Esophageal Squamous Cell Carcinoma Therapy Research Articles

TRIM33 promotes glycolysis through regulating P53 K48-linked ubiquitination to promote esophageal squamous cell carcinoma growth

Esophageal squamous cell carcinoma (ESCC) is a common fatal malignant tumor of the digestive tract; however, its pathogenic mechanism is unknown and lacks specific molecular diagnosis and treatment. Therefore, it is particularly important to identify new tumor biomarkers to enhance the early diagnosis and molecular-targeted therapy of ESCC. Here, we found that the E3 ubiquitin ligase Tripartitemotif-containing33 (TRIM33) is highly expressed in ESCC tissues and cell lines, and is associated with adverse clinical outcomes. We determined that TRIM33 drives aerobic glycolysis to promote tumor growth in vivo and in vitro. In terms of mechanism, TRIM33 binds to p53 to inhibit its stability and promote the expression of downstream glycolysis target genes GLUT1, HK2, PKM2, and LDHA. In addition, TRIM33 promotes the polyubiquitination of P53 K48-linked and proteasome degradation. Further studies have shown that the K351 site of P53 is the key site mediating the ubiquitination of P53 K48-linked to promote aerobic glycolysis in ESCC and tumor cell growth. Our results reveal that the TRIM33-P53 signal axis regulates glycolysis during ESCC and may provide a new perspective for the diagnosis and treatment of ESCC.

Functional heterogeneity of cancer-associated fibroblasts with distinct neoadjuvant immunotherapy plus chemotherapy response in esophageal squamous cell carcinoma

Novel neoadjuvant immunotherapy combined with chemotherapy (neoICT) has improved outcomes for patients with esophageal squamous-cell carcinoma (ESCC), but challenges persist in low response rates and therapy resistance. Little is known about the intra-tumoral heterogeneity in the ESCC tumor microenvironment (TME) that underlies differential responses to neoadjuvant therapy. We applied single-cell RNA sequencing (scRNA-seq) profiling and multiplexed immunofluorescence staining to thoroughly decipher the TME in ESCC specimens from a neoadjuvant anti-PD1 combination therapy clinical trial. The cancer-associated fibroblasts (CAFs) population showed the significant alteration in abundance following neoadjuvant therapy. Specifically, IL6 + CCL2 + immunomodulatory CAFs and a novel CD248 + mechanoresponsive CAFs subset exhibited increasing infiltration. Mechanistically, CD248 + mechanoresponsive CAFs approached and lined the tumor nest to physically block the infiltration of CD8 + T cells and drug delivery, while IL6 + CCL2 + immunomodulatory CAFs induced therapeutic resistance with distinct IL-6 expression. Among patients treated with neoICT, we observed prominent CAF-T cell interactions. In particular, the NECTIN2-TIGIT ligand-receptor pair was enriched in treated samples, and TIGIT was identified as the major inhibitory checkpoint of T cells. Our findings demonstrate distinct alterations in TME constituent responses to neoadjuvant immunotherapy and identify functional phenotypes of CAFs associated with unfavorable therapeutic responses in patients. This provides potential targets to enhance responses to neoadjuvant therapy in ESCC.

Safety and Efficacy of Conversion Therapy After Systemic Chemotherapy in Advanced Esophageal Cancer with Distant Metastases: A Multicenter Retrospective Observational Study.

Patients with esophageal squamous cell carcinoma (ESCC) with distant metastasis were treated with systemic chemotherapy. Recent advances in multimodal treatments have made conversion therapy a viable option for patients with incurable ESCC. We aimed to assess the safety and efficacy of conversion therapy for ESCC with distant metastases. Conversion therapy was defined as surgery or chemoradiotherapy (CRT) used to cure tumors that were previously considered incurable because of distant metastasis. We conducted a retrospective review of patients who underwent ESCC conversion therapy and assessed the treatment outcomes, including adverse events and survival rates. A total of 147 patients from 22 institutions were included. Systemic chemotherapy was initially administered to all patients. The most common M1 factor was the para-aortic lymph node, accounting for 55% of cases. Following the initial treatment, 116 patients underwent surgery, with 31 receiving CRT as conversion therapy. Postoperative complications in surgery patients included pneumonia (16%), anastomotic leakage (7%), and recurrent laryngeal nerve palsy (6%). During CRT, 18% of patients developed grade 3 or higher non-hematological toxicities. The 5-year overall survival (OS) rate was 31.7%. Pathological responders had significantly longer OS than non-responders (hazard ratio 0.493, p=0.012). The distribution of distant metastasis, regimen type, clinical response, and conversion therapy modality did not have a significant impact on OS. Conversion therapy can be safely performed for ESCC with distant metastasis and has a favorable prognosis.

Neoadjuvant radiation target volume definition in esophageal squamous cell cancer: a multicenter recommendations from Chinese experts

BackgroundThis study aimed to establish a consensus on the delineation of target volumes for neoadjuvant radiation therapy (nRT) in esophageal squamous cell carcinoma (ESCC) within China.MethodsFrom February 2020 to June 2021, nine ESCC patients who received nRT were retrospectively selected from Sun Yat-sen University Cancer Center and Shandong Cancer Hospital. A panel from eight cancer radiotherapy centers performed two rounds of nRT target volume delineation for these patients: the first round for cases 1–6 and the second for cases 7–9. Online meetings were held after each delineation round to discuss findings. The consistency of delineations across centers was compared using mean undirected Hausdorff distances (Hmean), dice similarity coefficients (DSC), and total volumes, analyzed with the Mann-Whitney U test.ResultsThe second round of delineations showed improved consistency across centers (total clinical target volume (CTVtotal): mean DSC = 0.76–0.81; mean Hmean = 2.11–3.14 cm) compared to the first round (CTVtotal: mean DSC = 0.63–0.64; mean Hmean = 5.66–7.34 cm; DSC and Hmean: P < 0.050 between rounds), leading to the formation of a consensus and an atlas for ESCC nRT target volume delineation. A proposal was reached through evaluating target volume delineations, analyzing questionnaire survey outcomes, and reviewing pertinent literature.ConclusionsWe have developed guidelines and an atlas for target volume delineation in nRT therapy for ESCC in China. These resources are designed to facilitate more consistent delineation of target volumes in both clinical practice and clinical trials.

Development and Validation of a Comprehensive Analysis of the Competing Endogenous circRNA/miRNA/mRNA Network for the Identification of Immune-Related Targets in Esophageal Squamous Cell Carcinoma.

Immunotherapy for esophageal squamous cell carcinoma (ESCC) exhibits notable variability in efficacy. Concurrently, recent research emphasizes circRNAs' impact on the ESCC tumor microenvironment. To further explore the relationship, we leveraged circRNA, microRNA, and mRNA sequence datasets to construct a comprehensive immune-related circRNA-microRNA-mRNA network, revealing competing endogenous RNA (ceRNA) roles in ESCC. The network comprises 16 circular RNAs, 13 microRNAs, and 1,560 mRNAs. Weighted gene co-expression analysis identified immune-related modules, notably cancer-associated fibroblast (CAF) and myeloid-derived suppressor cell modules, correlating significantly with immune and stemness scores. Among them, the CAF module plays a crucial role in extracellular matrix function and effectively discriminates ESCC patients. Four hub collagen family genes within CAF correlated robustly with CAF, macrophage infiltration, and T-cell exclusion. In-house sequencing and RT-qPCR validated their elevated expression. We also identified CAF module-targeting drugs as potential ESCC treatments. In summary, we established an immune-related circRNA-miRNA-mRNA network that not only illuminates ceRNA functionality but also highlights circRNAs' involvement in the CAF through collagen gene targeting. These findings hold promise to predict ESCC immune landscapes and therapy responses, ultimately aiding in more personalized and effective clinical decision-making.

Targeting Galectin-1 Overcomes Paclitaxel Resistance in Esophageal Squamous Cell Carcinoma.

Resistance to paclitaxel poses a major obstacle in esophageal squamous cell carcinoma (ESCC) treatment. A better understanding of the mechanisms underlying paclitaxel resistance could help identify prognostic biomarkers and improved therapeutic strategies. In this study, we established a patient-derived xenograft (PDX) model of acquired paclitaxel resistance and used RNA-sequencing to identify galectin-1, encoded by LGALS1, as a key mediator of resistance. Integrative analysis of clinical data and physiological studies indicated that serum galectin-1 levels were elevated in resistant patients and correlated with treatment outcomes before and during taxane therapy. Importantly, exposing cells to serum from resistant patients resulted in increased paclitaxel resistance compared to serum from sensitive patients, which was closely associated with galectin-1 concentrations in the serum. The specific clearance of galectin-1 from resistant patient serum significantly restored paclitaxel sensitivity, and inhibiting galectin-1, through knockdown or the pharmacologic inhibitor OTX008, increased sensitivity to paclitaxel. Galectin-1 inhibition reduced the activity of β-catenin, thereby inhibiting stem cell properties induced by the Wnt/β-catenin pathway. Furthermore, galectin-1 regulated MDR1 transcription through increased nuclear accumulation of β-catenin, thus increasing resistance to paclitaxel. Combining OTX008 with clinical taxane formulations effectively reversed paclitaxel resistance in vitro and in vivo. Elevated galectin-1 levels thus serve as an indicator of response to paclitaxel therapy in ESCC, offering a therapeutic intervention strategy to overcome drug resistance.

A single-arm, exploratory study of disitamab vedotin (DV) combined with camrelizumab and platinum-based chemotherapy as first-line treatment in patients with HER2 expressing locally advanced or metastatic esophageal squamous cell carcinoma (ESCC).

TPS217 Background: Esophageal cancer (EC) is one of the most common cancers worldwide with esophageal squamous cell carcinoma (ESCC) being the predominant histological subtype. PD-1 blockades combined with chemotherapy are now standard-of-care in first-line setting for patients with ESCC. HER2 expression, prevalent in an estimated 7.6% to 28.5% of ESCC cases, correlates with diminished overall survival, suggesting HER2-targeted therapy as a potential novel approach. To our knowledge, there is currently no clinical trial regarding anti-HER2 therapy in ESCC. Clinical evidence has suggested the potential synergistic anti-tumor effect of Disitamab Vedotin (DV) with PD-1 inhibitors in HER2-expressing gastric or gastroesophageal junction (G/GEJ) cancers. This single-arm, exploratory study aimed to evaluate the safety and preliminary anti-tumor activity of DV combined with camrelizumab and platinum-based chemotherapy in ESCC. Methods: This is a single-arm, exploratory study (NCT06055153). Approximately 20 patients will be enrolled in this study, phase 1 will initiate with a 3+3 dose-escalation scheme (2.5mg/kg to 2.0mg/kg) to identify dose-limiting toxicities (DLTs) and establish a safe dosage of DV. The expansion cohort will subsequently evaluate this dose for both efficacy and safety. The study population will included both male and female patients over the age of 18 with histologically confirmed ESCC, and locally advanced unresectable, recurrent unresectable or metastatic EC verified by imaging examination. This group also includes those patients who have experienced relapse or progression more than six months post the conclusion of last therapeutic regimen. Required HER2 expression levels in tumor specimens range from IHC 1+ to 3+. According to RECIST v1.1, participants must have at least one measurable lesion, an ECOG performance status of 0 or 1, and a minimum expected survival of 12 weeks with suitable organ function. Treatment protocol includes intravenous administration of DV (either 2.5 mg/kg or 2.0 mg/kg on day 1), Camrelizumab (200 mg on day 1), and Cisplatin (75mg/m2) or Nedaplatin (75mg/m2) on a 21-day cycle, continued until disease progression or the advent of unacceptable toxicity. The primary outcomes for evaluation are DLT for safety and the objective response rate (ORR) for efficacy. Adverse events will be monitored in accordance with the CTCAE 5.0 criteria. Secondary endpoints encompass progression-free survival (PFS), overall survival (OS), disease control rate (DCR), duration of response (DoR), and time to response (TTR). Clinical trial information: NCT06055153 .

Envafolimab plus chemotherapy as neoadjuvant therapy for resectable esophageal squamous cell carcinoma (ESCC): A prospective, single-arm, phase II study.

e14633 Background: Currently, chemotherapy plus PD-1 inhibitor immunotherapy has become a promising option in the neoadjuvant setting of resectable ESCC, nevertheless, the PD-ligand 1 (PD-L1) blockade-based combination has rarely been investigated to date. Envafolimab is the first and only globally approved subcutaneous (SC) PD-L1 inhibitor, which has shown satisfactory efficacy and safety profile in tumors. This Simon’s two-stage, phase II study explored the safety and feasibility of SC administration of envafolimab as an alternative to intravenous (IV) administration of PD-1/PD-L1 inhibitors in the neoadjuvant therapy of resectable ESCC. Here, we reported the results of the stage 1. Methods: In this Simon’s two-stage, phase II study, patients initially treated with locally advanced and resectable (stage II-IVA) ESCC were enrolled and received envafolimab (300mg, SC, day 1), albumin-bound paclitaxel (130mg/m2, IV, days 1 and 8), and carboplatin (AUC = 5, day 1) every 3 weeks for 3 cycles. The primary endpoints were safety and pathological complete response (pCR). A Simon’s two-stage design was utilized with following assumptions. Target accrual was 13 patients in the first stage (If 3 or more patients had pCR in stage 1, study could continue in stage 2), total 34 patients were enrolled in the stage 2. Secondary endpoints were major pathological response (MPR). Safety was assessed by adverse events (AEs) and postoperative complications. Results: Between October 2022 and April 2023, 13 (81.3%) of 16 enrolled patients completed neoadjuvant therapy and underwent surgery. All 13 (100%) surgical patients underwent successful R0 resection; of whom, 8 (61.5%) achieved pCR and 9 (69.2%) achieved MPR, respectively. Downstaging occurred in 10 (76.9%) of 13 patients. pCR and MPR were identified to be associated with tumor downstaging but not with PD-L1 expression. CPS levels in MPR and pCR group were significantly higher than that in Non- MPR and Non-pCR group. Grade 3-4 treatment-related AEs (TRAEs) and immune-related AEs (IrAEs) occurred in 12.5% and 37.5% of 16 patients, respectively. 10 (76.9%) of 13 patients reported postoperative complications. No treatment-related surgical delay or death occurred. Conclusions: Envafolimab plus chemotherapy as neoadjuvant therapy demonstrated promising antitumor activity for resectable ESCC with high rates of pCR, MPR and R0 resection, as well as acceptable tolerability. Based on the encouraging efficacy of our regimen in the first stage, we will continue the enrollment of patients in stage 2. Keywords: Esophageal squamous cell carcinoma; Envafolimab; Neoadjuvant therapy; Chemotherapy Clinical trial information: NCT05552651 .

Update results of anlotinib combined with TQB2450 (PD-L1 blockade) as first-line treatment for advanced esophageal squamous cell carcinoma (ESCC): A single-arm, multicenter, open-label phase II clinical trial.

e16035 Background: Recent studies have demonstrated encouraging efficacy in patients with ESCC when combining PD-1 blockades with chemotherapy in the first-line setting. However, the safety profile of conventional chemotherapy remains unsatisfactory, suggesting the chemotherapy-free regimen a promising strategy. Anlotinib, a novel multitarget TKI primarily targeting VEGFR1-3, has shown therapeutic activity as first-line combination therapy or second-line monotherapy for ESCC patients in China. TQB2450 is a novel PD-L1 blockade developed by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Nanjing, China). This study aims to investigate the efficacy and safety of anlotinib plus TQB2450 as first-line therapy for patients with advanced ESCC. Preliminary results were presented at the 2023 ASCO-GI Symposium (Abs 377), the 2023 ASCO (Abs 4041) and the 2023 ESMO (1531P), the consecutively updated results were presented in this report. Methods: Patients with previously untreated metastatic or locally advanced ESCC, whose age was between 18 and 75 years, with ECOG PS of 0 or 1 score and life expectancy of &gt; 3 months were inclusion criteria. Eligible patients were administered with anlotinib (12mg, po, d1~14, q3w) plus TQB2450 (1200mg, iv, d1, q3w) until disease progression or unacceptable toxicity. The tumor response was assessed according to RECIST 1.1 and iRECIST using CT scans every 2 cycles for the first 6 cycles, and every 3 cycles thereafter. Adverse events were recorded by severity in accordance with the NCI CTC AE Version 5.0. The predefined sample size was 46. The primary endpoint was ORR, secondary endpoints included safety, PFS, DCR, DoR and OS. Results: From Mar 2022 to Sep 2022, a total of 46 patients were enrolled. At the data cut-off date (Dec, 2023), there were 1 CR (2.2%), 31 PR (67.4%), 10 SD (21.7%), 1 PD (2.2%) and 3 NE (6.5%). Therefore, the preliminary ORR was 69.6% (95%CI: 54.2%~82.3%), DCR was 91.3% (95%CI: 79.2%~97.6%). At the data cut-off date, 16 patients discontinued treatment due to PD, the preliminary prognostic result exhibited that the median PFS of the 46 pts was 15.44 months (95%CI: NA~NA). Additionally, safety profile exhibited that the regimen was tolerable. The common grade ≥3 treatment-related adverse events in 46 patients were hypertension (8.7%), neutrophil count decreased (4.3%), hyponatremia (4.3%), lymphocyte count decreased (4.3%), white blood cell count decreased (2.2%), platelet count decreased (2.2%), hand-foot syndrome (2.2%), hemoptysis (2.2%) and toothache (2.2%). Conclusions: The preliminary results highlighted that anlotinib plus TQB2450 as a first-line therapy for advanced ESCC showed encouraging efficacy and manageable safety profile. Furthermore, these conclusions required to be confirmed in subsequent trials. Clinical trial information: NCT05038813 .

Update results of anlotinib combined with penpulimab and nab-paclitaxel as first-line treatment for advanced esophageal squamous cell carcinoma (ESCC): A single-arm, open-label phase II clinical trial.

e16017 Background: Recently, PD-1 blockades combined with dual chemotherapy in first-line setting exhibited encouraging efficacy for patients with ESCC. However, the safety profile of conventional dual chemotherapy remained unsatisfactory. Therefore, PD-1 blockades combined with anti-angiogenic tyrosine kinase inhibitors (TKIs) and single chemotherapy regimen might be a promising strategy. Anlotinib, a novel multitarget TKI primarily targeting VEGFR1-3, demonstrated promising therapeutic activity as first-line combination therapy or second-line monotherapy for patients with ESCC in China clinically. Therefore, this study was designed to explore the efficacy and safety of anlotinib combined with penpulimab (PD-1 blockade) and nab-paclitaxel as first-line therapy in advanced ESCC. Preliminary results were presented at the 2024 ASCO-GI Symposium (Abs 340), the consecutively updated results were presented in this report. Methods: Patients with previously untreated metastatic or locally advanced ESCC were recruited and treated with anlotinib (12mg, po, d1~14, q3w) and penpulimab (200mg, iv, d1, q3w) plus nab-paclitaxel (220mg/m2, iv, d1, q3w) until disease progression or unacceptable toxicity. The tumor response was assessed according to RECIST 1.1 using CT scans every two cycles. Adverse events were recorded by severity in accordance with the NCI CTC AE Version 5.0. The predefined sample size was 30. Primary endpoint was PFS and secondary endpoints included safety, ORR, DCR and OS. Results: From Jul 2022 to Dec 2023, a total of 27 patients were enrolled, 25 patients who had received first tumor response were included in this analysis. The best overall response indicated that there were 1 CR (4.0%), 18 PR (72.0%), 3 SD (12.0%) and 3 NE (12.0%). Therefore, the preliminary ORR was 76.0% (95%CI: 54.9%-90.6%), DCR was 88.0% (95%CI: 68.8%-97.5%). Median PFS of the 25 patients was not yet reached. Additionally, safety profile exhibited that the regimen was tolerable. The most common treatment-emergent adverse events among the 27 patients with the incidence &gt; 20% were anemia (40.7%), peripheral nerve toxicity (37.0) and white blood cell decreased (25.9%). Common grade ≥3 treatment-emergent adverse events were white blood cell decreased (7.4%), diarrhea (3.7%), glutamic-pyruvic transaminase was elevated (3.7%), glutamic oxalacetic transaminase increased (3.7%), hypertension (3.7%) and immune enteritis (3.7%). Conclusions: The combination of anlotinib plus penpulimab and nab-paclitaxel as first-line therapy for advanced ESCC demonstrated promising efficacy and manageable safety profile. And the conclusions needed to be confirmed in subsequent trials.

Conversion surgery for tislelizumab combined chemotherapy for T4a/N3 esophageal squamous cell carcinoma (ESCC): A prospective, single-arm, phase II study.

4069 Background: T4a/N3 ESCC is treated with neoadjuvant concurrent chemoradiotherapy plus surgery or radical chemoradiotherapy according to NCCN guidelines. This study aimed to evaluate the safety and efficacy of conversion therapy of Tislelizumab combined with chemotherapy in patients with T4a/N3 ESCC. Methods: This is a prospective, single-arm, single-center clinical trial. A total of 40 patients with newly diagnosed ESCC (cT4a/N3) received 2-4 cycles of paclitaxel (135 mg/m2) on day 1, and cisplatin (80 mg/m2) on day 1, Tislelizumab (200 mg) on day 2, every 3 weeks. After an efficacy evaluation, the patients will undergo radical esophagectomy resection within 4-6 weeks after conversion therapy. The primary endpoints are the safety and the R0 resection rate. The secondary endpoints include the objective response rate (ORR), a postoperative pathological complete response (pCR), major pathological response (MPR), overall survival (OS), disease-free survival (DFS). Exploratory endpoint is to examine immune biomarker of the efficacy of conversion therapy for ESCC. Results: 40 patients were enrolled, the ORR was 95%(38/40), and 60% (24/40) patients who completed conversion treatment underwent esophagectomy. R0 resection rate was 100% (24/24) with no reoperations or perioperative deaths. 70% (28/40) patients experienced at least one treatment-related adverse event (TRAE) and 12.5% (5/40) patients reported grade 3 and above TRAEs. For these surgical patients, the MPR and pCR rates were 33% (8/24) and 20.8% (5/24) respectively. Flow cytometry showed that the percentage of CD3+CD4+CD8-T cells was significantly increased in peripheral blood after conversion therapy (p-value 0.02). Conclusions: Tislelizumab combined with chemotherapy, followed by radical esophagectomy resection has promising efficacy and good safety for T4a/N3 ESCC. Clinical trial information: NCT05449483 .

Agaricus blazei murrill and Enteromorpha prolifera-derived polysaccharides gain therapeutic effects on esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC), the most common pathological type of esophageal cancer, is one of the leading causes of cancer death worldwide, but an effective therapeutic avenue is still absent. This study was to evaluate the therapeutic effect of Agaricus blazei Murrill polysaccharides (ABP) and Enteromorpha prolifera polysaccharides (EP) on ESCC. The results showed that both ABP and EP have therapeutic effects on multiple malignant processes of ESCC, such as proliferation, migration, invasion, and apoptosis, in vitro. Moreover, both ABP and EP retard tumor growth in vivo and the growth of ESCC-derived organoids. A series of genes and proteins changed upon treatments with ABP and EP after RNA sequencing and proteomics analysis. Autophagy, mitophagy and ferroptosis were enriched after treatments with these two polysaccharides, suggesting that the consumption of ABP and EP may represent a promising dietary supplement for ESCC therapy.

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

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. 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. 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. 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.

Development of a Specific Aptamer-Modified Nano-System to Treat Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a prevalent gastrointestinal cancer characterized by high mortality and an unfavorable prognosis. While combination therapies involving surgery, chemotherapy, and radiation therapy are advancing, targeted therapy for ESCC remains underdeveloped. As a result, the overall five-year survival rate for ESCC is still below 20%. Herein, ESCC-specific DNA aptamers and an innovative aptamer-modified nano-system is introduced for targeted drug and gene delivery to effectively inhibit ESCC. The EA1 ssDNA aptamer, which binds robustly to ESCC cells with high specificity and affinity, is identified using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX). An EA1-modified nano-system is developed using a natural egg yolk lipid nanovector (EA1-EYLNs-PTX/siEFNA1) that concurrently loads paclitaxel (PTX) and a small interfering RNA of Ephrin A1 (EFNA1). This combination counters ESCC's proliferation, migration, invasion, and lung metastasis. Notably, EFNA1 is overexpressed in ESCC tumors with lung metastasis and has an inverse correlation with ESCC patient prognosis. The EA1-EYLNs-PTX/siEFNA1 nano-system offers effective drug delivery and tumor targeting, resulting in significantly improved therapeutic efficacy against ESCC tumors. These insights suggest that aptamer-modified nano-systems can deliver drugs and genes with superior tumor-targeting, potentially revolutionizing targeted therapy in ESCC.

Development of an Interpretable Deep Learning Model for Pathological Tumor Response Assessment After Neoadjuvant Therapy.

Neoadjuvant therapy followed by surgery has become the standard of care for locally advanced esophageal squamous cell carcinoma (ESCC) and accurate pathological response assessment is critical to assess the therapeutic efficacy. However, it can be laborious and inconsistency between different observers may occur. Hence, we aim to develop an interpretable deep-learning model for efficient pathological response assessment following neoadjuvant therapy in ESCC. This retrospective study analyzed 337 ESCC resection specimens from 2020-2021 at the Pudong-Branch (Cohort 1) and 114 from 2021-2022 at the Puxi-Branch (External Cohort 2) of Fudan University Shanghai Cancer Center. Whole slide images (WSIs) from these two cohorts were generated using different scanning machines to test the ability of the model in handling color variations. Four pathologists independently assessed the pathological response. The senior pathologists annotated tumor beds and residual tumor percentages on WSIs to determine consensus labels. Furthermore, 1850 image patches were randomly extracted from Cohort 1 WSIs and binarily classified for tumor viability. A deep-learning model employing knowledge distillation was developed to automatically classify positive patches for each WSI and estimate the viable residual tumor percentages. Spatial heatmaps were output for model explanations and visualizations. The approach achieved high concordance with pathologist consensus, with an R^2 of 0.8437, a RAcc_0.1 of 0.7586, a RAcc_0.3 of 0.9885, which were comparable to two senior pathologists (R^2 of 0.9202/0.9619, RAcc_0.1 of 8506/0.9425, RAcc_0.3 of 1.000/1.000) and surpassing two junior pathologists (R^2 of 0.5592/0.5474, RAcc_0.1 of 0.5287/0.5287, RAcc_0.3 of 0.9080/0.9310). Visualizations enabled the localization of residual viable tumor to augment microscopic assessment. This work illustrates deep learning's potential for assisting pathological response assessment. Spatial heatmaps and patch examples provide intuitive explanations of model predictions, engendering clinical trust and adoption(Code and data will be available at https://github.com/WinnieLaugh/ESCC_Percentage once the paper has been conditionally accepted). Integrating interpretable computational pathology could help enhancethe efficiency and consistency of tumor response assessment and empower precise oncology treatment decisions.

Integrating bulk and single-cell sequencing data to construct a Scissor+ dendritic cells prognostic model for predicting prognosis and immune responses in ESCC

Esophageal squamous cell carcinoma (ESCC) is characterized by molecular heterogeneity with various immune cell infiltration patterns, which have been associated with therapeutic sensitivity and resistance. In particular, dendritic cells (DCs) are recently discovered to be associated with prognosis and survival in cancer. However, how DCs differ among ESCC patients has not been fully comprehended. Recently, the advance of single-cell RNA sequencing (scRNA-seq) enables us to profile the cell types, states, and lineages in the heterogeneous ESCC tissues. Here, we dissect the ESCC tumor microenvironment at high resolution by integrating 192,078 single cells from 60 patients, including 4379 DCs. We then used Scissor, a method that identifies cell subpopulations from single-cell data that are associated bulk samples with genomic and clinical information, to stratify DCs into Scissorhi and Scissorlow subtypes. We applied the Scissorhi gene signature to stratify ESCC scRNAseq patient, and we found that PD-L1, TIGIT, PVR and IL6 ligand-receptor-mediated cell interactions existed mainly in Scissorhi patients. Finally, based on the Scissor results, we successfully developed a validated prognostic risk model for ESCC and further validated the reliability of the risk prediction model by recruiting 40 ESCC clinical patients. This information highlights the importance of these genes in assessing patient prognosis and may help in the development of targeted or personalized therapies for ESCC.

En1 promotes cell proliferation and migration via Hedgehog signaling pathway in esophageal squamous cell carcinoma

Objective: To explore the function and mechanism of transcription factor En1 in esophageal squamous cell carcinoma (ESCC). Methods: The correlations of En1 with prognosis were analyzed using the overall survival data of 9 397 pan-cancer patients and progression-free survival data of 4 349 pan-cancer patients from The Cancer Genome Atlas (TCGA) database. The En1 expression data in 53 and 155 cases of ESCC and their paired adjacent tissues were from Gene Expression Omnibus (GEO) database and National Genomics Data Center-Genome Sequence Archive(NGDC-GSA)database. Lentivirus was used to generate En1 stable knockout cell lines KYSE180 and KYSE450. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The migration ability of the cells was detected by Transwell assay. The effect of En1 on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of En1, glioma-associated oncogene family zinc finger 1 (GLI1), glioma-associated oncogene family zinc finger 2 (GLI2) and smoothened (SMO). Results: Pan-cancer data from TCGA showed that patients with low En1 expression had longer overall survival and progression-free survival than patients with high En1 expression (P< 0.001). Data from GEO and GSA databases also showed a high expression level of En1 in ESCC tissues compared with paired tissues (P＜0.001). Proliferation was inhibited after knockout of En1 in KYSE180 and KYSE450 cells (P＜0.001). The colony formation numbers decreased. The colony formation numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 138.33±23.07 and 127.00±19.70, respectively, significantly lower than that of the shNC group 340.67±12.06 (P<0.001). The colony formation numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 65.33±2.52 and 9.00±3.00, respectively, significantly lower than that of the shNC group 139.00±13.00 (P<0.001). The migration numbers was inhibited after knockout of En1 [the Transwell numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 66.67±12.66 and 71.33±11.02, respectively, significantly lower than that of the shNC group 334.67±16.56 (P<0.001). The Transwell numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 112.33±14.57 and 54.33±5.51, respectively, significantly lower than that of the shNC group 253.33±21.03 (P<0.001)]. Xenograft model showed a slower growth rate of shEn1#1 and shEn1#2 cell lines (P＜0.001). The tumor weights of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were (0.046±0.026)g and (0.047±0.025)g, respectively, significantly lower than that of the shNC group (0.130±0.038)g (P＜0.001). After knockdown of En1, the relative expression levels of GLI1 in KYSE180 cells of the shEn1#1 group and the shEn1#2 group were 0.326±0.162 and 0.322±0.133, and the relative expression levels of GLI1 in KYSE450 cells of the shEn1#1 and shEn1#2 groups were 0.131±0.006 and 0.352±0.050, respectively, which were all lower than that in the shNC group (P＜0.01). After knockdown of En1, overexpression of GLI1 attenuated the inhibitory effect of knockdown of En1 on cell proliferation (P＜0.001), colony formation[the colony formation numbers of the shEn1#1-GLI1 group were 151.00±9.54, higher than 102.33±10.02 (P=0.004) of the shEn1#1-vector group] and migration [the migration numbers of the shEn1#1-GLI1 group were 193.67±10.07, higher than 109.33±11.50 (P<0.001) in the shEn1#1-vector group]. In clinical samples of ESCC, major regulatory factors of the Hedgehog pathway were up-regulated and the pathway was activated. Conclusion: En1 promotes the proliferation and migration of ESCC cells by regulating the Hedgehog pathway and can be used as a new potential target for targeted therapy of ESCC.

Artesunate-loaded solid lipid nanoparticles resist esophageal squamous cell carcinoma by inducing Ferroptosis through inhibiting the AKT/mTOR signaling

Esophageal squamous cell carcinoma (ESCC) is a severe malignancy with high incidence and mortality rate in China, while the application of standard chemotherapeutic drugs for ESCC meets the barriers of high toxicity and multiple drug resistance (MDR). In recent years, the anticancer effects of artesunate (ART), a Chinese medicine monomer have gained extensive attentions due to its characteristics of low toxicity, high potency, and reversal of MDR. In this study, we develop the artesunate-loaded solid lipid nanoparticles (SLNART) to overcome the poor water solubility and bioavailability of ART, further improving the efficiency of ART on ESCC treatment. Especially mentioned, SLNART is shown to present marked inhibitory effects on ESCC development based on the induction of ferroptosis by two pathways included upregulating TFR to increase Fe2+ ions and inhibiting the AKT/mTOR signaling to downregulate GPX4. Collectively, this study is the first to pave a promising approach for ESCC therapy based on a strategy of developing SLNART to induce ferroptosis by mediating Fe2+ ions and AKT/mTOR signaling.

The efficacy and safety of conversion therapy after initial systemic chemotherapy in advanced esophageal cancer with distant metastases: A multicenter retrospective observational study.

310 Background: Systemic chemotherapy, with or without radiation, has been the standard treatment for esophageal squamous cell carcinoma (ESCC) with distant metastasis. The increased efficacy of multimodality treatment allows for the consideration of conversion therapy with curative intent for initially unresectable ESCC after initial treatment. In the present study, we examined the safety and effectiveness of conversion therapy for ESCC. Methods: Conversion therapy was defined as surgery or chemoradiation therapy (CRT) aiming at cure after initial treatment for tumors that were initially unresectable due to distant metastasis. The patients who underwent conversion therapy for ESCC between 2011 and 2021 at participating institutions were retrospectively reviewed. Patient backgrounds, treatment outcomes, and overall survival (OS) were evaluated. Results: From 22 institutions, 149 patients were enrolled. As defined, all patients received systemic chemotherapy as an initial treatment. The distant metastatic sites in para-aortic lymph nodes/extra regional mediastinal lymph nodes/lung/liver/bone/others were 81(54%)/19 (13%)/14 (9%)/11 (7%)/4 (3%)/20 (14%), respectively. After the initial treatment, 116 patients underwent surgery and 33 patients received CRT as conversion therapy. Of 116 patients who underwent conversion surgery, the incidence rate of postoperative pneumonia/leakage/recurrent laryngeal nerve palsy was 16%/7%/6%, respectively. R0 resection was obtained in 87%. Regarding the non-hematological toxicities during conversion CRT (CTCAE grade 3 or higher), decreased appetite/dysphagia/esophagitis was 21%/15%/9%, respectively. No grade 4 or higher hematologic toxicities were observed. The 3-year OS (3y-OS) rate for all patients was 41.9%; no significant differences were found between surgery and CRT groups (43.3% vs 37.0%, p = 0.112). Pathological responders showed a significantly longer OS than non-responders (3y-OS, 56.4% vs 36.1%, p = 0.010). The distribution or number of distant metastases were not identified as prognostic factors. Conclusions: This study showed that conversion therapy for ESCC patients with distant metastasis could be conducted safely and have a favorable prognosis. Since its survival benefit would depend on the pathological response, it is important to develop an indicator to predict pathological response before surgery and carefully select appropriate candidates of conversion therapy in ESCC.

Anlotinib combined with penpulimab and nab-paclitaxel as first-line treatment for advanced esophageal squamous cell carcinoma (ESCC): A single-arm, open-label phase II clinical trial.

340 Background: Recently, PD-1 blockades combined with dual chemotherapy in first-line setting exhibited encouraging efficacy for patients with ESCC. However, the safety profile of conventional dual chemotherapy remained unsatisfactory. Therefore, PD-1 blockades combined with anti-angiogenic tyrosine kinase inhibitors (TKIs) and single chemotherapy regimen might be a promising strategy. Anlotinib, a novel multitarget TKI primarily targeting VEGFR1-3, demonstrated promising therapeutic activity as first-line combination therapy or second-line monotherapy for patients with ESCC in China clinically. Therefore, this study was designed to explore the efficacy and safety of anlotinib combined with penpulimab (PD-1 blockade) and nab-paclitaxel as first-line therapy in advanced ESCC. Methods: Patients with previously untreated metastatic or locally advanced ESCC were recruited and treated with anlotinib (12mg, po, d1~14, q3w) and penpulimab (200mg, iv, d1, q3w) plus nab-paclitaxel (220mg/m2, iv, d1, q3w) until disease progression or unacceptable toxicity. The tumor response was assessed according to RECIST 1.1 using CT scans every two cycles. Adverse events were recorded by severity in accordance with the NCI CTC AE Version 5.0. The predefined sample size was 30. Primary endpoint was PFS and secondary endpoints included safety, ORR, DCR and OS. Results: From Jul 2022 to Aug 2023, a total of 23 patients were enrolled, 19 patients who had received first tumor response were included in this analysis. The best overall response indicated that there were 14 PR (73.7%), 2 SD (10.5%) and 3 NE (15.8%). Therefore, the preliminary ORR was 73.7% (95%CI: 48.8%-90.9%), DCR was 84.2% (95%CI: 60.4%-96.6%). Median PFS of the 19 patients was not yet reached. Additionally, safety profile exhibited that the regimen was tolerable. The most common treatment-emergent adverse events among the 23 patients with the incidence &gt;20% were leukopenia (30%) and anemia (30%). Common grade ≥3 treatment-emergent adverse events were leukopenia (9%), glutamic-pyruvic transaminase was elevated (4%), glutamic oxaloacetic transaminase increased (4%), hypertension (4%) and diarrhea (4%). Conclusions: The combination of anlotinib plus penpulimab and nab-paclitaxel as first-line therapy for advanced ESCC demonstrated promising efficacy and manageable safety profile. The conclusions needed to be confirmed in subsequent trials.