot of description of the statistical analyses conducted. I'd\nrecommend clearly including analyses description under the \"Statistical Analysis\"\nsection, which is currently located after line 149. Related to my first comment I'd\nalso recommend clearly distinguishing within this section what analysis were originally\nplanned in the SAP (primary) and what were post-hoc (or secondary).\nResponse: Thank you for this valuable recommendation. This information has been\nincluded in the Statistical Analysis section, clearly distinguishing what analyses were\noriginally planned in the SAP and what were post hoc.\n- Line 110. When was PCR assessed? Over time? What was the follow-up, planned and\nobserved?\nResponse: PCR results were evaluated within one month after surgery. The initial\nfollow-up was conducted one month posttreatment, followed by subsequent\nassessments at three-month intervals for a duration of two years and, thereafter, at six-\n8\nmonth intervals. This information has been added to the Methods section.\n- Line 144. I'd recommend including more details (maybe in supplement) of these\nanalyses. For example, how was the k=20 selected? Through cross validation? The\nnumber of groups seem pretty high given the number of individuals.\nResponse: Thank you for this suggestion. The detailed methodologies for the cellular\nneighbourhood (CN) analysis are now provided in the supplementary methods. The\nmethodology for defining Cellular Neighborhoods (CNs) using graph-based clustering\non spatial single-cell data, including the range of k values explored, draws directly from\nestablished work in the field, notably the foundational study by Schürch et al. (Cell,\n2020, doi: 10.1016/j.cell.2020.07.005.) titled \"Coordinated Cellular Neighborhoods\nOrchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front\". This and\nsubsequent studies utilizing similar technologies (e.g., IMC, CODEX, PhenoCycler)\nroutinely explore a range of k values, commonly including k=5, 10, 15, 20 (Mol Ther.\n2024, doi: 10.1016/j.ymthe.2024.03.023.). In our study, the selection of k=20 was not\ndetermined solely by cross-validation in the classical machine learning sense. Instead,\nit was the result of empirical evaluation across multiple k values (k=5, 10, 15, 20 were\ntested), guided by the biological interpretability and spatial coherence of the resulting\nCNs, as well as their correlation with known tissue morphology. Crucially, the CN\nstructure derived at k=20 provided the best morphological correspondence with the\nH&E images, suggesting it most accurately reflected the underlying tissue architecture\ncomplexity in our samples. This alignment with gold-standard pathology was a primary\nfactor in our final selection.\nAdditional analytical content has also been added to the supporting materials, including\ndata preprocessing, cellular clustering and annotation, and cell‒cell interaction scoring.\n- Line 159. SPSS is included in software description but several R packages are also\nmentioned throughout the text. Please clarify what software was used for each analysis.\nResponse: The software programs used have been added to the Statistical Analysis\n9\nsection.\n- Line 178-181. Given that the Authors stress in the Methods section that both men and\nwomen were included, the result that all participants were actually men needs some\nfurther explanation. How does this affect generalizability?\nResponse: The study did not specifically assess or report sex or gender. Individuals of\nboth sex who met the defined inclusion and exclusion criteria were allowed to\nparticipate in the trial. There was no analysis or post hoc sex- or gender-based analysis\nbecause of the small sample size of the study. This has been clarified in the reporting\nsummary checklist.\n- Line 199 (and other instances). The heading of table 1 says \"baseline\", but it looks\nlike this information on who achieved complete response is not a baseline\ncharacteristics. Please clarify in table and text what variables represent baseline\ninformation and what refer to later assessment.\nResponse: Thank you for your careful review. Tables 1 and 2 have been reformatted.\n- Line 200. I do not see this result in Table 2 as indicated in the text.\nResponse: The reference to Table 2 has been deleted in this sentence and moved to the\nnext sentence.\n- Line 210. Figure 2C also include a p-value that is not described nor discussed in the\ntext. Is this a logrank test? What is its interpretation?\nResponse: Yes, it is a log-rank test and has been added to the Statistical Analysis section.\nThe p value is described in the Results section.\n- Lines 222-223. This result is expected since they are matching by propensity score.\nWhat would be useful to include, instead, is some information on what variables were\nincluded in the propensity score model and how these were selected.\nThis can be added in a supplement.\nResponse: We apologize for the previously employed methodology, which was neither\nmeaningful nor appropriate, as reviewer 1 noted. As the number of patients in clinical\nstage III was significantly higher in the non-PCR group compared to the PCR group,\nour intention was to minimize selection bias to the greatest extent possible; selection\nbias is an unavoidable challenge in small-sample clinical studies. We have removed the\nmethodology and have provided a detailed discussion of selection bias in the limitations\nsection.\n- The caption of Figure 1 could be expanded a bit.\nResponse: The caption of Figure 1 has been expanded.\nReviewer #3: Thank you for your kind suggestions, which were very valuable to us. We\nhave revised our manuscript according to your suggestions.\nThe paper presents the results a single-arm phase 2 trial assessing serplulimab, a fully\nhuman anti-PD1 antibody, combined to chemotherapy for the neoadjuvant treatment of\nlocally- advanced resectable esophageal SCC (ESCC). They further explored functional\nrelationships between spatial multicellular interaction patterns and pathological\nresponse (pCR or not), using imaging mass cytometry.\nSerplulimab has already shown favorable anti-tumor activity combined to\nchemotherapy in the treatment of advanced PD-L1 positive (CPS>1) ESCC in a\nChinese randomized, double-blind phase 3 study (ASTRUM007), showing\nimprovement of OS as compared to placebo plus chemotherapy arm, and was approved\nin China following this study in first line setting of advanced ESCC.\nThis study was a single-arm, phase 2 trial aiming at assessing the pathological complete\nresponse rate of patients with locally advanced ESCC undergoing three cycles of\nneoadjuvant therapy comprising serplulimab, carboplatin, and albumin paclitaxel.\nAmong the 45 patients who underwent oesophagostomy, all patients achieved R0\nresection, and 14 patients (31.1%, 95% CI, 18.2–46.6%) achieved PCR.\nUsing Imaging mass cytometry (IMC), authors further compared the heterogeneity of\nthe tumour microenvironment (TME) between non-PCR and PCR patients. Compared\nwith PCR patients, authors claimed that non-PCR patients presented\nincreased TME cell density, immunosuppressive molecule expression, enriched regulatory T cells/M2\nmacrophages, spatially confined CD8⁺ T cells, and expanded immunosuppressive\nfibroblast neighborhoods.\nThis paper show interesting anti-tumor activity and efficacy with the neo-adjuvant\ntreatment assessed in this phase II including serplulimab.\nSome caveats need to be underlined :\nneeds to be rephrased for better intelligibility.\nResponse: We apologize for these highly speculative conclusions raised by the IMC\nresults. This text has been deleted.\nThe results should be interpreted with caution, as some statements made by the author\nappear to overstate the findings, especially regarding the results with IMC analyses.\nFor each part of the results in the manuscript, there is no conclusion made. Especially\nfor IMC results, the results are mainly descriptive results, presented as descriptive data\nwithout any bibliography, interpretation or conclusion on these results, making it\ndifficult to find the value of these results.\nResponse: We are sorry these highly speculative conclusions raised by the IMC results\nobserved and this part has been deleted.\nOther team have assessed IMC to elucidate the dynamics within the tumor\nmicroenvironment and correlation with pathological response to neo-adjuvant\nchemotherapy plus immunotherapy :\nSpatial proteomic profiling elucidates immune determinants of neoadjuvant chemo-\nimmunotherapy in esophageal squamous cell carcinoma. Oncogene . 2024\nSep;43(37):2751-2767. doi: 10.1038/s41388-024-03123-z.\nManuscript would need major revisions at least before new revision, and could not be\npublished as it is in Nature Communication.\nResponse: Our manuscript has been revised according to your and the other reviewers’\nsuggestions. Thank you again for your valuable suggestions.\nIn more details:\n-Abstract needs to be formatted including secondary endpoints.\nResponse: The secondary endpoints have been added to the Abstract.\n13\n-Authors need to cite paper describing other data with IMC analyses describing the\nTME in SCC of the esophagus, like this paper : Spatial proteomic profiling elucidates\nimmune determinants of neoadjuvant chemo-immunotherapy in esophageal squamous\ncell carcinoma. Oncogene . 2024 Sep;43(37):2751-2767. doi: 10.1038/s41388-024-\n03123-z.\nResponse: Thank you for highlighting this relevant work. We have now cited the\nsuggested paper by Wu et al. (Oncogene, 2024) in the revised manuscript in the\nIntroduction and Discussion sections [These findings align with those of Wu et al., who\nidentified S100A9⁺ macrophages in fibrotic regions as drivers of therapy resistance,\nemphasizing that an immunosuppressive TME spatially orchestrates treatment failure.].\nThis study provides valuable complementary spatial proteomic (IMC) insights into the\ntumour microenvironment of oesophageal squamous cell carcinoma (ESCC) under\nneoadjuvant chemoimmunotherapy, further strengthening the context of our findings.\n- Table 1 and 2 should be structured with one table resuming baseline characteristics,\nand one table resuming efficacy results after neo-adjuvant treatment\nResponse: Thank you for your careful review. Tables 1 and 2 have been structured with\none table resuming baseline characteristics and one table resuming efficacy results after\nneoadjuvant treatment.\nSome precision needs to be added:\n- At what timepoint CT-scan are done to evaluate objective response rate before surgery ?\nResponse: Three weeks after the last neoadjuvant, enhanced thoracic computed\ntomography was performed. This information has been added to the Methods section.\n- Delay of the planned surgery need to be added as a surgical outcome\nResponse: There were no surgical delays related to neoadjuvant treatment. This has\nbeen clarified in the Results section.\n14\n- pCR rate according to clinical stage needs to be detailed\nResponse: The clinical stage for each patient has been added to Figure 2A.\n- line 90 “who were receiving antiviral therapy » : to which antiviral therapy this\nsentence refer ?\nResponse: According to the protocol, patients who were receiving antiviral therapy (i.e.,\nchronic hepatitis B virus carriers who needed antiviral therapy) at the time of this study\nwere excluded. This information has been added to the Methods section.\n- line 93 : the sentence needs to be removed, it should not be stated like that in the\nmethods : The sex of the participants was determined on the basis of the identity\ninformation provided by the patients. The study did not specifically assess or report\ngender, which is influenced by social and cultural contexts. Both male and female\npatients were included in the study.\nResponse: This sentence has been deleted.\n- line 156 : this is not a randomized study, authors cannot claim that they are planning\nto demonstrate the superiority of serplulimab in combination with chemotherapy over\nchemotherapy alone\nResponse: The expression deemed inappropriate has been removed.\n-line 191 : « Following surgery, 46.7% of patients opted for maintenance therapy with\nserplulimab, 37.8% of patients selected a combination of serplulimab and\nchemotherapy, and 4.4% of patients selected a combination of serplulimab and\nradiotherapy as adjuvant treatment » : the choice was leaved to the patient ? there were\nno standard of care following guidelines ? On what was based the decision of adjuvant\ntreatment ? the pCR rate versus not ? the clinical stage ? (some patients with pCR still\nreceived adjuvant treatment). This needs to be detailed.\npathological response, and the initial clinical stage.\nResponse: Thank you for this valuable suggestion. Based on the final pathological\nassessment, the investigator and the participant could collaboratively determine the\nappropriate subsequent treatment. Patients were then eligible to receive serplulimab for\na period of up to one year. For patients who achieved PCR, a single administration of\nserplulimab was recommended. In contrast, patients who did not achieve PCR were\nadvised to undergo an additional cycle of chemotherapy along with continued\nimmunotherapy. Furthermore, for patients with positive lymph nodes, radiotherapy was\nrecommended. This text has been added to the Methods section, and a discussion of\nadjuvant treatment has been added to the Discussion section.\n- line 208, results should be presented with statistics, this result being non-significant.\n- in the section “Treatment-related AEs\", authors need to precise if there were any\nimmune-related AEs.\nResponse: In this study, no immune-related AEs, including myositis, pneumonia,\nhypothyroidism, or pituitary dysfunction, were observed. The AEs did not cause dose\nreductions or treatment discontinuation for immune therapy. This information has been\nadded to the Results section.\n-line 222: it seems in Sup table 1 that stage II were more frequent among pCR patient\nthan non pCR patients.\nResponse: We tried to minimize selection bias to the greatest extent possible,\nacknowledging that it remains an unavoidable challenge in small-sample clinical\nstudies. This has been described in detail in the limitations section. Additionally, for\npatients used for IMC, there was no significant difference in clinical stage was observed\nbetween patients who achieved PCR and those who did not.\n- line 225: when authors state that results are significant, numerical values plus statistics\n16\nneeds to be detailed.\nResponse: We completely agree that claims of significance must be supported with\nexplicit numerical values and statistical details. The p values have been added to the\nfigures.\n- Figure S1A: a precision could be made to categorize the canonical markers : like\nmyeloid, lymphocytes, epithelial, stroma cells\nResponse: We sincerely appreciate these insightful suggestions to enhance the clarity\nand scholarly rigor of our manuscript. We have implemented the following revisions:\nWe have now added a clear categorization system in the revised Figure S1A. All the\nmarkers are grouped into six major cell lineages:\nEpithelial markers: Pan-CK, E-cadherin;\nStromal markers: CD31, LEVE-1, vimentin, collagen I, FAP, and ɑSMA;\nPanimmune markers: CD45;\nLymphoid markers: CD3, CD4, CD8, CD20, CD45RO, Foxp3, and TCF1;\nMyeloid markers: CD11b, CD1c, CD11c, HLA-DR, CD14, CD16, CD68, CD163, and\nS100A9;\nFunctional markers: KI67, HLA-I, GranzymeB, PD1, PDL1, TOX, CD27, CD38,\nCD44, CD57, CD103, CXCL10, HIF1A, HSP90, MMP9, and CCR7.\n- part showing IMC results should globally be enriched with bibliography.\nResponse: We have revised the Discussion section and enriched the bibliography\nthroughout the IMC results.\n- figure 3: results should be presented with quantification on the total of patients\nanalyzed (10 in pCR versus 10 in non-pCR), for example for figure 3 D to 3G : results\nshould be presented with quantifications; authors do not show sufficient data to\nclaim the results they describe for figure 3D to 3G concern all the pCR vers non pCR groups,\nbecause what is shown is only the spatial distribution for 2 pCR patients and 2 non pCR\n17\npatients.\nResponse: We sincerely appreciate your constructive critique regarding the need for\ncohort-level quantification in Figure 3. To address this thoroughly, we have now\nperformed comprehensive quantitative analyses across all 20 patients (10 PCR patients\nvs. 10 non-PCR patients) with the key results presented in Supplementary Figure S2B-\nE. This validation confirmed the following:\n(1) Both groups showed predominant stromal-localized immune cell infiltration (Figure\n3D-E, and Figure S2B-C).\n(2) Despite the abundance of immune cells had no significant difference between the\ntwo groups (Figure S2D), the dense spatial distribution of immune cells was associated\nwith elevated expression levels of immunosuppressive molecules in the non-PCR group\nwhen compared with PCR group, including HIF1A, CD163, and FoxP3 (Figure 3F and\nG, Figure S2E).\n- Figure 5 A : legend should be more explicit, what are the statistical analyzes ?\nResponse: We have comprehensively revised the legend to Figure 5A and expanded the\ndescription of the statistical methodology to increase clarity.\n- line 247 for figure 5A: BOX3 do not highlight interaction with CD8 T cells as it is\nstated in the text.\nResponse: We sincerely apologize for the oversight and imprecise description. We have\nmodified the highlighted region in Figure 5A and amended the text as follows:\nBOX3 highlights a tightly interconnected network centred on CD44⁻FAP⁺ fibroblast\nsubsets, demonstrating strong interactions with Ki67⁺ Tregs, M2 macrophages, and\nCD8⁺ TRMs in non-PCR patients. These interactions were attenuated or absent in PCR\npatients.\n- line 249 : for BOX4, it is difficult to interpret that Tregs particularly interact with M2\nmacrophages, Tregs in BOX4 also interact with Neutrophils, B cells, CD8 TRM cells.\nThe text has been modified to accurately reflect the interaction patterns:\nBOX4 reveals that Tregs engaged in significant interactions with multiple immune cell\npopulations, including neutrophils, B cells, and CD8⁺ TRMs and M2 macrophages. This\ntype of cellular crosstalk, particularly through the Treg–M2 macrophage axis, may\nsustain the immunosuppressive microenvironment in non-PCR patients.\n- Figure 5A: it is difficult from this figure to understand what the differences of cell-\ncell interactions between the two groups of pCR are versus non pCR patients.\nResponse: We sincerely apologize for any lack of clarity in our presentation. This study\nemployed imaging mass cytometry (IMC) to investigate the mechanisms underlying\nsuboptimal responses to chemotherapy–immunotherapy combination treatment in non-\nPCR patients compared with PCR patients. Accordingly, our analysis of four annotated\ninteraction networks (BOX1–4) revealed consistently stronger cell‒cell interaction\nintensities in non-PCR patients:\nBOX1: Epithelial cell interaction network;\nBOX2: Macrophage interaction network;\nBOX3: FAP⁺ fibroblast interaction network;\nBOX4: Treg interaction network;\nThese cell populations have been previously implicated in establishing tumour\nimmunosuppressive microenvironments. By characterizing the interaction patterns\namong these immunosuppressive cells, we aimed to elucidate the underlying\nmechanisms of treatment resistance and guide subsequent research directions.\n- line 254, FIGURE 5B: results refer to CD8 T cell infiltration between group of patients\nwith pCR versus non pCR? However, figure 5B show an example of one FFPE tissue\nfrom one patient of each group, so how can author conclude on the whole group of pCR\nversus non pCR patients ?\nResponse: We sincerely appreciate this critical inquiry and apologize for the limitations\n19\nin our original sample presentation. Figure 5B was indeed designed to demonstrate\ndifferential CD8⁺ T-cell infiltration within the tumour microenvironment of PCR versus\nnon-PCR patients. In the initial manuscript, we presented the most representative single\nFFPE tissue section from each group. To address this concern, we have now included\nadditional results from supplementary patients in Supplementary Figure S5. These new\ndata exhibit consistent spatial distribution patterns with those of the original cases,\ncollectively demonstrating the following:\nPCR patients show diffuse CD8⁺ T-cell infiltration;\nNon-PCR patients display spatial exclusion of CD8⁺ T cells by immunosuppressive cell\naggregates surrounding tumour nests;\nThis reproducible spatial pattern supports our cohort-level conclusions in the main text.\n- line 257 : CN6 do not reflect only the FAP+ fibroblast cells, it is difficult to understand\nwhy authors are emphasizing this population, from figure S3A CN6 represents a group\nof CD8 TRM cells, Tregs, M2 cells.\nResponse: The text regarding CN6 has been revised as follows: the immunosuppressive\ncellular neighbourhood CN6, centred on FAP⁺ fibroblasts as spatial organizers. This\ndomain was coenriched with Ki67⁺ Tregs, TCF1hi Tregs, and PD-L1hi M2\nmacrophages (Figure S4A), collectively forming a functional immunosuppressive hub.\n- line 262: results also show an increase in FOXP3 molecule reflecting Tregs in the non-\nPCR group.\nResponse: As suggested, we have incorporated FOXP3 into the molecular profiling\nanalysis with expanded functional annotations, as follows: Nevertheless, functional\nprofiling indicated that TLSs in the non-PCR group presented elevated expression\nlevels of immunosuppressive molecules, including FOXP3, HIF1A, CD38, PD1, and\nHSP90 (Figure S4B).\n- line 267: patients were treated with immunotherapy plus chemotherapy, authors\n20\ncannot state that the results they observe in the TME are due to immunotherapy\ntreatment only, there is no comparison arm with patients treated with chemotherapy\nalone.\nResponse: The expression deemed inappropriate has been removed.\n- in the first part of discussion, authors should also discuss other publications assessing\nanti-PD1 molecules combined to chemotherapy as neoadjuvant treatment to describe\npCR rates (like this paper : Spatial proteomic profiling elucidates immune determinants\nof neoadjuvant chemo-immunotherapy in esophageal squamous cell carcinoma, and the\npapers cited after describing survival rates ref 24, ref 25: what are the pCR rates in the\npublications ?).\nResponse: The PCR rates for other publications assessing anti-PD1 molecules\ncombined with chemotherapy as neoadjuvant treatment have been added to the\nDiscussion.\n-line 290 : “ In addition, previous research has suggested that a decrease in viable\ntumour cells following neoadjuvant therapy is associated with improved survival\noutcomes in cancer patients (20)”. Authors are stating that, but without putting\nthese findings in perspective with their results.\nResponse: This finding in perspective with our results has been added.\n- line 358 : results stated in the sentence are not clearly evidenced in the results\npresented in the paper. Figure 5B show sequestered spatial distribution of cD8 T cells\nin one sample of FPFPE slide from one non-pCR patient, no results show the same\ntendency across all the non-pCR patients.\nResponse: We sincerely appreciate your rigorous critique regarding the sample\nrepresentation in Figure 5B. To address this concern comprehensively, we have now\nincluded multiple additional FFPE tissue sections from other patients in Supplementary\nFigure S5. These new results consistently validate the spatial distribution pattern\n21\noriginally observed. We thank you for this insightful comment, which has strengthened\nour evidence presentation.\n-for the reproductivity of the results, authors should detailed the antibodies used for\nIMC analyses in the method\n22"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors address most of the comments I asked about. However they still make no comment about the uncontrolled nature of post surgery treatment. Their results appear little to no different than the results of the NEXT trial giving triplet chemotherapy alone. Results in this study are similar to other phase 2 studies and the agent studied here appears to offer no advantage over other agents. Biomarker studies investigated only in patients getting investigational therapy are impossible to interpret as similar observations of biomarker analysis might be oberseved in non responders to neoadjuvant treatment in a control group not getting immunotherapy.", "reviewer_2": "All my comments have been successfully and comprehensively addressed.", "reviewer_4": "I enjoyed reading this article reporting the results of this interesting prospective phase II trial to investigate the efficacy of neoadjuvant immunochemotherapy using serplulimab and chemotherapy in patients with resectable ESCC. The primary endpoint was pathological complete response (PCR) rate for both the primary tumor and lymph nodes, which was 31.1%. They found that the neoadjuvant regimen had promising antitumor activity and an acceptable safety profile. They also evaluated changes in tumor microenvironment between non-PCR and PCR patients by using imaging mass cytometry, this will provide useful clinical information for resectable ESCC patients.\nThe manuscript is logically structured and clearly communicates the study's findings. I have outlined some minor points for your consideration to ensure the manuscript reaches its full potential upon publication.\n1.For the description of age in Line 99 and Table 1, would the use of median and range be more appropriate, given that the age data distribution is typically non-normal?\n2.The results presented in Lines 104-106 and Table 2 indicate that 62.2% of patients underwent the Ivor-Lewis procedure and 37.8% the McKeown procedure. However, the tumor location (upper, middle, or lower) is not presented. It is recommended to add these data. Additionally, the criteria for surgical procedure selection should be included in the Surgery subsection of the Methods.\n3.In lines 223-224 of the Discussion section, the order in which the studies are listed does not correspond to the order of their pCR rates. The true order is 43.2% for NEOCRTEC5010 trial, 38.5% for JCOG1109 trial, and 33.3%for FFCD9901 trial, respectively.\n4.In the 4 paragraph (line 230) of the Discussion section, where the prognosis of patients achieving pCR is mentioned. It is recommended to cite studies demonstrating that pCR patients have significantly better outcomes than non-pCR patients in esophageal cancer, such as:\nShen J, Kong M, Yang H, et al. Pathological complete response after neoadjuvant treatment determines survival in esophageal squamous cell carcinoma patients (NEOCRTEC5010). Ann Transl Med 2021; 9(20): 1516.\n5.The Abstract is structured in accordance with the journal's requirements. However, I have one minor concern: it should be mentioned in the final sentence of the Abstract that a phase Ⅲ randomized trial and longer follow-up are still needed."}, "author_response": "REVIEWERS' COMMENTS\nFirst, we thank all the reviewers for their time and kind advice. The revised portions are\nmarked in red (Track changes on). The main corrections in the paper and the responses\nto the reviewers’ comments are as follows and are marked in blue.\nReviewer #1 (Remarks to the Author):\nThe authors address most of the comments I asked about. However they still make no\ncomment about the uncontrolled nature of post surgery treatment. Their results appear\nlittle to no different than the results of the NEXT trial giving triplet chemotherapy alone.\nResults in this study are similar to other phase 2 studies and the agent studied here\nappears to offer no advantage over other agents. Biomarker studies investigated only in\npatients getting investigational therapy are impossible to interpret as similar\nobservations of biomarker analysis might be oberseved in non responders to\nneoadjuvant treatment in a control group not getting immunotherapy.\nResponse: Although adjuvant therapy remains a critical determinant of long-term\nsurvival, the completion of adjuvant chemotherapy presents substantial challenges for\npatients who often experience compromised health following radical oesophagectomy,\nas described in the JCOG1109 NEXT study. The CheckMate577 study demonstrated\nthat a 12-month regimen of nivolumab is efficacious for patients with residual disease\nafter neoadjuvant chemoradiotherapy and oesophagectomy. As an adjuvant treatment,\nnivolumab significantly enhanced disease-free survival (hazard ratio 0.69, 95% CI\n0.56–0.86, p<0.001) while maintaining manageable toxicity and exerting minimal\nimpact on health-related quality of life compared with placebo. Consequently, its use is\nnow recommended, as single-agent immune checkpoint inhibitors, in contrast to\ncytotoxic chemotherapy, present an appealing option in the adjuvant setting. Given the\nexploratory nature of this study, the primary objective was to investigate neoadjuvant\ntreatment. Like other neoadjuvant immunotherapy approaches, adjuvant therapy is not\nrestricted. Immunotherapy may be administered for one year to all patients without\nrestrictions to improve survival outcomes. This text has been added to the Discussion\nsection.\nThis investigator-initiated exploratory study aims to assess the efficacy and safety of\ncombining serplulimab with chemotherapy, followed by minimally invasive\noesophagectomy, for the treatment of locally advanced ESCC. Serplulimab has\ndemonstrated superior efficacy in advanced ESCC, as evidenced by the ASTRUM-007\ntrial, and is extensively utilized in China. Our objective is to contribute to the\ndevelopment of multimodal neoadjuvant therapeutic strategies for this condition.\nThe current study utilized IMC to provide a comprehensive analysis of the mechanisms\nunderlying immunity in conjunction with chemotherapy resistance, specifically in the\ncontext of comparing patients with pathological complete response (PCR) to those\nwithout (non-PCR). Identifying biomarkers that differentiate between responders and\nnon-responders prior to the initiation of treatment would facilitate the targeted\nadministration of therapy to patients likely to respond, while simultaneously offering\nalternative regimens to those unlikely to benefit, thereby preventing disease progression.\nTo rigorously assess these findings, phase 3 randomized trials with larger sample sizes\nare now warranted could contribute to the optimization of more reliable neoadjuvant\nimmunotherapy combined with chemotherapy treatment strategies for locally advanced\nESCC. This has been mentioned in the limitation part.\nReviewer #2 (Remarks to the Author):\nAll my comments have been successfully and comprehensively addressed.\nResponse: Thank you for reviewing our manuscript.\nReviewer #4 (Remarks to the Author):\nI enjoyed reading this article reporting the results of this interesting prospective phase\nII trial to investigate the efficacy of neoadjuvant immunochemotherapy using\nserplulimab and chemotherapy in patients with resectable ESCC. The primary endpoint\nwas pathological complete response (PCR) rate for both the primary tumor and lymph\nnodes, which was 31.1%. They found that the neoadjuvant regimen had promising\nantitumor activity and an acceptable safety profile. They also evaluated changes in\ntumor microenvironment between non-PCR and PCR patients by using imaging mass\ncytometry, this will provide useful clinical information for resectable ESCC patients.\nThe manuscript is logically structured and clearly communicates the study's findings. I\nhave outlined some minor points for your consideration to ensure the manuscript\nreaches its full potential upon publication.\n1. For the description of age in Line 99 and Table 1, would the use of median and range\nbe more appropriate, given that the age data distribution is typically non-normal?\nResponse: Thank you for this suggestion. The distribution of age is normal in this study.\nOne-Sample Kolmogorov-Smirnov Test\nAGE\nN 48\nNormal Parametersa,b Mean 64.52\nStd. Deviation 6.277\nMost Extreme Absolute .078\nDifferences Positive .076\nNegative -.078\nTest Statistic .078\nAsymp. Sig. (2-tailed) .200c,d\na. Test distribution is Normal.\nb. Calculated from data.\nc. Lilliefors Significance Correction.\nd. This is a lower bound of the true significance.\n2.The results presented in Lines 104-106 and Table 2 indicate that 62.2% of patients\nunderwent the Ivor-Lewis procedure and 37.8% the McKeown procedure. However, the\ntumor location (upper, middle, or lower) is not presented. It is recommended to add\nthese data. Additionally, the criteria for surgical procedure selection should be included\nin the Surgery subsection of the Methods.\nResponse: Thank you for this suggestion. Tumour location is presented in the Table 1. And the\ncriteria for surgical procedure have been added in the Methods part.\n3.In lines 223-224 of the Discussion section, the order in which the studies are listed\ndoes not correspond to the order of their pCR rates. The true order is 43.2% for\nNEOCRTEC5010 trial, 38.5% for JCOG1109 trial, and 33.3%for FFCD9901 trial,\nrespectively.\nResponse: Thank you for your carefully review. These has been revised as: trials such as\nNEOCRTEC5010 trial, JCOG1109 NExT trial and FFCD9901 trial reported PCR rates of\n43.2%, 38.5% and 33.3%, respectively\n4.In the 4 paragraph (line 230) of the Discussion section, where the prognosis of\npatients achieving pCR is mentioned. It is recommended to cite studies demonstrating\nthat pCR patients have significantly better outcomes than non-pCR patients in\nesophageal cancer, such as:\nShen J, Kong M, Yang H, et al. Pathological complete response after neoadjuvant\ntreatment determines survival in esophageal squamous cell carcinoma patients\n(NEOCRTEC5010). Ann Transl Med 2021; 9(20): 1516.\nResponse: This article has been added as references 23.\n5.The Abstract is structured in accordance with the journal's requirements. However, I\nhave one minor concern: it should be mentioned in the final sentence of the Abstract\nthat a phase Ⅲ randomized trial and longer follow-up are still needed.\nResponse: This has been added in the Abstract."}]

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