Biliary tract cancers (BTCs) are aggressive malignancies associated with a poor prognosis. Although molecular profiling is recommended to guide therapeutic decision-making, real-practice data on the prevalence and prognostic significance of genomic alterations in European BTC cohorts remain limited. This retrospective cohort study characterized the molecular landscape of BTCs in a Belgian real-practice setting and evaluated its prognostic relevance. Patients with BTC who underwent informative molecular testing as part of routine diagnostics at Antwerp University Hospital between 2016 and 2024 were included. Demographic, clinical, and molecular data were extracted from electronic health records. Survival outcomes were analyzed using Kaplan-Meier methods and Cox proportional hazards regression. Among 224 included patients, genomic alterations were identified in 59.4% of tumors, with clear subtype-specific patterns. IDH1 mutations (17.3%) and FGFR2 fusions (13.9%) were exclusively observed in intrahepatic cholangiocarcinoma, KRAS mutations were most prevalent in extrahepatic cholangiocarcinoma (42.9%), and ERBB2 amplification was enriched in gallbladder cancer (16.7%). Co-occurring alterations were present in 7.8% of cases. KRAS mutation was the most frequent alteration overall (20.4%) and remained significantly associated with worse overall survival in multivariate analysis adjusted for tumor subtype and tumor stage (HR = 1.54, 95% CI: 1.03-2.31, p = 0.04). Although 32.6% of tumors harbored a potentially actionable alteration, only 31.6% of eligible patients received matched targeted therapy. These findings underscore the clinical value of routine molecular profiling in BTC and highlight its importance for identifying therapeutic opportunities in clinical practice.

Chimeric antigen receptor (CAR) therapies have shown great success in hematological malignancies but remain largely ineffective against solid tumors such as pancreatic ductal adenocarcinoma (PDAC). A key obstacle among various aspects, is the dense stromal barrier formed by cancer-associated fibroblasts (CAFs), providing a rationale for simultaneously targeting stroma and tumor cells. Using immunohistochemistry of primary PDAC tumors and liver metastases, we confirmed high mesothelin (MSLN) expression on tumor cells, and CD70 expression on tumor cells and predominantly CAFs. Based on these results and the favorable safety profile of CAR natural killer (NK) cells over CAR T cells, we generated MSLN- and CD70-targeting IL-15-armored CAR NK cells. Both constructs mediated cytotoxicity against different pancreatic cancer and CAF cell lines with varying antigen expression in vitro, demonstrating that both, the CAR-molecule and IL-15 were required to increase functionality against more treatment-resistant cell lines. Interestingly, pooled MSLN- and CD70-CAR NK cells did not significantly improve cytolysis compared to monotherapies in an advanced 3D in vitro model or in vivo. Together these findings highlight the limitations of dual-targeting approaches and underscore the need for advanced engineering strategies to improve CAR NK cells beyond antigen targeting and cytokine support in the PDAC microenvironment.

The management of locally-advanced, resectable head and neck squamous cell carcinoma (HNSCC) is undergoing a major shift driven by the integration of neoadjuvant immunotherapy (nIO). The rationale for nIO lies in its administration within an immunologically active, treatment-naïve microenvironment that enhances immune priming and anti-tumor response. Despite encouraging clinical data, including the pivotal KEYNOTE-689 trial and multiple phase II studies, methodological heterogeneity in trial design, endpoint definitions, and response criteria currently hampers data comparability and the establishment of new standards of care. This expert narrative review proposes a structured framework for standardizing clinical, pathologic, imaging, and translational endpoints in HNSCC nIO trials, highlighting harmonized definitions of pathologic response, practical reporting templates, and methods to evaluate immune priming. Standardization of response evaluation, biomarker integration, and trial methodology is essential to accelerate the translation of neoadjuvant immunotherapy into routine clinical practice for HNSCC.

In oral squamous cell carcinoma (OSCC) surgery, inadequate tumor margins are reported in up to 85% of cases, adversely affecting outcomes. In this prospective, single-center study (n = 31; NCT04191460), we evaluated the safety and imaging feasibility of cRGD-ZW800-1, a near-infrared fluorescent integrin-targeted tracer. The secondary objective was to determine whether intraoperative fluorescence imaging could identify inadequate resection margins and inform surgical decision-making. Patients received 0.01, 0.025, or 0.05 mg/kg cRGD-ZW800-1, and tracer uptake was quantified using in vivo multi-diameter single-fiber reflectance and single-fiber fluorescence spectroscopy to optimize dosing and timing. All doses were well tolerated, achieving tumor-to-background ratios exceeding 4.5, with optimal performance at 0.025 mg/kg. Fluorescence imaging detected all 23 inadequate margins, including nine undetected by conventional assessment (sensitivity 100% versus 70%), altered surgical plans in five cases, and avoided adjuvant radiotherapy in three cases. These findings demonstrate that cRGD-ZW800-1 is safe, tumor-specific, and facilitates intraoperative margin assessment in OSCC. Incomplete tumor removal during oral cancer surgery remains a major clinical challenge. Here, the authors show, in a feasibility trial, that fluorescence imaging using the integrin-targeted tracer cRGD-ZW800 is safe, achieved a patient-level sensitivity of 100%, enabled additional fluorescence-guided resections and prevented postoperative radiotherapy in some patients with oral squamous cell carcinoma.

Abstract Background Despite its viral etiology and immunogenic features, cervical cancer shows limited and often short-lived benefit from programmed cell death protein 1 blockade, currently the only approved immunotherapy for this disease. This limitation highlights the need for a deeper understanding of its immune microenvironment to uncover alternative or complementary immunotherapeutic targets that may improve outcomes. Methods We integrated spatial proteomic and bulk transcriptomic profiling of the cervical cancer immune landscape. Immune subset markers (CD8, CD4, CD68, FoxP3, NKp46) and clinically actionable immune checkpoint molecules (HLA-E, CD47, CD73, CD276, CD155, Gal-9, PD-L1, CD70, LAG-3) were assessed by immunohistochemistry in 65 resected tumors, spatially resolved across tumor stroma and tumor epithelium niches. Expression patterns and correlations with clinicopathological variables, immunotypes, and survival were systematically analyzed. Key findings were cross-validated in The Cancer Genome Atlas cohort, and tumor-killing assays were conducted to evaluate the therapeutic potential of identified checkpoint axes. Results Immune infiltration was predominantly localized to the tumor stroma, with CD8+ and CD4+ T cells as dominant subsets. Within the tumor epithelium, CD8+ T cells and CD68+ macrophages were most abundant. Among checkpoints, HLA-E and CD47 showed the highest and most widespread expression, whereas CD276 and CD155 were enriched in the tumor epithelium, and CD73 and CD70 in the tumor stroma. Squamous cell carcinoma showed a stronger immunologic profile than adenocarcinoma. Immunotype stratification revealed distinct expression profiles and prognostic patterns. Elevated niche-defined expression of CD8, CD4, CD4-FoxP3, and NKp46 associated with improved survival. CD155 emerged as the only checkpoint consistently linked with poor survival across niches and cohorts, and associated with chemotherapy resistance. Notably, CD155 was the most promising functional target and was highly and selectively enriched in the tumor epithelium across all immunotypes, including immune-desert tumors where other immune markers were scarce. Conclusion This study reveals a complex, niche-specific and immunotype-specific immunoregulatory architecture in cervical cancer that extends well beyond programmed death-ligand 1. CD155 stands out as a compelling and underused therapeutic target, supporting a paradigm shift in targeting the T cell immunoreceptor with Ig and ITIM domains (TIGIT) axis. Its functional impact and selective enrichment in the tumor epithelium positions CD155 as a promising therapeutic target for both checkpoint inhibition and epithelial-directed approaches in cervical cancer.

Purpose In pancreatic ductal adenocarcinoma (PDAC), immune checkpoint inhibitors have shown limited efficacy, and the role of the TIGIT axis remains underexplored. This study aimed to characterize TIGIT axis components on protein level and their relationship to PD-1/PD-L1 expression in matched blood and tumor samples from PDAC patients to identify immunosuppressive mechanisms and fuel future strategies for immune checkpoint co-targeting in PDAC patients. Experimental design Fresh tumor and peripheral blood samples were collected from PDAC patients undergoing surgical resection. Flow cytometry was performed on tumor-infiltrating lymphocytes and PBMCs to assess expression of TIGIT, DNAM-1, TACTILE, and PD-1. Ligands CD111, CD112, CD113, and CD155 were analyzed using immunohistochemistry. Additional RNA expression analysis (TCGA/GTEx) was used to evaluate ligand distribution and gene expression profiles. Results TIGIT was highly upregulated on intratumoral CD8⁺ T cells and regulatory T cells, frequently co-expressed with PD-1. DNAM-1 expression was significantly reduced in tumors. However, contrasting pattern emerges with Tregs, which uniquely upregulate DNAM-1 in the PDAC TME. In addition, CD112 and CD155 were broadly expressed, including novel stromal CD112 localization. NK cells were nearly absent intratumorally, correlating with DNAM-1 downregulation. Conclusions Our findings identify TIGIT as a promising immunotherapeutic target in PDAC and suggest that dual checkpoint blockade (TIGIT/PD-1), alongside restoration of DNAM-1 signaling, may overcome immune suppression. These results provide mechanistic rationale to inform future clinical trials in PDAC. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-026-04343-w.

KRAS mutations are among the most prevalent oncogenic alterations in colorectal, lung, and pancreatic cancer, yet their detection remains analytically challenging in the presence of an overwhelming wild-type (WT) background. Here, we report a photoelectrochemical (PEC) genotyping platform that integrates clamp-inhibited loop-mediated isothermal amplification (C-LAMP) with enzyme-free singlet oxygen (1O2)-driven PEC transduction for mutation-selective KRAS detection. Locked nucleic acid (LNA) clamp probes selectively suppress WT amplification during isothermal amplification, enriching mutant alleles and enabling single-nucleotide variant (SNV) discrimination with high selectivity. Amplified products are magnetically captured and transduced into photocurrent via visible-light-induced 1O2 redox cycling, eliminating enzymatic reporters and reducing background interference. The C-LAMP/PEC platform achieves a limit of detection of 35 copies µL-1 (58 aM) and a minimum detectable variant allele frequency (VAF) of 4.8% in heterogeneous mutant/WT genomic DNA mixtures. Analytical performance was validated in cancer cell lines and in patient-derived fresh frozen tissues, showing complete concordance with Nanopore sequencing and droplet digital PCR (ddPCR) within the evaluated cohort (n = 16). This work introduces a robust and modular PEC biosensing strategy that combines molecular WT suppession with enzyme-free photoelectrochemistry, offering an economically competitive and instrumentation-simplified approach for clinically relevant KRAS mutation analysis toward decentralized testing.

Ki67 is a well-established proliferation marker in breast cancer. Current clinical use focuses on the proportion of Ki67-positive cells, ignoring spatial heterogeneity in expression. However intra-tumoral heterogeneity has demonstrated to be associated with worse outcome. We hypothesized that spatial Ki67 heterogeneity carries clinical information beyond conventional scoring and aimed to evaluate its added value for predicting pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) and for stratifying recurrence risk using genomic expression profiling (GEP). Using digital image analysis (DIA), precise and spatial quantification of biomarker distribution is possible. We analyzed two retrospective breast cancer cohorts using an AI-assisted DIA pipeline. Tumor sections stained for ER, PR, Ki67, and HER2 were digitized and analyzed in QuPath. Using AI, individual tumor cells were recognized and four tumor regions (0.5mm x 0.5mm) with the highest tumor/stroma ratio were selected for analysis. Spatial Ki67 heterogeneity was quantified using the Morisita-Horn Index (MHI) after the Ki67-positive and Ki67-negative tumor cells were mapped using XY-coordinates and square tessellation (100×100 µm tiles) was applied. The MHI was used to compare the similarity in cell composition between all pairs of tiles within a region. MHI values range from 0 to 1, with higher values indicating a more uneven distribution of Ki67+ cells. We used logistic regression and model comparison with Akaike Information Criterion (AIC), likelihood ratio test (LRT) or Vuong test, to evaluate the predictive value of Ki67 heterogeneity. In the first cohort (n=45), spatial heterogeneity was assessed on pretreatment biopsies from patients treated with NACT. In the second cohort (n=79), heterogeneity was evaluated in HR+/HER2- breast cancer patients stratified as high or low risk of recurrence based on GEP. In the GEP cohort, both a higher proportion of Ki67- positive cells and greater Ki67 heterogeneity were significantly associated with high genomic risk. The median MHI was 0.24 (0.03–0.35) in the high-risk group compared to 0.14 (0.01–0.43) in the low-risk group (P = 0.008). This higher MHI indicates more heterogeneous regionally clustered Ki67 expression, suggesting biologically distinct proliferative zones. In multivariate models, Ki67 heterogeneity remained a significant predictor of high-risk classification (OR 0.22, P = 0.036). Furthermore, in nested model comparison using LRT, addition of Ki67 heterogeneity significantly improved the model for predicting genomic risk (P = 0.034). These findings were consistent across biopsy and resection specimens, highlighting the robustness of heterogeneity measures. In the NACT cohort, Ki67 heterogeneity was higher in patients who achieved pCR (median MHI 0.26 [0.17–0.35]) compared to those who did not (median MHI 0.22 [0.12–0.40], P = 0.023). In multivariate modeling, Ki67 heterogeneity emerged as an independent predictor of pCR (OR 23.5, P = 0.038), outperforming Ki67 density and improving model fit (AIC 31.6 vs. 36.1; P = 0.038). Finally, in both cohorts, DIA-derived Ki67 models slightly outperformed traditional pathologist scoring, although Vuong tests did not show a statistically significant difference. Spatial Ki67 heterogeneity provides additional prognostic and predictive value beyond conventional Ki67 scoring. This heterogeneity indicates distinct areas of higher proliferation, clinically relevant biological variation, not captured by simple percentage positivity. Although validation in larger, prospective cohorts is necessary before clinical implementation, DIA provides a more objective and reproducible alternative to manual scoring, particularly when incorporating spatial heterogeneity. C. Van Berckelaer, K. Zwaenepoel, L. Cox, D. Charlotte, D. Julie, E. Louise, H. Fleur, L. Evy, G. R. Devi, A. Ramadhan, S. Koljenovic, P. Van Dam. Ki67 Spatial Heterogeneity as a Predictive and Prognostic Marker in Breast Cancer: A Spatial Image Analysis Approach [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-08-19.

PURPOSE OF REVIEW Achieving adequate resection margins (i.e., ≥5 mm of healthy tissue surrounding the tumor) in oral cavity squamous cell carcinoma (OCSCC) is difficult. This review discusses recent developments to guide surgical resection. It highlights the transition from the subjective conventional approaches to emerging, objective photonics-based methods. RECENT FINDINGS Specimen-driven intraoperative assessment of resection margins (IOARM) has significantly improved surgical outcomes. However, IOARM is subjective; moreover, it lacks widespread adoption due to reliance on a dedicated team of specialists. Raman spectroscopy is an objective, fast, nondestructive, and label-free technique that is suitable for IOARM. The latest Raman-based prototype demonstrates high precision. SUMMARY Integrating Raman-guided IOARM into the surgical-pathological workflow offers a practical, scalable approach to real-time, objective margin assessment, thereby improving patient outcomes.