Background Esophageal squamous cell carcinoma (ESCC) is a major cause of cancer mortality in Asia, where histopathological diagnosis of endoscopic biopsy specimens remain challenging. Methods To address this challenge, we developed an AI-based ESCC diagnostic system in biopsies (AI-EDS) using 1 104 H&E-stained whole slide images (WSIs) from one center, which were divided into three datasets at the patient level: 515 WSIs (including 226 malignancies) for training, 50 WSIs (22 malignancies) for validation, and 539 WSIs (149 malignancies) for internal testing. Lesion areas were annotated using an iPad-based system, and the DeepLab-v3 model, supported by a ResNet-50 backbone, was trained for analysis. AI-EDS was subsequently applied to 945 esophageal biopsy WSIs (351 malignancies) from four additional hospitals. Finally, we also assessed the model's performance on surgical resection and endoscopic submucosal dissection (ESD) specimens with a total of 173 WSIs (131 malignancies). Results The AI-EDS demonstrated high accuracy in distinguishing malignant from benign lesions in an internal test dataset, achieving an AUC of 0.986 (95.36% accuracy, 99.49% sensitivity, and 84.56% specificity). In multicenter validation across four external hospitals (945 WSIs), the system maintained strong performance (AUC range: 0.966-0.998). Notably, the AI-EDS matched diagnostic accuracy of junior pathologists (89%) while reducing interpretation time by more than half. Furthermore, the system demonstrated robust generalization capability in surgical resection and endoscopic submucosal dissection specimens (173 WSIs, AUC = 0.827). Conclusions This multicenter validation confirms that the AI-EDS serves as a reliable, interpretable tool for computer-aided ESCC diagnosis, particularly valuable in resource-limited regions.

Background and Objective:Microbiota and their metabolites form a dynamic regulatory network that modulates the tumor microenvironment (TME) and immune cell functionality, emerging as critical targets in cancer immunotherapy. Despite remarkable advances in immune checkpoint inhibitors (ICIs), clinical efficacy remains limited by primary or acquired resistance in a substantial proportion of patients. Accumulating evidence indicates that the gut microbiota-metabolite-immune axis is a critical determinant of ICI responsiveness, but the underlying molecular mechanisms and tumor-specific regulatory patterns remain incompletely elucidated. This narrative review aims to systematically dissect the mechanistic interplay of this axis across diverse cancer types and synthesize current microbiota-targeted therapeutic strategies for improved immunotherapy outcomes. Methods:We conducted a systematic literature search of PubMed for studies published between January 2019 and December 2025, with a particular focus on basic and translational research elucidating the roles of gut microbiota and their metabolites in cancer immunotherapy across various malignancies. Key Content and Findings:This review elucidates the molecular mechanisms by which core metabolites regulate antitumor immunity, synthesizes characteristic microbial signatures across gastrointestinal and non-gastrointestinal cancers. Furthermore, we evaluate the translational potential of intervention strategies, including fecal microbiota transplantation, probiotic supplementation, and engineered microbes, as adjuvants to enhance ICI efficacy and overcome resistance. Conclusions:Microbiota-based personalized therapeutic strategies are increasingly recognized as promising tools to modulate antitumor immunity and improve immunotherapy outcomes. Systematic profiling of the gut microbiota-metabolite-immune axis across cancer types will facilitate the development of precision interventions to overcome ICI resistance. Future research integrating multi-omics approaches and large-scale clinical trials is pivotal to translate mechanistic insights into clinically viable therapies, ultimately revolutionizing the landscape of cancer immuno-oncology.

This study reports an exceptionally rare clinical encounter of synchronous multiple primary cancers involving high-grade serous ovarian carcinoma (HGSOC) and well-differentiated appendiceal mucinous adenocarcinoma in a 73-year-old woman. Presenting with abdominal distension and complex abdominopelvic masses, the patient's ascitic fluid revealed 2 morphologically distinct malignant cell populations. Following cytoreductive surgery, histopathological and immunohistochemical (IHC) analyses confirmed a dual-origin malignancy: ovarian lesions exhibited PAX8, WT1, and mutant-pattern TP53 expression, while the appendiceal lesion demonstrated strong keratin 20 positivity. Crucially, targeted gene sequencing validated these findings by identifying mutually exclusive mutational profiles-the HGSOC harbored a TP53 c.549del mutation, whereas the well-differentiated appendiceal mucinous adenocarcinoma carried KRAS p.G12D, GNAS p.R201C, and ATR p.R1951* mutations. Beyond simple coexistence, this patient had extensive cross-metastasis and tumor-to-tumor metastasis within both primary sites and metastatic deposits, likely facilitated by mucinous ascitic dissemination and lymphovascular invasion. Such diagnostic complexity necessitates a multidimensional approach integrating histomorphology, IHC, and molecular profiling to prevent misdiagnosis. Our findings emphasize that when imaging or cytology suggests multiorigin components, clinicians should pursue thorough intraoperative exploration, multisite biopsies, and prophylactic appendectomy. Ultimately, the management of such patients requires highly individualized surgical and chemotherapeutic strategies that account for the divergent biological behaviors and therapeutic sensitivities of both HGSOC and well-differentiated appendiceal mucinous adenocarcinoma to optimize oncological outcomes.

412 Background: The optimal treatment strategy for locally advanced potential/borderline resectable esophageal squamous cell carcinoma (LAPBR-ESCC) remains controversial. In this phase II study, we aim to evaluate the safety and efficacy of immunomodulatory low-dose radiotherapy (iRT) combined with chemoimmunotherapy (iCT) as conversion therapy for LAPBR-ESCC. Methods: This prospective study plans to enroll 43 patients (pts) with LAPBR-ESCC. Treatment begins with iRT (2 Gy × 3 fractions or 3 Gy × 2 fractions) combined with chemotherapy (nab-paclitaxel plus carboplatin) and tislelizumab. If the tumor becomes resectable, surgery is performed; if still unresectable, definitive chemoradiotherapy (dCRT) is administered. The primary endpoint is R0 resection rate. Secondary endpoints include safety, conversion rate, objective response rate (ORR), pathological complete response (pCR), major pathologic response (MPR), 2-year event-free survival (EFS), and overall survival (OS). Exploratory endpoints involve efficacy-related biomarkers and predictive models. Results: From April 2024 to May 2025, 25 pts were enrolled. The cohort consisted of 21 males and 4 females, with a median age of 66 years (range: 53–77). Among them, 20 pts (80.0%) were initially diagnosed with stage IVA disease, and 3 pts (12.0%) had stage IVB disease (limited to supraclavicular lymph node metastasis). As of May 9, 2025, all 25 pts were evaluable for efficacy: 5 achieved complete response (CR), 16 achieved partial response (PR), and 4 had stable disease (SD), resulting in an ORR of 84.0% (21/25). Surgery was performed on 23 pts, with R0 resection achieved in 21 pts. The R0 resection rate was 84.0% in the intention-to-treat (ITT) population and 91.3% in the surgical cohort (21/25 and 21/23, respectively), exceeding the prespecified threshold of 65%. Among the 23 resected cases, pCR was observed in 5 pts (21.7%), and MPR was achieved in 10 pts (43.5%). Primary lesion pCR and MPR were observed in 6 (26.1%) and 15 pts (65.2%), respectively. All pts (25/25) experienced at least one treatment-related adverse event (TRAE), and 17 pts (68.0%) had at least one grade 3/4 TRAE. The most common TRAEs were leukopenia (92.0%), lymphopenia (88.0%), neutropenia (88.0%), alopecia (88.0%), and anemia (84.0%). The most common grade ≥3 TRAEs were leukopenia and neutropenia, each occurring in 40.0% of pts (10/25). No grade 5 events or perioperative deaths were observed. Conclusions: Interim results from this phase II trial demonstrate that iRT combined with iCT followed by surgery is feasible and safe for patients with LAPBR-ESCC. Clinical trial information: ChiCTR2400083999 .

Intraductal carcinoma (IDC) and salivary duct carcinoma (SDC) are both rare tumors of the salivary glands that most frequently occur in the parotid glands. We present an example of apocrine IDC with a frankly invasive SDC component in the parotid gland. The clinicopathologic and molecular features were analyzed in this study. A 76-year-old female patient was admitted to hospital with a parotid mass. The biopsy result showed SDC, and radical resection surgery was performed. The tumor was characterized by an intraductal proliferation forming solid and cribriform structures with abundant eosinophilic cytoplasm, accompanied by comedo necrosis and apocrine secretion, some of which showed infiltrative growth without myoepithelial cells surrounding. Identifiable intravascular tumor thrombi and extensive lymph node (12 of 30) metastases were observed. The tumor cells were positive for androgen receptor and HER2, while negative for S100 and SOX10. The final diagnosis was apocrine IDC with an invasive SDC component. Next-generation sequencing revealed MET gene fusions and other gene mutations in the tumor cells. The rare MET gene fusions have not been reported in either IDC or SDC, which may enhance our understanding of the genetic alterations in a subset of SDCs that derive from IDC.

As an emerging biomarker, tumor mutational burden (TMB) has attracted increasing attention from clinicians in predicting the efficacy of tumor immunotherapy. Currently, TMB is detected primarily by whole-exome sequencing or targeted panel sequencing on high-throughput sequencing platforms. However, the lack of uniformity in detection methods, threshold settings, and reporting formats, as well as the significant differences in TMB values among different cancer types, have hindered the standardized application of this biomarker in clinical practice. This consensus focuses on the definition, standardization of detection, clinical significance, and limitations of TMB, and provides consensus recommendations for the clinical application of TMB in real-world practice in China. This consensus is aimed at helping clinicians and laboratory personnel understand the clinical significance and testing standards of TMB, promoting more accurate interpretation of test results, and improving patient care.

BACKGROUND:Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is challenging to diagnose accurately from pathological images due to its complex histological features. This study aims to develop an artificial intelligence (AI) model, IBDAIM, to assist pathologists in quickly and accurately diagnosing IBD by analyzing whole-slide images (WSIs) of intestinal biopsies. METHODS:This retrospective cohort study used data from two institutions, Nanjing Drum Tower Hospital (NDTH) and Zhujiang Hospital (ZJH). The NDTH dataset was randomly divided into a model development set and an internal test set, while the ZJH dataset served as an external validation set. We developed a weakly supervised deep learning model, IBDAIM, that uses WSI-level diagnostic labels without detailed annotation. The model integrates features from patch-level predictions using Patch Likelihood Histogram (PLH) and Bag of Words (BoW) to build WSI-level representations. Performance was evaluated using area under the receiver operating characteristic curve (AUROC), accuracy (ACC), sensitivity, and specificity. Probability plots and heatmaps were generated to analyze and visualize the diagnostic labels and organizational results of WSIs. Additionally, the model was applied to assist pathologists in diagnosis, and the improvement in diagnostic performance was assessed. RESULTS:In the normal intestinal mucosa vs. IBD task, the internal test cohort achieved an AUROC of 0.998 (95% CI 0.995-1.000) and ACC of 0.982, while the external test cohorts achieved an AUROC of 0.967 (95% CI 0.939-0.995) and ACC of 0.934. For the CD vs. UC task, the internal test cohort achieved an AUROC of 0.972 (95% CI 0.942-1.000) and ACC of 0.901, and the external test cohorts achieved an AUROC of 0.952 (95% CI 0.923-0.982) and ACC of 0.949. The model's performance exceeded that of five pathologists, and AI assistance significantly improved diagnostic accuracy across all pathologists. CONCLUSION:The IBDAIM model demonstrates high performance in diagnosing IBD biopsy pathological images and can effectively assist pathologists in identifying normal intestinal mucosa, CD, and UC tissues. This AI tool enhances diagnostic efficiency and accuracy, supporting better clinical decision-making and patient outcomes.

Microsatellite instability-high (MSI-H) is a favorable prognostic factor for neoadjuvant chemotherapy in several solid tumors, and large datasets are needed to provide robust evidence of its prognostic value in esophageal carcinoma (EC). We aimed to determine the incidence of MSI-H tumors in patients with EC and identify genomic alterations in MSI-H tumors. Data were obtained from three centers in China: Nanjing Drum Tower Hospital, Beijing Cancer Hospital, and the Cancer Hospital Chinese Academy of Medical Sciences. From June 2023 to December 2024, 1485 patients with EC were enrolled in this study. We investigated the incidence of MSI-H tumors and performed whole-exome sequencing of MSI-H tumor tissues. There were three patients with MSI-H from three centers (3/614, 0.49%) in Nanjing Drum Tower Hospital, five (5/644, 0.78%) in Beijing Cancer Hospital, and four (4/227, 1.76%) in the Cancer Hospital Chinese Academy of Medical Sciences. Among the 12 MSI-H tumor tissues, 6 were esophageal squamous cell carcinoma (ESCC) and 6 were esophageal adenocarcinoma (EAC). All MSI-H tumor tissues showed loss of PMS2 expression (100%), with concurrent loss of MLH1 in 9 (75%). Among the MSI-H tumors subjected to Whole Exome Sequencing (WES), all demonstrated high tumor mutation burden (TMB: 19.12-32.68 mutations/Mb) and harbored multiple somatic mutations. The consistency of MSI-H and high TMB scores showed that MSI-H could be an indicator of receiving immunotherapy in EC patients.

Esophageal squamous cell carcinoma (ESCC) has a significantly low survival rate, primarily due to the lack of diagnostic markers for early diagnosis and effective therapies. Recently, CD98 has been proposed as a specific marker of mature esophageal basal cells, which may be associated with esophageal carcinogenesis. Therefore, we aimed to investigate the clinical significance and biological function of CD98 in ESCC progression, as well as the value of CD98 as a potential new marker for the early diagnosis of ESCC. Through MassARRAY system spectroscopy, DIA proteomics analysis, immunohistochemical and functional experiments, we found hypomethylation-linked upregulation of CD98 expression, which was associated with poor prognosis, promoted cell proliferation by regulating the cell cycle in ESCC. Furthermore, we not only demonstrated that the range of CD98 expression was consistent with that of dysplastic cells in 73.81% of low-grade intraepithelial neoplasia (LGIN) and 83.08% of high-grade intraepithelial neoplasia (HGIN) cases, but also confirmed the expression level of CD98 was positively correlated with the classical diagnosis marker P53. Compared to using P53 alone, the combination of the immunohistochemical markers CD98 and P53 (either one was positive) provided more accurate diagnostic data for LGIN (92.86% vs. 88.10%, p < 0.01) and HGIN (93.85% vs. 73.85%, p < 0.01). In summary, we propose that CD98 is involved in a crucial step in ESCC carcinogenesis, and when combined with P53, may serve as a diagnostic marker for ESCC precancerous lesions.

Background:The incidence of esophageal squamous cell carcinoma (ESCC) is high and the prognosis is poor. It has become one of the important factors threatening the economic development and social stability of the region. The lack of effective early diagnostic biological indicators is the main reason for the late visit time and high mortality rate of clinical patients with ESCC. This study aims to investigate the role of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in predicting the prognosis and survival of patients with ESCC. Methods:Prognosis-related genes were studied by analyzing RNA sequencing data of ESCC in The Cancer Genome Atlas (TCGA) database, and they were divided into a training cohort (n=83) and a test cohort (n=76). A risk score model was constructed and prognosis-related markers were verified. Results:Nine prognosis-related genes (RP11-51F16.1, FABP3, RP11-4K3-A.3, GNG12-AS1, RP11-2N1.2, PRICKLE2-AS1, KB-1254G8.1, AC132825.1, and RP11-294N21.3) were screened out. Further construction of the risk score model revealed that the risk score was an independent prognostic factor. In addition, this study integrated prognostic markers and combined clinical features to develop a nomogram for predicting the overall survival (OS) rate for 1 to 3 years. Through quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) staining experiments, this study found that FABP3 was upregulated in ESCC, and patients with high expression of FABP3 had a shorter OS (P=0.04), and the upregulation of FABP3 was significantly correlated with the degree of differentiation of ESCC (P=0.04). Gene Set Enrichment Analysis (GSEA) indicated that the functions related to metabolism were mainly concentrated in the high-risk group. From a metabolic perspective, they played a role in regulating the tumorigenesis process. The analysis of changes in chemotherapy drug resistance found that the half maximal inhibitory concentration (IC50) of 25 drugs was higher in the high-risk group, which confirmed that this feature played an important role in the prognosis and treatment of ESCC. Conclusions:This study identified a new set of characteristics of nine prognostic genes related to ESCC, which may provide ideas for prognosis prediction and new therapeutic methods for ESCC patients.

This investigation aims to elucidate the effects of Timosaponin B-II (TB-II) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) through both in vitro experiments and an in vivo orthodontic tooth movement model utilizing rats. The primary objective is to clarify the mechanisms by which TB-II influences the remodeling of periodontal tissue under biomechanical stress, thereby providing insights into its potential role in reducing relapses after orthodontic tooth movement. hPDLSCs were isolated and characterized via flow cytometry and multilineage differentiation assays (osteogenic and adipogenic induction). The impact of TB-II on the expression levels of osteogenic genes and proteins, including runt-related transcription factor-2 (RUNX-2), alkaline phosphatase (ALP), and collagen type 1 (COL-1), was evaluated through quantitative real-time PCR (qRT-PCR) and Western blotting. Alizarin Red Staining (ARS) was utilized to assess the formation of mineralized nodules. Additionally, the involvement of the phosphatidylinositol - 3 – kinase (PI3K)/ protein kinase B(AKT)/ glycogen synthase kinase - 3β(GSK3β) signaling pathway in TB-II-mediated osteogenesis was explored using pharmacological inhibitors (LY294002 for PI3K/AKT and CHIR-99021 for GSK3β). Western blot analysis identified key osteogenic markers (GSK3β, p-GSK3β, AKT, p-AKT) in treated cells. For in vivo validation, eighteen male Wistar rats were randomly divided into TB-II-treated and saline-control groups. Micro-computed tomography (micro-CT) evaluated tooth movement and alveolar bone structural changes. Histological assessment included hematoxylin–eosin (HE) staining, Masson trichrome staining, and tartaric-resistant acid phosphatase (TRAP) staining to analyze periodontal tissue morphology. Immunohistochemical (IHC) analysis assessed osteogenic markers (RUNX-2, ALP, COL-1) and the osteoclastogenic regulator RANKL to evaluate tissue remodeling. All statistical analyses were performed using GraphPad Prism 8. Comparisons between groups were conducted via one-way/two-way ANOVA with Tukey’s post-hoc test. Values of p < 0.05 were regarded as statistically significant. In vitro studies revealed that TB-II at 20 μM significantly enhanced the proliferation, ALP activity, and mineralized nodule formation of hPDLSCs, accompanied by markedly elevated expression of RUNX-2, ALP, COL-1, p-AKT and p-GSK3β. Pharmacological inhibition of the PI3K/AKT pathway via LY294002 abolished TB-II’s osteogenic effects, while treatment with CHIR99021 indicated that GSK3β activity was downstream and regulated by the PI3K/AKT signaling axis. In vivo, TB-II administration in a rat orthodontic tooth movement (OTM) model upregulated RUNX-2, ALP, and COL-1 expression on the tension side of tooth roots, while simultaneously reducing TRAP + osteoclast numbers and inhibiting RANKL expression. TB-II stimulates the proliferation and osteogenic maturation of hPDLSCs in vitro by activation of the PI3K/AKT/GSK3β signaling axis. In vivo investigations using OTM model further demonstrate that TB-II enhances periodontal tissue regeneration. Collectively, these results highlight the therapeutic potential of TB-II in preventing relapses following OTM, positioning it as a viable candidate for clinical strategies aimed at stabilizing orthodontic outcomes.

Background Radiotherapy and immunotherapy can be synergistically combined, and studies on immunotherapy combined with neoadjuvant chemoradiotherapy (nCRT) have achieved pathologic complete response (pCR) rates that exceed those of conventional nCRT. However, no data have confirmed that a pCR increase leads to overall survival (OS) improvements. The low-dose radiotherapy (LDRT) immunomodulatory function has attracted much attention in recent years. The purpose of this study is to investigate the efficacy and safety of low-dose immunomodulatory radiotherapy (iRT) combined with chemoimmunotherapy to convert locally advanced borderline/potential resectable esophageal squamous cell carcinoma (ESCC).Methods Forty-three pathologically confirmed thoracic ESCC patients with stage cT3-4NxM0 whose clinical primary lesions were not completely (R0) resectable and/or stage cT2-4N+M0 with unresectable metastatic lymph nodes were enrolled. The participants will receive the following radiotherapy combined with chemoimmunotherapy. The iRT prescribed doses for cycles 1−2 will be 6 Gy (2 Gy/Fr × 3 Fr), days 1−3, 22−24, and 6 Gy (3 Gy/Fr × 2 Fr) on days 1−2, 22−23 once a day. An intravenous drip of tislelizumab at 200 mg will be used after radiotherapy on the last day of iRT (on days 3 and 24 (2 Gy/Fr × 3 Fr regimen) or days 2 and 23 (3 Gy/Fr × 2 Fr regimen) at cycles 1–2, and at the third cycle be used on the second day after chemotherapy (on day 44). Chemotherapy (nanoparticle albumin-bound (nab-) paclitaxel at 175−260 mg/m2 + carboplatin area under the curve (AUC) = five days will be used on the 1st, 22nd, and 43rd days at cycles 1−3). Patients feasible for R0 resection following the third treatment cycle will be identified as successful conversions and undergo radical surgery. Patients with non-pathological complete response (pCR) results after esophagectomy will receive adjuvant chemotherapy combined with immunotherapy (nab-paclitaxel plus carboplatin and tislelizumab) for two cycles. Patients with postoperative stage ypT3-4N0 or N+ or R1/R2 resection will be considered to receive supplemental postoperative adjuvant radiotherapy according to the Chinese radiotherapy ESCC guideline. Unsuccessful conversion patients may receive definitive chemoradiotherapy based on the guideline (radiotherapy dose: 50 Gy/25 Fr, once a day, five times a week; nab-paclitaxel plus carboplatin, three-weekly regimen × two cycles). It will be determined whether immunotherapy could be combined with postoperative adjuvant therapy after a full pneumonia risk assessment. The primary endpoint of this study is the R0 resection rate. The secondary endpoints include safety, the conversion rate, the objective response rate (ORR), the pCR rate, the major pathologic response (MPR) rate, the event-free survival (EFS), and the overall survival (OS).Discussion This protocol was reviewed and approved by the Ethics Committee of the Nanjing Drum Tower Hospital (No. 202416802). This prospective clinical trial will be used to investigate the safety and efficacy of iRT combined with chemoimmunotherapy for conversion therapy of locally advanced borderline/potential resectable ESCC. We hypothesize that iRT combined with chemoimmunotherapy for conversion therapy will be a promising therapeutic strategy that could provide the required R0 resection rate in patients with borderline/potential resectable ESCC.

Abstract Phospholipase C epsilon 1 (PLCE1) is a well-established susceptibility gene for esophageal squamous cell carcinoma (ESCC). Identification of the underlying mechanism(s) regulated by PLCE1 could lead to a better understanding of ESCC tumorigenesis. In this study, we found that PLCE1 enhances tumor progression by regulating the replicative helicase MCM7 via two pathways. PLCE1 activated PKCα-mediated phosphorylation of E2F1, which led to the transcriptional activation of MCM7 and miR-106b-5p. The increased expression of miR-106b-5p, located in intron 13 of MCM7, suppressed autophagy and apoptosis by targeting Beclin-1 and RBL2, respectively. Moreover, MCM7 cooperated with the miR-106b-25 cluster to promote PLCE1-dependent cell-cycle progression both in vivo and in vitro. In addition, PLCE1 potentiated the phosphorylation of MCM7 at six threonine residues by the atypical kinase RIOK2, which promoted MCM complex assembly, chromatin loading, and cell-cycle progression. Inhibition of PLCE1 or RIOK2 hampered MCM7-mediated DNA replication, resulting in G1–S arrest. Furthermore, MCM7 overexpression in ESCC correlated with poor patient survival. Overall, these findings provide insights into the role of PLCE1 as an oncogenic regulator, a promising prognostic biomarker, and a potential therapeutic target in ESCC. Significance: PLCE1 promotes tumor progression in ESCC by activating PKCα-mediated phosphorylation of E2F1 to upregulate MCM7 and miR-106b-5p expression and by potentiating MCM7 phosphorylation by RIOK2.

Esophageal basaloid squamous cell carcinoma (BSCC) is a heterogenous entity with multilineage differentiation. It lacks systematical analysis on submucosal gland differentiation (SGD) due to the histological diversity and low incidence of esophageal BSCC. This study aims to find the correlation of SGD and clinicopathological features. A total of 152 esophageal BSCCs were separated into three histological groups: pure, mixed, and borderline group. The clinicopathological features were compared between different groups. The prevalence of SGD was also compared between cases of different groups. A panel of antibodies were used to identify SGD. The pure group differed from the mixed and borderline groups in many aspects, lymph node metastasis (LNM), cancer embolus, perineural invasion, and advanced stage occurred less frequently in the pure group ( P <0.01). The pure group had a better but statistically insignificant overall survival ( P =0.097). The squamous cell carcinoma (SCC) component or focal squamous differentiation was present in metastatic lymph nodes in almost all mixed BSCCs (95.7%, 22/23) with LNM. The LNM rate of superficial (T1b) BSCCs (17.6%, 6/34) was comparable to that of superficial (T1b) SCCs (18.5%, 57/308). However, LNM exclusively occurred in superficial mixed (3/5) and borderline (3/10) BSCCs. The IHC results demonstrated a prevalence of SGD in pure group (77%, 43/56). SGD is considered to be a favorable factor, while the squamous differentiation or invasive SCC component is an adverse factor in esophageal BSCCs. Refinement of classification is a promising way to improve patient management.

Supplementary Methods and Materials, supplementary tables, supplementary figure legends

e16016 Background: Our prior investigations have demonstrated that PLCE1 actively promotes tumorigenesis, angiogenesis, and cell cycle progression in ESCC. However, the precise role of PLCE1 in glycolysis and its influence on the tumor immune microenvironment (TIME) remain elusive. Methods: To elucidate potential pathways regulated by PLCE1, we applied Affymetrix GeneChip, Affymetrix ClariomD Array, and IP-MS techniques to identify molecules interacting with PLCE1. Subsequently, we validated the involement of PLCE1 in glycolysis through assessments of glycolytic product production in both in vivo and in vitro assays. The underlying mechanisms by which PLCE1 influences glycolysis were further probed through a combination of IP assays, tumor growth experiments, and ubiquitination assays. Next, we conducted multi-color immunofluorescence staining to examine the infiltration of immune cells in human ESCC tissues. We utilized a transgenic PLCE1 -/- C57BL/6 mice model to investigate the immune cell distribution in TIME. Results: PLCE1 emerges as a pivotal regulator in glycolytic processes within ESCC cell lines. Knockdown of PLCE1 instigates a notable attenuation of glycolysis, achieved through the modulation of ENO1 expression, a key enzyme in glycolysis. This effect was also observed in vivo. We have elucidated a novel pathway in which PLCE1 orchestrates a reduction in the expression of FBXW7, an E3 ubiquitin ligase, thereby attenuating FBXW7-mediated ubiquitination of ENO1 and fostering ENO1 stability. Based on mutations identified through sequencing of ESCC tissue and drawing from previous studies, we have pinpointed the specific interacting sites between FBXW7 and ENO1. Substrate phosphorylation is necessary for the recognition of FBXW7. We unveil a mechanism wherein PLCE1 interacts with CDK2 to augment ENO1 phosphorylation, potentiating FBXW7 recognition. Nonetheless, CDK2-mediated ENO1 phosphorylation fails to elicit FBXW7-mediated ubiquitination due to limited FBXW7 expression in ESCC. Elevated tumor glycolysis induced by PLCE1 via ENO1 engenders heightened lactate production, thereby dampening cytokine production and CD8 + T cell activation. In human ESCC tissues, a diminished CD8+ T cell population is observed proximal to PLCE1 + ENO1 + tumor cells, concomitant with an augmented accumulation of exhausted CD8 + PD1 + T cells in their vicinity. In an ESCC murine model, a more robust infiltration and cytokine production, coupled with diminished PD-1 expression on both CD4 + and CD8 + T cells, is noted in the PLCE1 -/- genotype. This effect was particularly prominent upon ENO1 inhibitor administration. Conclusions: Our investigation elucidates that PLCE1 orchestrates the glycolytic pathway in ESCC by upregulating the expression of ENO1, consequently hindering the infiltration and activation of CD8 + T cells, thus fostering an immunosuppressive TIME.

Background and Aims Inflammatory bowel disease (IBD) is a global disease that is evolving with increasing incidence. However, there are few works on computationally assisted diagnosis of IBD based on pathological images. Therefore, based on the UK and Chinese IBD diagnostic guidelines, our study established an artificial intelligence-assisted diagnostic system for histologic grading of inflammatory activity in ulcerative colitis (UC). Methods We proposed an efficient deep-learning (DL) method for grading inflammatory activity in whole-slide images (WSIs) of UC pathology. Our model was constructed using 603 UC WSIs from Nanjing Drum Tower Hospital for model train set and internal test set. We collected 212 UC WSIs from Zhujiang Hospital as an external test set. Initially, the pre-trained ResNet50 model on the ImageNet dataset was employed to extract image patch features from UC patients. Subsequently, a multi-instance learning (MIL) approach with embedded self-attention was utilized to aggregate tissue image patch features, representing the entire WSI. Finally, the model was trained based on the aggregated features and WSI annotations provided by senior gastrointestinal pathologists to predict the level of inflammatory activity in UC WSIs. Results In the task of distinguishing the presence or absence of inflammatory activity, the Area Under Curve (AUC) value in the internal test set is 0.863 (95% confidence interval [CI] 0.829, 0.898), with a sensitivity of 0.913 (95% [CI] 0.866, 0.961), and specificity of 0.816 (95% [CI] 0.771, 0.861). The AUC in the external test set is 0.947 (95% confidence interval [CI] 0.939, 0.955), with a sensitivity of 0.889 (905% [CI] 0.837, 0.940), and specificity of 0.858 (95% [CI] 0.777, 0.939). For distinguishing different levels of inflammatory activity in UC, the average Macro-AUC in the internal test set and the external test set are 0.827 (95% [CI] 0.803, 0.850) and 0.908 (95% [CI] 0.882, 0.935). the average Micro-AUC in the internal test set and the external test set are 0.816 (95% [CI] 0.792, 0.840) and 0.898 (95% [CI] 0.869, 0.926). Conclusions Comparative analysis with diagnoses made by pathologists at different expertise levels revealed that the algorithm reached a proficiency comparable to the pathologist with 5 years of experience. Furthermore, our algorithm performed superior to other MIL algorithms.

This study aimed to explore the potential mechanism by which sulfasalazine (SAS) inhibits esophageal cancer cell proliferation. A cell counting kit-8 (CCK-8) assay was used to detect the effect of SAS (0, 1, 2, and 4 mM) on the proliferation of TE-1 cells. Subsequently, TE-1 cells were divided into control group, SAS group, SAS + ferrostatin-1 (ferroptosis inhibitor) group, and SAS + Z-VAD (OH)-FMK (apoptosis inhibitor) group, and cell proliferation was measured using a CCK-8 assay. Real-time quantitative polymerase chain reaction and western blotting were used to determine the expression of solute carrier family member 7 11 (SLC7A11, also called xCT), glutathione peroxidase 4 (GPX4), and acyl-CoA synthase long-chain family member 4 (ACSL4) in TE-1 cells. Measurement of ferroptosis in TE-1 cells was achieved by flow cytometry. Compared with the control group (0 mM SAS), the proliferation of TE-1 cells was significantly inhibited by different concentrations of SAS for different time lengths, and 4 mM SAS treatment for 48 h could obtain the maximum inhibition rate (53.9%). In addition, SAS treatment caused a significant decrease in the mRNA and protein expression of xCT and GPX4, and a significant increase in ACSL4 expression in TE-1 cells treated with SAS. Flow cytometry results showed that the ferroptosis level was significantly increased after SAS treatment. However, the activation of ferroptosis by SAS was partially eliminated by treatment with ferrostatin-1 or Z-VAD (OH)-FMK. In conclusion, SAS inhibits the proliferation of esophageal carcinoma cells by activating the ferroptosis pathway.

BackgroundPrevious clinical trials indicated the addition of anti-PD-1 antibody remarkably improved the efficacy of trastuzumab and chemotherapy in patients with HER2-positive gastric/gastroesophageal junction (GEJ) cancer. However, no real-world experiences have been reported yet. MethodsWe retrospectively analyzed 1212 patients with gastric/GEJ cancer treated at Nanjing Drum Tower Hospital between 2019 and 2022. Among 138 patients with HER2-positive gastric/GEJ cancer, 47 patients receiving at least two doses of the combination regimen with anti-PD-1 antibody, trastuzumab, and chemotherapy were recruited in the study population, and 38 out of 47 patients with measurable disease were included in the efficacy population. Progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and toxicity profiles were reported. ResultsIn the study population, 37 (78.7%) received the study therapy as a first-line treatment. In the efficacy population, the ORR and DCR were 76.3% and 94.7%, respectively. The overall median PFS was 9.1 months (95% confidence interval [CI] 6.3-11.9 months). For the first-line treatment, the mPFS was 10 months, and 7 months for the second-line. Among 14 patients who failed the study treatment, three (21.4%) developed brain metastasis as the first failure site. No significant association was found between PFS and the expression of PD-L1. 22.2% of patients developed grade 3 treatment-related adverse events (TRAEs). No treatment-related grade >= 4 adverse events or deaths occurred. ConclusionThis real-world study validated the combination regimen's high efficacy and good tolerance in patients with HER2-positive gastric/GEJ cancer. An increased incidence of brain metastasis was observed in patients who failed this regimen.