[Porphyromonas gingivalis infection facilitates immune escape of esophageal cancer by enhancing YTHDF2-mediated Fas degradation].

OBJECTIVE: To investigate the effect of Porphyromonas gingivalis (Pg) infection on immune escape of oesophageal cancer cells and the role of YTHDF2 and Fas in this regulatory mechanism. METHODS: We examined YTHDF2 and Fas protein expressions in esophageal squamous cell carcinoma (ESCC) tissues with and without Pg infection using immunohistochemistry and in Pg-infected KYSE150 cells using Western blotting. The interaction between YTHDF2 and Fas was investigated by co-immunoprecipitation (Co-IP). Pg-infected KYSE150 cells with lentivirus-mediated YTHDF2 knockdown were examined for changes in expression levels of YTHDF2, cathepsin B (CTSB), Fas and FasL proteins, and the effect of E64 (a cathepsin inhibitor) on these proteins were observed. After Pg infection and E64 treatment, KYSE150 cells were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the expressions of T cell-related effector molecules were detected by flow cytometry. RESULTS: ESCC tissues and cells with Pg infection showed significantly increased YTHDF2 expression and lowered Fas expression. The results of Co-IP demonstrated a direct interaction between YTHDF2 and Fas. In Pg-infected KYSE150 cells with YTHDF2 knockdown, the expression of CTSB was significantly reduced while Fas and FasL expressions were significantly increased. E64 treatment of KYSE150 cells significantly decreased the expression of CTSB without affecting YTHDF2 expression and obviously increased Fas and FasL expressions. Flow cytometry showed that in Pg-infected KYSE150 cells co-cultured with PBMCs, the expressions of Granzyme B and Ki67 were significantly decreased while PD-1 expression was significantly enhanced. CONCLUSION: Pg infection YTHDF2-dependently regulates the expression of Fas to facilitate immune escape of esophageal cancer and thus promoting cancer progression, suggesting the key role of YTHDF2 in regulating immune escape of esophageal cancer.

Correlation of PD-L1 expression with CD8+ T cells and oxidative stress-related molecules NRF2 and NQO1 in esophageal squamous cell carcinoma.

Oxidative stress and the immune microenvironment both contribute to the pathogenesis of esophageal squamous cell carcinoma (ESCC). However, their interrelationships remain poorly understood. We aimed to examine the status of key molecules involved in oxidative stress and the immune microenvironment, as well as their relationships with each other and with clinicopathological features and prognosis in ESCC. The expression of programmed death-ligand 1 (PD-L1), CD8, nuclear factor erythroid-2 related factor-2 (NRF2), and NAD(P)H quinone oxidoreductase 1 (NQO1) was detected using immunohistochemistry in tissue samples from 176 patients with ESCC. We employed both combined positive score (CPS) and tumor proportion score (TPS) to evaluate PD-L1 expression and found a positive correlation between CPS and TPS. Notably, PD-L1 expression, as assessed by either CPS or TPS, was positively correlated with both NRF2 nuclear score and NQO1 score in stage II-IV ESCC. We also observed a positive correlation between the density of CD8+ T cells and PD-L1 expression. Furthermore, high levels of PD-L1 CPS, but not TPS, were associated with advanced TNM stage and lymph node metastases. Moreover, both PD-L1 CPS and the nuclear expression of NRF2 were found to be predictive of shorter overall survival in stage II-IV ESCC. By using the Mandard-tumor regression grading (TRG) system to evaluate the pathological response of tumors to neoadjuvant chemotherapy (NACT), we found that the TRG-5 group had higher NRF2 nuclear score, PD-L1 CPS, and TPS in pre-NACT biopsy samples compared with the TRG-3 + 4 group. The NQO1 scores of post-NACT surgical specimens were significantly higher in the TRG-5 group than in the TRG 3 + 4 group. In conclusion, the expression of PD-L1 is associated with aberrant NRF2 signaling pathway, advanced TNM stage, lymph node metastases, and unfavorable prognosis. The dysregulation of PD-L1 and aberrant activation of the NRF2 signaling pathway are implicated in resistance to NACT. Our findings shed light on the complex interrelationships between oxidative stress and the immune microenvironment in ESCC, which may have implications for personalized therapies and improved patient outcomes.

Rechallenge with immune checkpoint inhibitors for advanced esophageal squamous cell carcinoma.

BACKGROUND: Despite the widespread use of immune checkpoint inhibitors (ICIs) in cancer treatment, disease progression remains common in the majority of patients and subsequent therapeutic options for this population are limited. ICI rechallenge has been validated favorably in terms of efficacy and safety in many cancer types, while data in esophageal squamous cell carcinoma (ESCC) are still lacking. METHODS: Clinical and pathological characteristics of advanced ESCC patients who received ICI rechallenge were collected retrospectively. The primary outcomes of interest were the disease control rate (DCR) and progression-free survival (PFS). Treatment-related adverse events were also recorded. We categorized patients into primary resistance and secondary resistance based on a 6-month disease control duration following the initial immunotherapy and further conducted exploratory analyses. RESULTS: A retrospective cohort study spanning January 2018 and October 2023, at Peking University Cancer Hospital, scrutinized 45 advanced ESCC patients undergoing two lines of ICI-based therapies (ICI-1 and ICI-2). The initial therapeutic approach involved combining ICIs with chemotherapy, and the ICI rechallenge primarily comprised ICIs and angiogenesis inhibitors. The median PFS for ICI-1 was 6.7 months with a disease control rate of 88.9 %. Following the ICI rechallenge, the median PFS and disease control rate remained at 3.2 months and 73.3 %, respectively. It is noteworthy that patients with secondary resistance to ICI-1 exhibited a higher 6-month PFS rate (29.6 % v.s. 11.1 %) in the ICI-2 stage. Any grade of treatment-related adverse events was observed in 29 (64.4 %) and 18 (40.0 %) patients at ICI-1 and ICI-2. The incidence of treatment-related adverse events in grades 3-4 was 9.1 % at ICI-1 and 9.1 % at ICI-2. CONCLUSION: ICI rechallenge may offer a potential survival benefit and a favorable safety profile for patients with ESCC who have progressed after initial immunotherapy. Patients exhibiting acquired resistance during initial immunotherapy are more likely to achieve prolonged disease control after undergoing rechallenge therapy. Prospective studies are required to further explore the optimal combined therapy and select targeted population.

Progress in second-line antibody therapies for advanced esophageal squamous cell carcinoma.

INTRODUCTION: The prognosis of advanced esophageal squamous cell carcinoma (ESCC) is poor. Although cytotoxic drugs have been widely used in advanced ESCC, several antibody agents have recently been reported to be effective. AREAS COVERED: Nivolumab and pembrolizumab are anti-PD-1 antibodies that improve immunosuppression by binding to programmed death-1 (PD-1), leading to an antitumor effect. Randomized phase III trials have found these immune checkpoint inhibitors (ICIs) to be effective as second-line treatment. ATTRACTION-3, which compared nivolumab monotherapy with taxane monotherapy in patients with previously treated advanced ESCC, reported prolonged overall survival in the nivolumab group. KEYNOTE-181 found that overall survival was longer in patients with PD-L1-positive ESCC who received second-line treatment with pembrolizumab than in those who received chemotherapy. Sym004 and amivantamab are antibodies that target the epidermal growth factor receptor and have demonstrated efficacy in the treatment of other tumors in recent phase I studies. Furthermore, clinical trials on antibody-drug conjugates such as enfortumab vedotin and DS-7300 for solid tumors are currently ongoing. EXPERT OPINION: The standard first-line treatments for patients with advanced ESCC contain ICIs. Therefore, drugs with different mechanisms of action that can overcome resistance to ICIs are needed as second-line or later-line treatments to improve clinical outcomes in these patients.

Enhancer demethylation-regulated gene score identified molecular subtypes, inspiring immunotherapy or CDK4/6 inhibitor therapy in oesophageal squamous cell carcinoma.

BACKGROUND: The 5-year survival rate of oesophageal squamous cell carcinoma (ESCC) is approximately 20%. The prognosis and drug response exhibit substantial heterogeneity in ESCC, impeding progress in survival outcomes. Our goal is to identify a signature for tumour subtype classification, enabling precise clinical treatments. METHODS: Utilising pre-treatment multi-omics data from an ESCC dataset (n = 310), an enhancer methylation-eRNA-target gene regulation network was constructed and validated by in vitro experiments. Four machine learning methods collectively identified core target genes, establishing an Enhancer Demethylation-Regulated Gene Score (EDRGS) model for classification. The molecular function of EDRGS subtyping was explored in scRNA-seq (n = 60) and bulk-seq (n = 310), and the EDRGS's potential to predict treatment response was assessed in datasets of various cancer types. FINDINGS: EDRGS stratified ESCCs into EDRGS-high/low subtypes, with EDRGS-high signifying a less favourable prognosis in ESCC and nine additional cancer types. EDRGS-high exhibited an immune-hot but immune-suppressive phenotype with elevated immune checkpoint expression, increased T cell infiltration, and IFNγ signalling in ESCC, suggesting a better response to immunotherapy. Notably, EDRGS outperformed PD-L1 in predicting anti-PD-1/L1 therapy effectiveness in ESCC (n = 42), kidney renal clear cell carcinoma (KIRC, n = 181), and bladder urothelial carcinoma (BLCA, n = 348) cohorts. EDRGS-low showed a cell cycle-activated phenotype with higher CDK4 and/or CDK6 expression, demonstrating a superior response to the CDK4/6 inhibitor palbociclib, validated in ESCC (n = 26), melanoma (n = 18), prostate cancer (n = 15) cells, and PDX models derived from patients with pancreatic cancer (n = 30). INTERPRETATION: Identification of EDRGS subtypes enlightens ESCC categorisation, offering clinical insights for patient management in immunotherapy (anti-PD-1/L1) and CDK4/6 inhibitor therapy across cancer types. FUNDING: This study was supported by funding from the National Key R&D Program of China (2021YFC2501000, 2020YFA0803300), the National Natural Science Foundation of China (82030089, 82188102), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-018, 2022-I2M-2-001, 2021-I2M-1-067), the Fundamental Research Funds for the Central Universities (3332021091).

ΔNp63-restricted viral mimicry response impedes cancer cell viability and remodels tumor microenvironment in esophageal squamous cell carcinoma.

Tumor protein p63 isoform ΔNp63 plays roles in the squamous epithelium and squamous cell carcinomas (SCCs), including esophageal SCC (ESCC). By integrating data from cell lines and our latest patient-derived organoid cultures, derived xenograft models, and clinical sample transcriptomic analyses, we identified a novel and robust oncogenic role of ΔNp63 in ESCC. We showed that ΔNp63 maintains the repression of cancer cell endogenous retrotransposon expression and cellular double-stranded RNA sensing. These subsequently lead to a restricted cancer cell viral mimicry response and suppressed induction of tumor-suppressive type I interferon (IFN-I) signaling through the regulations of Signal transducer and activator of transcription 1, Interferon regulatory factor 1, and cGAS-STING pathway. The cancer cell ΔNp63/IFN-I signaling axis affects both the cancer cell and tumor-infiltrating immune cell (TIIC) compartments. In cancer cells, depletion of ΔNp63 resulted in reduced cell viability. ΔNp63 expression is negatively associated with the anticancer responses to viral mimicry booster treatments targeting cancer cells. In the tumor microenvironment, cancer cell TP63 expression negatively correlates with multiple TIIC signatures in ESCC clinical samples. ΔNp63 depletion leads to increased cancer cell antigen presentation molecule expression and enhanced recruitment and reprogramming of tumor-infiltrating myeloid cells. Similar IFN-I signaling and TIIC signature association with ΔNp63 were also observed in lung SCC. These results support the potential application of ΔNp63 as a therapeutic target and a biomarker to guide candidate anticancer treatments exploring viral mimicry responses.

The role of JMJD2A in immune evasion and malignant behavior of esophageal squamous cell carcinoma.

OBJECTIVE: This study aimed to investigate the role of JMJD2A in radiotherapy tolerance of esophageal squamous cell carcinoma (ESCC). METHODS: The levels of H3K9me3 modification were analyzed in anti-PD-1 therapy non-responder or responder patients, and the expression differences of H3K9me3-related modifying enzymes were assessed in TCGA-ESCC and ICGC cohorts. Subsequently, JMJD2A was knocked down in ESCC cells using CRISPR-Cas9 or lentivirus-mediated shRNA, and changes in malignant behavior of ESCC cells were observed. RNA-seq, ATAC-seq, and ChIP-seq analyses were then conducted to investigate the genes and downstream signaling pathways regulated by JMJD2A, and functional validation experiments were performed to analyze the role of downstream regulated genes and pathways in ESCC malignant behavior and immune evasion. RESULTS: JMJD2A was significantly overexpressed in ESCC and anti-PD-1 therapy non-responders. Knockdown or deletion of JMJD2A significantly promoted the malignant behavior and immune evasion of ESCC. JMJD2A facilitated the structural changes in chromatin and promoted the binding of SMARCA4 to super-enhancers, thereby inducing the expression of GPX4. This resulted in the inhibition of radiation-induced DNA damage and cell ferroptosis, ultimately promoting the malignant behavior and immune evasion of ESCC cells. CONCLUSION: JMJD2A plays an indispensable role in the malignant behavior and immune evasion of ESCC. It regulates the binding of SMARCA4 to super-enhancers and affects the chromatin's epigenetic landscape, thereby promoting the expression of GPX4 and attenuating iron-mediated cell death caused by radiotherapy. Consequently, it triggers the malignant behavior and immune evasion of ESCC cells.

Prognostic value of genomic mutation signature associated with immune microenvironment in southern Chinese patients with esophageal squamous cell carcinoma.

The genomic landscape of esophageal squamous cell cancer (ESCC), as well as its impact on the regulation of immune microenvironment, is not well understood. Thus, tumor samples from 92 patients were collected from two centers and subjected to targeted-gene sequencing. We identified frequently mutated genes, including TP53, KMT2C, KMT2D, LRP1B, and FAT1. The most frequent mutation sites were ALOX12B (c.1565C > T), SLX4 (c.2786C > T), LRIG1 (c.746A > G), and SPEN (c.6915_6917del) (6.5%). Pathway analysis revealed dysregulation of cell cycle regulation, epigenetic regulation, PI3K/AKT signaling, and NOTCH signaling. A 17-mutated gene-related risk model was constructed using random survival forest analysis and showed significant prognostic value in both our cohort and the validation cohort. Based on the Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression (ESTIMATE) algorithm, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm, and the MCPcounter algorithm, we found that the risk score calculated by the risk model was significantly correlated with stimulatory immune checkpoints (TNFSF4, ITGB2, CXCL10, CXCL9, and BTN3A1; p < 0.05). Additionally, it was significantly associated with markers that are important in predicting response to immunotherapy (CD274, IFNG, and TAMM2; p < 0.05). Furthermore, the results of immunofluorescence double staining showed that patients with high risk scores had a significantly higher level of M2 macrophage than those with low risk scores (p < 0.05). In conclusion, our study provides insights into the genomic landscape of ESCC and highlights the prognostic value of a genomic mutation signature associated with the immune microenvironment in southern Chinese patients with ESCC.

Construction of a Novel Mitochondria-Associated Gene Model for Assessing ESCC Immune Microenvironment and Predicting Survival.

Esophageal squamous cell carcinoma (ESCC) is among the most common malignant tumors of the digestive tract, with the sixth highest fatality rate worldwide. The ESCC-related dataset, GSE20347, was downloaded from the Gene Expression Omnibus (GEO) database, and weighted gene co-expression network analysis was performed to identify genes that are highly correlated with ESCC. A total of 91 transcriptome expression profiles and their corresponding clinical information were obtained from The Cancer Genome Atlas database. A mitochondria-associated risk (MAR) model was constructed using the least absolute shrinkage and selection operator Cox regression analysis and validated using GSE161533. The tumor microenvironment and drug sensitivity were explored using the MAR model. Finally, in vitro experiments were performed to analyze the effects of hub genes on the proliferation and invasion abilities of ESCC cells. To confirm the predictive ability of the MAR model, we constructed a prognostic model and assessed its predictive accuracy. The MAR model revealed substantial differences in immune infiltration and tumor microenvironment characteristics between high- and low-risk populations and a substantial correlation between the risk scores and some common immunological checkpoints. AZD1332 and AZD7762 were more effective for patients in the low-risk group, whereas Entinostat, Nilotinib, Ruxolutinib, and Wnt.c59 were more effective for patients in the high-risk group. Knockdown of TYMS significantly inhibited the proliferation and invasive ability of ESCC cells in vitro. Overall, our MAR model provides stable and reliable results and may be used as a prognostic biomarker for personalized treatment of patients with ESCC.

HLA-A+ tertiary lymphoid structures with reactivated tumor infiltrating lymphocytes are associated with a positive immunotherapy response in esophageal squamous cell carcinoma.

BACKGROUND: Immune checkpoint blockade (ICB) therapy provides remarkable clinical benefits for multiple cancer types. However, the overall response rate to ICB therapy remains low in esophageal squamous cell carcinoma (ESCC). This study aimed to identify biomarkers of ICB therapy for ESCC and interrogate its potential clinical relevance. METHODS: We investigated gene expression in 42 treatment-naïve ESCC tumor tissues and identified differentially expressed genes, tumor-infiltrating lymphocytes and immune-related genes signatures associated with differential immunotherapy responses. We systematically assessed the tumor microenvironment using the NanoString GeoMx digital spatial profiler, single-cell RNA-seq and multiplex immunohistochemistry in ESCC. Finally, we evaluated the associations between HLA-A-positive tertiary lymphoid structures (TLSs) and patients' responses to ICB in 60 ESCC patients. RESULTS: Tumor infiltrating B lymphocytes and several immune-related gene signatures, such as the antigen presenting machinery (APM) signature, are significantly elevated in ICB treatment responders. Multiplex immunohistochemistry identified the presence of HLA-A+ TLSs and showed that TLS-resident cells increasingly express HLA-A as TLSs mature. Most TLS-resident HLA-A+ cells are tumor-infiltrating T (TIL-T) or tumor-infiltrating B (TIL-B) lymphocytes. Digital spatial profiling of spatially distinct TIL-T lymphocytes and single-cell RNA-seq data from 60 ESCC tumor tissues revealed that CXCL13-expressing exhausted TIL-Ts inside TLSs are reactivated with elevated expression of the APM signature as TLSs mature. Finally, we demonstrated that HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, are associated with a clinical benefit from ICB treatment for ESCC. CONCLUSIONS: HLA-A+ TLSs are present in ESCC tumor tissues. TLS-resident TIL-Ts with elevated expression of the APM signature may be reactivated. HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, may serve as biomarkers for ICB-treated ESCC patients.

CD276 regulates the immune escape of esophageal squamous cell carcinoma through CXCL1-CXCR2 induced NETs.

BACKGROUND: CD276 (B7-H3), a pivotal immune checkpoint, facilitates tumorigenicity, invasiveness, and metastasis by escaping immune surveillance in a variety of tumors; however, the underlying mechanisms facilitating immune escape in esophageal squamous cell carcinoma (ESCC) remain enigmatic. METHODS: We investigated the expression of CD276 in ESCC tissues from patients by using immunohistochemistry (IHC) assays. In vivo, we established a 4-nitroquinoline 1-oxide (4NQO)-induced CD276 knockout (CD276wKO) and K14cre; CD276 conditional knockout (CD276cKO) mouse model of ESCC to study the functional role of CD276 in ESCC. Furthermore, we used the 4NQO-induced mouse model to evaluate the effects of anti-CXCL1 antibodies, anti-Ly6G antibodies, anti-NK1.1 antibodies, and GSK484 inhibitors on tumor growth. Moreover, IHC, flow cytometry, and immunofluorescence techniques were employed to measure immune cell proportions in ESCC. In addition, we conducted single-cell RNA sequencing analysis to examine the alterations in tumor microenvironment following CD276 depletion. RESULTS: In this study, we elucidate that CD276 is markedly upregulated in ESCC, correlating with poor prognosis. In vivo, our results indicate that depletion of CD276 inhibits tumorigenesis and progression of ESCC. Furthermore, conditional knockout of CD276 in epithelial cells engenders a significant downregulation of CXCL1, consequently reducing the formation of neutrophil extracellular trap networks (NETs) via the CXCL1-CXCR2 signaling axis, while simultaneously augmenting natural killer (NK) cells. In addition, overexpression of CD276 promotes tumorigenesis via increasing NETs' formation and reducing NK cells in vivo. CONCLUSIONS: This study successfully elucidates the functional role of CD276 in ESCC. Our comprehensive analysis uncovers the significant role of CD276 in modulating immune surveillance mechanisms in ESCC, thereby suggesting that targeting CD276 might serve as a potential therapeutic approach for ESCC treatment.

Molecular Landscape and Clinical Implication of CCNE1-amplified Esophagogastric Cancer.

Cyclin E overexpression as a result of CCNE1 amplification is a critical driver of genomic instability in gastric cancer, but its clinical implication is largely unknown. Thus, we integrated genomic, transcriptomic, and immune profiling analysis of 7,083 esophagogastric tumors and investigated the impact of CCNE1 amplification on molecular features and treatment outcomes. We identified CCNE1 amplification in 6.2% of esophageal adenocarcinoma samples, 7.0% of esophagogastric junction carcinoma, 4.2% of gastric adenocarcinoma samples, and 0.8% of esophageal squamous cell carcinoma. Metastatic sites such as lymph node and liver showed an increased frequency of CCNE1 amplification relative to primary tumors. Consistent with a chromosomal instability phenotype, CCNE1 amplification was associated with decreased CDH1 mutation and increased TP53 mutation and ERBB2 amplification. We observed no differences in immune biomarkers such as PD-L1 expression and tumor mutational burden comparing CCNE1-amplified and nonamplified tumors, although CCNE1 amplification was associated with changes in immune populations such as decreased B cells and increased M1 macrophages from transcriptional analysis. Real-world survival analysis demonstrated that patients with CCNE1-amplified gastric cancer had worse survival after trastuzumab for HER2-positive tumors, but better survival after immunotherapy. These data suggest that CCNE1-amplified gastric cancer has a distinct molecular and immune profile with important therapeutic implications, and therefore further investigation of CCNE1 amplification as a predictive biomarker is warranted. SIGNIFICANCE: Advanced gastric cancer has a relatively dismal outcome with a 5-year overall survival of less than 10%. Furthermore, while comprehensive molecular analyses have established molecular subtypes within gastric cancers, biomarkers of clinical relevance in this cancer type are lacking. Overall, this study demonstrates that CCNE1 amplification is associated with a distinct molecular profile in gastric cancer and may impact response to therapy, including targeted therapy and/or immunotherapy.

Immune cell patterns before and after neoadjuvant immune checkpoint blockade combined with chemoradiotherapy in locally advanced esophageal squamous cell carcinoma.

BACKGROUND: Neoadjuvant immune checkpoint blockade (ICB) combined with chemoradiotherapy offers high pathologic complete response (pCR) rate for patients with locally advanced esophageal squamous cell carcinomas (ESCC). But the dynamic tumor immune microenvironment modulated by such neoadjuvant therapy remains unclear. PATIENTS AND METHODS: A total of 41 patients with locally advanced ESCC were recruited. All patients received neoadjuvant toripalimab combined with concurrent chemoradiotherapy. Matched pre- and post-treatment tissues were obtained for fluorescent multiplex immunohistochemistry (mIHC) and IHC analyses. The densities and spatial distributions of immune cells were determined by HALO modules. The differences of immune cell patterns before and after neoadjuvant treatment were investigated. RESULTS: In the pre-treatment tissues, more stromal CD3 + FoxP3 + Tregs and CD86+/CD163 + macrophages were observed in patients with residual tumor existed in the resected lymph nodes (pN1), compared with patients with pCR. The majority of macrophages were distributed in close proximity to tumor nest in pN1 patients. In the post-treatment tissues, pCR patients had less CD86 + cell infiltration, whereas higher CD86 + cell density was significantly associated with higher tumor regression grades (TRG) in non-pCR patients. When comparing the paired pre- and post-treatment samples, heterogeneous therapy-associated immune cell patterns were found. Upon to the treatment, CD3 + T lymphocytes were slightly increased in pCR patients, but markedly decreased in non-pCR patients. In contrast, a noticeable increase and a less obvious decrease of CD86 + cell infiltration were respectively depicted in non-pCR and pCR patients. Furthermore, opposite trends of the treatment-induced alterations of CD8 + and CD15 + cell infiltrations were observed between pN0 and pN1 patients. CONCLUSIONS: Collectively, our data demonstrate a comprehensive picture of tumor immune landscape before and after neoadjuvant ICB combined with chemoradiotherapy in ESCC. The infiltration of CD86 + macrophage may serve as an unfavorable indicator for neoadjuvant toripalimab combined with chemoradiotherapy.

Immunotherapy for esophageal cancer: Where are we now and where can we go.

Immune checkpoint inhibitor therapy has dramatically improved patient prognosis, and thereby transformed the treatment in various cancer types including esophageal squamous cell carcinoma (ESCC) in the past decade. Monoclonal antibodies that selectively inhibit programmed cell death-1 (PD-1) activity has now become standard of care in the treatment of ESCC in metastatic settings, and has a high expectation to provide clinical benefit during perioperative period. Further, anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) monoclonal antibody has also been approved in the treatment of recurrent/metastatic ESCC in combination with anti-PD-1 antibody. Well understanding of the existing evidence of immune-based treatments for ESCC, as well as recent clinical trials on various combinations with chemotherapy for different clinical settings including neoadjuvant, adjuvant, and metastatic diseases, may provide future prospects of ESCC treatment for better patient outcomes.

The single cell immunogenomic landscape after neoadjuvant immunotherapy combined chemotherapy in esophageal squamous cell carcinoma.

Neoadjuvant immunotherapy represents promising strategy in the treatment of esophageal squamous cell carcinoma (ESCC). However, the mechanisms underlying its impact on treatment sensitivity or resistance remain a subject of controversy. In this study, we conducted single-cell RNA and T/B cell receptor (scTCR/scBCR) sequencing of CD45+ immune cells on samples from 10 patients who received neoadjuvant immunotherapy and chemotherapy. We also validated our findings using multiplexed immunofluorescence and analyzed bulk RNA-seq from other cohorts in public database. By integrating analysis of 87357 CD45+ cells, we found GZMK + effector memory T cells (Tem) were relatively enriched and CXCL13+ exhausted T cells (Tex) and regulator T cells (Treg) decreased among responders, indicating a persistent anti-tumor memory process. Additionally, the enhanced presence of BCR expansion and somatic hypermutation process within TNFRSF13B + memory B cells (Bmem) suggested their roles in antigen presentation. This was further corroborated by the evidence of the T-B co-stimulation pattern and CXCL13-CXCR5 axis. The complexity of myeloid cell heterogeneity was also particularly pronounced. The elevated expression of S100A7 in ESCC, as detected by bulk RNA-seq, was associated with an exhausted and immunosuppressive tumor microenvironment. In summary, this study has unveiled a potential regulatory network among immune cells and the clonal dynamics of their functions, and the mechanisms of exhaustion and memory conversion between GZMK + Tem and TNFRSF13B + Bmem from antigen presentation and co-stimulation perspectives during neoadjuvant PD-1 blockade treatment in ESCC.

To develop a prognostic model for neoadjuvant immunochemotherapy efficacy in esophageal squamous cell carcinoma by analyzing the immune microenvironment.

Objective: The choice of neoadjuvant therapy for esophageal squamous cell carcinoma (ESCC) is controversial. This study aims to provide a basis for clinical treatment selection by establishing a predictive model for the efficacy of neoadjuvant immunochemotherapy (NICT). Methods: A retrospective analysis of 30 patients was conducted, divided into Response and Non-response groups based on whether they achieved major pathological remission (MPR). Differences in genes and immune microenvironment between the two groups were analyzed through next-generation sequencing (NGS) and multiplex immunofluorescence (mIF). Variables most closely related to therapeutic efficacy were selected through LASSO regression and ROC curves to establish a predictive model. An additional 48 patients were prospectively collected as a validation set to verify the model's effectiveness. Results: NGS suggested seven differential genes (ATM, ATR, BIVM-ERCC5, MAP3K1, PRG, RBM10, and TSHR) between the two groups (P < 0.05). mIF indicated significant differences in the quantity and location of CD3+, PD-L1+, CD3+PD-L1+, CD4+PD-1+, CD4+LAG-3+, CD8+LAG-3+, LAG-3+ between the two groups before treatment (P < 0.05). Dynamic mIF analysis also indicated that CD3+, CD8+, and CD20+ all increased after treatment in both groups, with a more significant increase in CD8+ and CD20+ in the Response group (P < 0.05), and a more significant decrease in PD-L1+ (P < 0.05). The three variables most closely related to therapeutic efficacy were selected through LASSO regression and ROC curves: Tumor area PD-L1+ (AUC= 0.881), CD3+PD-L1+ (AUC= 0.833), and CD3+ (AUC= 0.826), and a predictive model was established. The model showed high performance in both the training set (AUC= 0.938) and the validation set (AUC= 0.832). Compared to the traditional CPS scoring criteria, the model showed significant improvements in accuracy (83.3% vs 70.8%), sensitivity (0.625 vs 0.312), and specificity (0.937 vs 0.906). Conclusion: NICT treatment may exert anti-tumor effects by enriching immune cells and activating exhausted T cells. Tumor area CD3+, PD-L1+, and CD3+PD-L1+ are closely related to therapeutic efficacy. The model containing these three variables can accurately predict treatment outcomes, providing a reliable basis for the selection of neoadjuvant treatment plans.

Molecular and immunological characteristics of postoperative relapse in lymph node-positive esophageal squamous cell cancer.

BACKGROUND: The molecular and immunological characteristics of primary tumors and positive lymph nodes in esophageal squamous cell carcinoma (ESCC) are unknown and the relationship with recurrence is unclear, which this study attempted to explore. METHODS: A total of 30 ESCC patients with lymph node positive (IIB-IVA) were enrolled. Among them, primary tumor and lymph node specimens were collected from each patient, and subjected to 551-tumor-targeted DNA sequencing and 289-immuno-oncology RNA panel sequencing to identify the different molecular basis and immunological features, respectively. RESULTS: The primary tumors exhibited a higher mutation burden than lymph nodes (p < 0.001). One-year recurrent ESCC exhibited a higher Mucin16 (MUC16) mutation rate (p = 0.038), as well as univariate and multivariate analysis revealed that MUC16 mutation is independent genetic factor associated with reduced relapse-free survival (univariate, HR: 5.39, 95% CI: 1.67-17.4, p = 0.005; multivariate, HR: 7.36, 95% CI: 1.79-30.23, p = 0.006). Transcriptomic results showed non-relapse group had higher cytolytic activity (CYT) score (p = 0.025), and was enriched in the IFN-α pathway (p = 0.036), while those in the relapsed group were enriched in the TNF-α/NF-κB (p = 0.001) and PI3K/Akt pathway (p = 0.014). CONCLUSION: The difference in molecular characteristics between primary lesions and lymph nodes may be the cause of the inconsistent clinical outcomes. Mutations of MUC16 and poor immune infiltration are associated with rapid relapse of nodes-positive ESCC.

Combination of high anti-SKI and low anti-TMED5 antibody levels is preferable prognostic factor in esophageal carcinoma.

Given that esophageal cancer is highly malignant, the discovery of novel prognostic markers is eagerly awaited. We performed serological identification of antigens by recombinant cDNA expression cloning (SEREX) and identified SKI proto-oncogene protein and transmembrane p24 trafficking protein 5 (TMED5) as antigens recognized by serum IgG antibodies in patients with esophageal carcinoma. SKI and TMED5 proteins were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens. The serum anti-SKI antibody (s-SKI-Ab) and anti-TMED5 antibody (s-TMED5-Ab) levels were significantly higher in 192 patients with esophageal carcinoma than in 96 healthy donors. The presence of s-SKI-Abs and s-TMED5-Abs in the patients' sera was confirmed by western blotting. Immunohistochemical staining showed that the TMED5 protein was highly expressed in the cytoplasm and nuclear compartments of the esophageal squamous cell carcinoma tissues, whereas the SKI protein was localized predominantly in the nuclei. Regarding the overall survival in 91 patients who underwent radical surgery, the s-SKI-Ab-positive and s-TMED5-Ab-negative statuses were significantly associated with a favorable prognosis. Additionally, the combination of s-SKI-Ab-positive and s-TMED5-Ab-negative cases showed an even clearer difference in overall survival as compared with that of s-SKI-Ab-negative and s-TMED5-Ab-positive cases. The s-SKI-Ab and s-TMED5-Ab biomarkers are useful for diagnosing esophageal carcinoma and distinguishing between favorable and poor prognoses.

SPP1 represents a therapeutic target that promotes the progression of oesophageal squamous cell carcinoma by driving M2 macrophage infiltration.

BACKGROUND: Tumour-associated macrophages (TAMs) are an important component of the tumour microenvironment (TME). However, the crosstalk between oesophageal squamous cell carcinoma (ESCC) cells and TAMs remains largely unexplored. METHODS: Clinical samples and the TCGA database were used to evaluate the relevance of SPP1 and TAM infiltration in ESCC. Mouse models were constructed to investigate the roles of macrophages educated by SPP1 in ESCC. Macrophage phenotypes were determined using qRT‒PCR and immunohistochemical staining. RNA sequencing was performed to elucidate the mechanism. RESULTS: Increasing expression of SPP1 correlated with M2-like TAM accumulation in ESCC, and they both predicted poor prognosis in the ESCC cohort. Knockdown of SPP1 significantly inhibited the infiltration of M2 TAMs in xenograft tumours. In vivo mouse model experiments showed that SPP1-mediated education of macrophages plays an essential role in the progression of ESCC. Mechanistically, SPP1 recruited macrophages and promoted M2 polarisation via CD44/PI3K/AKT signalling activation and then induced VEGFA and IL6 secretion to sustain ESCC progression. Finally, blockade of SPP1 with RNA aptamer significantly inhibited tumour growth and M2 TAM infiltration in xenograft mouse models. CONCLUSIONS: This study highlights SPP1-mediated crosstalk between ESCC cells and TAMs in ESCC. SPP1 could serve as a potential target in ESCC therapy.

Development of a Novel Prognostic Model for Esophageal Squamous Cell Carcinoma: Insights into Immune Cell Interactions and Drug Sensitivity.

Esophageal squamous cell carcinoma (ESCC) presents a five-year survival rate below 20%, underscoring the need for improved prognostic markers. Our study analyzed ESCC-specific datasets to identify consistently differentially expressed genes. A Venn analysis followed by gene network interactions revealed 23 key genes, from which we built a prognostic model using the COX algorithm (p = 0.000245, 3-year AUC = 0.967). This model stratifies patients into risk groups, with high-risk individuals showing worse outcomes and lower chemotherapy sensitivity. Moreover, a link between risk scores and M2 macrophage infiltration, as well as significant correlations with immune checkpoint genes (e.g., SIGLEC15, PDCD1LG2, and HVCR2), was discovered. High-risk patients had lower Tumor Immune Dysfunction and Exclusion (TIDE) values, suggesting potential responsiveness to immune checkpoint blockade (ICB) therapy. Our efficient 23-gene prognostic model for ESCC indicates a dual utility in assessing prognosis and guiding therapeutic decisions, particularly in the context of ICB therapy for high-risk patients.