Background:Alcohol and tobacco are established carcinogens, which promote field carcinogenesis for esophageal squamous cell carcinoma (ESCC). This study aimed to evaluate the long-term effects of alcohol and tobacco cessations, and background mucosal status, on risk for metachronous ESCC (mESCC) after endoscopic resection (ER). Methods:This was a multicentre prospective cohort study of patients with intramucosal ESCC treated by ER. All participants received structured education on cessation, and underwent regular endoscopic surveillance. Patients were stratified by Lugol-voiding lesion (LVL) grade (A: none, B: 1-9, C: ≥10). The impacts of alcohol and smoking cessation on field carcinogenesis were assessed. Findings:Among 331 enrolled patients, the median follow-up was 120 months (range: 1.3-176.9). The cumulative incidences of mESCC were 10.4%, 27.2%, and 61.8% in grades A, B, and C, respectively. An increment of 1 unit (22 g ethanol) of alcohol consumption and higher LVL grade independently increased the risk for mESCC. Alcohol or smoking cessation reduced this risk (hazard ratio [HR] 0.52, 95% confidence interval [CI]: 0.31-0.88; HR 0.44, 95% CI: 0.25-0.78, respectively), and combined cessation had the greatest impact (HR 0.21, 95% CI: 0.07-0.65). Complete cessation, rather than partial reduction, was necessary to achieve meaningful risk reduction. Interpretation:Alcohol and tobacco exposure, and a large number of LVL, are major determinants of mESCC. Complete cessation markedly reduces risk, underscoring the importance of behavioural interventions for secondary prevention of field carcinogenesis after ER. Funding:National Cancer Center Research and Development Fund 36, Japan.

ABSTRACT Transgelin is an actin‐binding protein that promotes cancer progression via activation of cancer‐associated fibroblasts and has been identified as a prognostic marker. However, its distribution and functional role in colon cancer remain unclear. In this study, we aimed to elucidate the mechanistic role of transgelin in colon cancer progression by focusing on its functional impact in cancer‐associated fibroblasts. Tissue microarrays from 359 human colon cancer tissues were investigated to elucidate the clinical importance of transgelin expression in cancer stroma. We focused on transgelin in fibroblasts and investigated its functional role in stromal activation using in vitro knockdown experiments and in vivo co‐transplantation models. Primary cultures of human colon fibroblasts were evaluated for their biological function. Our data showed that transgelin expression is predominant in activated cancer‐associated fibroblasts in colon cancer tissues. Stimulation by cancer‐cell‐conditioned medium (CM) significantly upregulated transgelin, ACTA2, COL1A1, and TNC expression in colonic fibroblasts. Additionally, transgelin knockdown (KD) in fibroblasts did not influence the upregulation except for transgelin itself. Transgelin KD in fibroblasts did not result in drastic alterations in gene expression profiles. Transgelin KD suppressed collagen gel contractility. Furthermore, co‐transplantation experiments of cancer cells and colonic fibroblasts into immunodeficient mice revealed that transgelin KD inhibited tumor growth in fibroblasts. In conclusion, stromal transgelin expression in colon cancer strongly correlated with distant metastasis and served as a prognostic factor for colon cancer. Mechanistically, transgelin in cancer‐associated fibroblasts promotes tumor growth by regulating stromal contractility, suggesting transgelin as a potential therapeutic target.

Immune checkpoint inhibitors show insufficient efficacy against pancreatic ductal adenocarcinoma (PDAC). The tumor microenvironment (TME) has a remarkable influence on responsiveness to cancer immunotherapy. The aim of this study was to investigate immunosuppressive characteristics of TME in PDAC tissues. The flow cytometry (FCM) of PDAC surgical specimens revealed that the profile of tumor-infiltrating leukocytes was classified into myeloid cell- and T-cell-dominant subtypes; the myeloid subtype was associated with poorer patient outcomes. Myeloid-derived suppressor cells (MDSCs) showed the highest hazard ratio among various myeloid cell types. Single-cell RNA sequencing and FCM revealed that most MDSCs, but not lymphocytes, in PDAC tissues characteristically express CD74. Macrophage migration inhibitory factor (MIF), a CD74 ligand, was highly expressed in cancer-associated fibroblasts (CAFs) and cancer cells. Spatial transcriptomics demonstrated that the MIF-CD74+ myeloid cell interaction was recognized in CAF-dominant areas in PDAC tissue. CAFs expressing immune suppressor molecules such as MFAP5 and LRRC15 were consistent with MIF+ CAFs. Furthermore, MIF+ CAFs enhanced the migratory activity of MDSCs and promoted MDSC induction and activation. In the murine model, MDSCs were significantly increased in MIF-expressing PDAC tumors, as were CD74+ M-MDSCs per M-MDSC, confirming in vivo interaction between CD74 and MIF. MDSCs play a crucial role in creating an immunosuppressive TME in PDAC; the MIF-CD74 axis drives interactions between MDSCs and CAFs.

3573 Background: The phase 3 PARADIGM trial showed overall survival (OS) and tumor response advantages of anti-EGFR (panitumumab, PAN) over anti-VEGF (bevacizumab, BEV) therapy with FOLFOX6 in first-line metastatic colorectal cancer (mCRC) despite similar progression-free survival (PFS), highlighting limitations of imaging-based PFS as a surrogate for clinical benefit. We investigated whether ctDNA dynamics provide more refined prognostic information complementary to baseline (BL) ctDNA. Methods: Plasma samples collected from 556 patients (pts) with mCRC paired at pre-treatment (BL) and post-treatment (after treatment discontinuation) were profiled for ctDNA. Distribution of the highest variant allele fraction was modeled using a Gaussian mixture model. At pre- and post-treatment, pts were classified into 3 groups—High, Mid, and Low—according to ctDNA levels. OS, post-treatment survival (PTS), and PFS were compared across 9 ctDNA dynamic categories (eg, High→Low, High→Mid, High→High) based on changes from pre- to post-treatment. Results: Lower BL ctDNA was significantly associated with longer OS (median OS [95% CI]: Low, n = 97, 50.7 mo [44.0–65.2]; Mid, n = 159, 34.1 mo [26.9–37.1]; High, n = 300, 27.8 mo [26.2–31.3]). BL ctDNA level also correlated with tumor volume and mutation burden. Pts with lower ctDNA at treatment discontinuation had longer PTS. Incorporating ctDNA dynamics provided additional prognostic value. Regardless of BL ctDNA level, pts whose post-treatment ctDNA decreased to Low had the best survival (eg, High→Low: 50.9 mo [35.8–61.0]), while pts with persistently high ctDNA showed the poorest survival (High→High: 23.3 mo [18.2–27.8]). The prognostic value of ctDNA dynamics was consistent across treatment arms. Among pts with High BL ctDNA (PAN, n = 152; BEV, n = 148), a greater proportion achieved Low ctDNA at post-treatment with PAN vs BEV (29 [19.1%] vs 16 [10.8%], p = 0.053), whereas the proportions achieving Mid ctDNA were similar (52 [34.2%] vs 58 [39.2%], p = 0.40). In the High→Low/Mid ctDNA subsets, treatment discontinuation following curative resection was more frequent with PAN vs BEV (High→Low: 18/29 [62.1%] vs 7/16 [43.8%]; High→Mid: 11/52 [21.2%] vs 5/58 [8.6%]). OS and PTS trends were favorable with PAN vs BEV (OS; High→Low: 61.0 vs 43.1 mo, HR 0.49, 95% CI 0.23–1.04; High→Mid: 33.6 vs 26.8 mo, HR 0.74, 95% CI 0.50–1.11, PTS; High→Low: 55.1 vs 39.0 mo, HR 0.48, 95% CI 0.22–1.03; High→Mid: 20.6 vs 19.4 mo, HR 0.72, 95% CI 0.48–1.07). Conclusions: ctDNA dynamics add prognostic value beyond BL ctDNA level. In pts with high BL ctDNA, PAN yielded more conversions into low ctDNA levels and discontinuations following curative resection, with OS/PTS trends favoring PAN. These findings suggest that ctDNA dynamics may better reflect long-term clinical benefit than PFS, although standardized thresholds and prospective validation are needed. Clinical trial information: NCT02394834 ; NCT02394795 .

Papillary adenocarcinoma (Pap) is a relatively rare histological subtype of differentiated adenocarcinoma, and its biological behavior has not been fully elucidated. The present study aimed to clarify the clinicopathological characteristics of Pap. This study retrospectively included 1701 patients with differentiated-type gastric cancer who underwent gastrectomy at our institution between 2009 and 2021. Clinicopathological characteristics, recurrence patterns, and survival outcomes were compared between the two groups. Patients in the Pap group were older and showed higher rates of venous—and lymphatic invasion, as well as more advanced clinical and pathological stages. In the multivariate analysis of cumulative recurrence, histological type was identified as an independent predictor (HR 2.051, p = 0.037). The most common initial site of recurrence was the liver in both groups, with a significantly higher incidence observed in the Pap group (p < 0.001). In the multivariate analysis of overall survival, histological type was also identified as an independent prognostic factor (HR 1.962, p = 0.044). Pap showed poorer survival than the Tub group, with a particularly higher incidence of liver metastasis recurrence.

Abstract Background A new circulating biomarker superior to carbohydrate antigen 19–9 (CA19-9) is needed for diagnosing pancreatobiliary cancer (PBca). The aim of this study was to identify serum microRNA (miRNA) signatures comprising reproducible and disease-related miRNAs. Methods This multicenter study involved patients with treatment-naïve PBca and healthy participants. The optimized serum processing conditions were evaluated using t-distributed stochastic neighbor embedding (t-SNE) visualization. Serum miRNA candidates for disease association were selected using weighted gene coexpression network analysis (WGCNA). A miRNA signature combining multiple serum miRNAs was tested in exploratory, validation, and independent validation sets. The synthesis and secretion of diagnostic miRNAs were evaluated using human pancreatic cancer cells. Results In total, 284 (150 healthy and 134 PBca) of 827 serum samples were processed within 2 h of blood collection before freezing, distributed in the same area as that in the t-SNE map, and assigned to an exploratory set. The 193 optimized samples were assigned to either the validation (50 healthy, 47 PBca) or independent validation (50 healthy, 46 PBca) set. Index-1, a combination of five serum miRNAs (hsa-miR-1343-5p, hsa-miR-4632-5p, hsa-miR-4665-5p, hsa-miR-665, and hsa-miR-6803-5p) with disease association in WGCNA, showed a sensitivity and specificity of > 80% and an AUC outperforming that of CA19-9 in the exploratory, validation, and independent validation sets. The AUC of Index-1 was superior to that of CA19-9 (0.856 vs. 0.649, p = 0.038) for detecting T1 tumors. miR-665, a component of Index-1, was expressed in human pancreatic cancer cells, and its transfection inhibited cell growth. Conclusions The serum miRNA signature Index-1 is useful for detecting PBca and could facilitate the early diagnosis of PBca. These findings can help improve clinical PBca detection by providing an optimized biomarker that overcomes the limitations of the current standard.

Supplementary Figure S3. Method to generate macrophages and the phenotype of the macrophages.

Gastric and gastroesophageal junction cancer (G/GEJC) is a heterogeneous and complex disease characterized by histologic and molecular subtypes. Although a growing number of treatments have improved survival outcomes in the advanced setting, the greatest therapeutic benefits are observed among patient populations eligible for biomarker-directed therapies. Fibroblast growth factor receptor 2 isoform IIIb (FGFR2b) is an emerging biomarker under phase 3 clinical investigation for G/GEJC with the novel monoclonal antibody bemarituzumab. FGFR2b protein overexpression in gastric cancer, together with its function in various oncogenic signaling pathways, makes it an attractive target for precision medicine and thereby has gained clinical interest for its potential prognostic role in G/GEJC. Thus, to explore the potential role of FGFR2b, this narrative review summarizes the role and mechanism of FGFR2b in advanced G/GEJC, describes appropriate detection methodology for FGFR2b protein overexpression, and discusses future considerations for precision treatment in advanced G/GEJC with respect to FGFR2b protein overexpression and the emergence of other biomarkers.

Supplementary Figure S6. The strategy of MDSC induction by cancer cells.

Supplementary Figure S2. CD8+ T cell-attractant chemokine gene expression in UMAP plots of single-cell RNA-sequencing analysis.

Tyrosine kinase inhibitors are initially efficacious against anaplastic lymphoma kinase (ALK) fusion gene-positive lung adenocarcinoma, but acquired resistance inevitably occurs. Therefore, alternative treatment strategies are needed for tyrosine kinase inhibitor-resistant cases. Although the use of immune checkpoint inhibitors (ICI) has improved the prognosis of patients with lung cancer, patients with ALK+ lung adenocarcinoma exhibit little or no response to immunotherapy and the underlying resistance mechanisms remain unknown. In this study, we explored the immunologic status of the tumor microenvironment (TME) in ALK+ lung adenocarcinoma tissues. Tumor-infiltrating leukocyte analysis revealed reduced numbers of effector T cells and increased myeloid-derived suppressor cells (MDSC) relative to ALK- lung adenocarcinoma cases, indicating that ALK+ lung adenocarcinoma has a myeloid cell-dominant immunosuppressive TME. Single-cell RNA sequencing analysis identified a subset of macrophages that expressed most T cell-attractant chemokines (CXCL9, CXCL10, and CXCL11), and the macrophages were inactivated in ALK+ lung adenocarcinoma. In contrast, ALK+ lung adenocarcinoma expressed high levels of MDSC-attractant chemokines (CXCL1 and CXCL8). In addition, ALK+ lung adenocarcinoma showed higher levels of IL6, an MDSC-inducing cytokine, than ALK- lung adenocarcinoma. An IL6R inhibitor transformed the TME in a murine ALK+ lung adenocarcinoma model, shifting it from an immunosuppressive to a T cell-dominant status. Although ICI monotherapy lacked antitumor effects, a combination of ICI and the IL6R inhibitor had significant antitumor effects in mice. Our findings illustrate the molecular basis of fusion gene-mediated immunosuppressive TMEs, providing a rationale for a novel combination immunotherapy for ALK+ lung adenocarcinoma. See related Spotlight by Vitale and Bria, p.1326.

Despite recent advances in gastric cancer therapy, chemotherapy resistance and lack of methods for selecting combination regimens remain major problems. Organoids, which recapitulate the in vivo micro-environment, can be established from surgical specimens with a high success rate, and are widely used for drug sensitivity assays. Cancer organoids are also known to contain abundant amounts of cancer stem cells (CSCs), a subpopulation of tumor cells with self-renewal and differentiation capabilities. CSCs are thought to contribute to tumor initiation, metastasis, and acquiring resistance to chemotherapy by altering their phenotype during the treatment.In this study, we aimed to discover a novel biomarker for predicting multidrug resistance using gastric cancer organoids (GCOs), which provide in vivo-like culture system that more closely resembles tumor cell organization than traditional cell lines, to understand the mechanism of multidrug resistance in cells, including CSCs, under in vivo conditions and to identify novel biomarkers to predict chemoresistance.We evaluated 5-fluorouracil or oxaliplatin-resistant GCOs to find novel biomarkers that reflect multidrug resistance in gastric cancer. To examine the resistance mechanisms, RNA-seq analysis and ex vivo drug sensitivity testing were performed. The association of biomarkers with patient prognosis and chemotherapy efficacy was evaluated using three original cohorts with a total of 230 cases. The results were also validated with two independent public cohorts and a single-cell RNA sequence data. Increased expression of PI3/Elafin was detected in all 5-fluorouracil or oxaliplatin-resistant GCOs. Our findings suggest a potential association of PI3/Elafin expression with ribosome biosynthesis and RNA metabolism under organoid conditions. We also found that PI3/Elafin overexpression promotes 5-fluorouracil/oxaliplatin/cisplatin resistance but not paclitaxel resistance. Immunohistochemical evaluation of Elafin expression revealed that the Elafin-positive gastric cancer group had a poorer outcome, especially in terms of time-to-recurrence. Elafin positivity was also an independent predictor of relapse after chemotherapy with DNA-damaging agents. PI3/Elafin promotes DNA-damaging drug resistance through multiple downstream regulations related to RNA and ribosomal metabolism. We demonstrated that PI3/Elafin contributes to the acquisition of resistance to 5-fluorouracil and platinum drugs in in vivo-like environments and identified GOs related to ribosome and RNA metabolism as novel PI3/Elafin downstream targets. Our results provide new insights into multidrug resistance mechanisms and highlight PI3/Elafin as a novel tool for precise stratification of patients with gastric cancer. Kenji Harada, Naoya Sakamoto, Shingo Sakashita, Motohiro Kojima, Kazuaki Tanabe, Hideki Ohdan, Kohei Shitara, Takahiro Kinoshita, Genichiro Ishii, Wataru Yasui, Atsushi Ochiai, Shumpei Ishikawa. Identification of a novel marker gene predicting recurrence after chemotherapy with DNA-damaging agents by using drug-resistant gastric cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4420.

The incidence of esophagogastric junction (EGJ) cancer is increasing worldwide. Siewert type II EGJ cancer encompasses intestinal and gastric phenotypes; however, the molecular profiles and clinicopathological features remain unclear. Overall, 922 patients who underwent surgical resection for EGJ or gastric cancer from 2014 to 2023 were analyzed. The tumors were classified into intestinal and gastric phenotypes using immunohistochemistry. Molecular profiling was conducted using whole-exome sequencing, and clinicopathological features, mutational patterns, immune responses, and survival outcomes were investigated. The intestinal phenotype exhibited frequent TP53 mutations and high NOX1 expression. High NOX1 expression was correlated with increased CD4 + and CD20 + lymphocyte infiltration. The intestinal phenotype was associated with better relapse-free survival (RFS) than the gastric phenotype. Metastatic patterns varied, with peritoneal and lymph node metastases being more common in the gastric and intestinal phenotypes, respectively. High NOX1 expression was an independent prognostic factor for RFS. EGJ cancers with intestinal and gastric phenotypes demonstrate distinct molecular and immune profiles that influence prognosis. The intestinal phenotype, characterized by TP53 mutations, high NOX1 expression, increased immune cell infiltration, and better survival outcomes, may impact EGJ cancer prognosis and could guide future diagnostic and therapeutic approaches.

Supplementary Figure S9. Immunohistochemical (IHC) staining of PD-1+ and PD-L1+ cells, and macrophages in the tumor.

Despite recent advances in gastric cancer therapy, chemotherapy resistance and lack of methods for selecting combination regimens remain major problems. Organoids, which provide a culture system that more closely resembles tumor cell organization than traditional cell lines, can be established from surgical specimens with a high success rate and are widely used for drug sensitivity assays. In this study, we aimed to identify a novel biomarker for predicting multidrug resistance using gastric cancer organoids (GCOs). We evaluated 5-fluorouracil or oxaliplatin-resistant GCOs to find novel biomarkers that reflect multidrug resistance in gastric cancer. To examine the resistance mechanisms, RNA-sequencing analysis and ex vivo drug sensitivity testing were performed. The association of biomarkers with patient prognosis and chemotherapy efficacy was evaluated using three original cohorts with a total of 230 cases. The results were also validated with two independent public cohorts and single-cell RNA sequence data. Increased expression of peptidase inhibitor 3 (PI3) was detected in all 5-fluorouracil or oxaliplatin-resistant GCOs. Our findings suggest a potential association of PI3 expression with ribosome biosynthesis and RNA metabolism under organoid conditions. We also found that PI3 overexpression promoted 5-fluorouracil/oxaliplatin/cisplatin resistance but not paclitaxel resistance. Immunohistochemical evaluation of PI3 expression revealed that the PI3-positive gastric cancer group had a poorer outcome, especially in terms of time to recurrence. PI3 positivity was also an independent predictor of relapse after chemotherapy with DNA-damaging agents. PI3 promotes DNA-damaging drug resistance through multiple downstream regulations related to RNA and ribosomal metabolism. PI3 may be useful as a biomarker for the therapeutic selection of non-DNA-damaging agents. (c) 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Supplementary Figure S8. Gating strategies for mouse immune cell types.

Supplementary Figure S5.MDSC-abundant TME in fusion gene-positive LUAD.

Supplementary Figure S7. The suppressive activity of induced MDSC.