Journal of Experimental & Clinical Cancer Research

BACKGROUNDWe have recently described SARS-COV-2 antigens showing sequence and conformational homology to tumor associated antigens (TAAs). Moreover, cross-reactive T cells have been identified in individuals either infected by the SARS-CoV-2 virus or vaccinated with the BNT162b2 preventive vaccine. In the present study, we analyzed the specific cross-binding TCRs by single cell RNA TCR sequencing. METHODS AND RESULTSThe paired SARS-CoV-2 epitope LLLDDFVEI (VIR) and the PRDX5 tumor associated antigen LLLDDLLVS (TAA) were selected to elicit cross-reacting T cells ex vivo. PBMCs from 5 healthy individuals were cultured for 10 days with 10 ug every 3 days of one of the two peptides and cells were selected for single cell RNA TCR sequencing. Results in CD8+ T Effector cells (TTE) showed the amplification or the de novo identification of a handful number of TRAV/TRBV genes and of CDR3{beta} motifs upon treatment ex vivo with both epitopes, which are specific for each subject in the analysis. The very same clonotypes were identified also in the CD8+ T proliferating subset, confirming that both epitopes induced a highly activated and plastic state. Conformational prediction analyses of pMHC-TCR complexes showed perfect structural overlap, supporting the functional cross-reaction of CD8+ T cells with both the viral and the tumor antigens. CONCLUSIONSOur results describe for the first time the TCR CDR3{beta} motifs amplified or de novo expanded by induction with a viral antigen showing a molecular mimicry with a tumor antigen. They are strictly individual and do not match with any motif in the publicly available TCR repository. However, considering the significant degeneracy in the TCR binding to the same epitope, the finding of identical TCR CDR3{beta} motifs elicited by two homologous epitopes is of the highest functional relevance. Such results provide a clear experimental validation proof that microbial epitopes mimicking TAAs can be used to develop off-the-shelf preventive/therapeutic vaccine formulations. Indeed, such non-self antigens are much stronger immunogens and may elicit a potent cross-reacting anti-cancer T cell response.

BackgroundImmune checkpoint inhibitors (ICIs) targeting the programmed death (ligand)-1 (PD-1/PD-L1) axis, like pembrolizumab, have significantly improved survival in non-small cell lung cancer (NSCLC). However, less than 50% of patients respond. Identifying early-response biomarkers is crucial to personalize therapy, thereby preventing ineffective, expensive and potentially harmful treatment. MethodsWe applied a novel ex vivo immunopharmacological bioassay to assess pembrolizumab-dependent T cell signalling in baseline peripheral blood mononuclear cells (PBMCs) from 64 NSCLC patients. PBMCs were stimulated with anti-CD3/CD28 with or without pembrolizumab, and phosphorylation states of PD-1-dependent T cell receptor (TCR) signalling pathways were measured by spectral flow cytometry. A composite signalling score was calculated representing the net pembrolizumab-induced phosphorylation response and patients were classified as low, optimal and high modulation responders based on this signalling score. Associations with progression-free survival and overall survival (OS) were evaluated using univariate Cox regression. ResultsPatients with optimal baseline pembrolizumab-induced signalling scores exhibited significantly higher signalling score outcomes than those with low modulation (p < 0.0001) and lower than patients with excessive modulation (p < 0.01) and had significantly longer OS (HR = 2.83, p = 0.013; and HR = 12.05, p = 0.003, respectively). Notably, conventional pharmacodynamic parameters, including half-maximal effective concentration (EC50) for PD-1 receptor occupancy and maximum IL-2 production (Emax), were not associated with clinical outcomes, underscoring the unique predictive value of the phosphorylation-based signalling score. In vivo, pembrolizumab-induced T cell activation changes and TCR signalling inhibition post-treatment correlated with shorter survival (HRs = 1.33-1.95), consistent with our ex vivo findings. ConclusionsWe demonstrate that a pretreatment signalling score derived from ex vivo pembrolizumab-modulated T cell phosphorylation identifies clinical response in NSCLC. This functional bioassay offers a novel approach to identify patients most likely to benefit from ICI therapy, potentially enabling personalised treatment decisions before therapy initiation. Graphical abstract textOur findings reveal that pretreatment, pembrolizumab-dependent modulation of T cell phosphorylation identifies clinical response in NSCLC. Furthermore, we introduce an overall signalling score, reflecting the net phosphorylation profile, which could serve as a potential predictive biomarker to distinguish responders from non-responders, thereby supporting biomarker-driven therapeutic strategies.

The JAK-STAT pathway (JSP) is a well-known oncogenic cascade; however, recent clinical trials have detected JSP upregulation in breast cancer following anti-PD1 immunotherapy. This paradoxical observation warrants further investigation into JSPs intercellular heterogeneity, tumor dynamics, molecular mechanisms, and clinical implications for immunotherapy. JSP expression showed dynamic shifts during breast cancer progression, with higher levels in T cells and para-cancerous epithelial cells. In tumor cells, elevated JSP highly correlated with malignant phenotypes. JSP-high tumor cells overexpressed oncogenic pathways, while exhibiting increased immunosuppressive signaling via MIF-CD74 signaling axis. In T cells, higher JSP levels were associated with enhanced cytotoxic activity, improved differentiation, and reduced exhaustion, reflecting robust anti-tumor immunity. Analysis of immunotherapy datasets revealed that higher JSP levels were associated with improved responses towards PD-1 inhibitors, particularly in triple-negative breast cancer (TNBC) patients, with JSP serving as a predictive biomarker for immunotherapy sensitivity. As a key JSP component, STAT4 exerts dual roles in breast cancer: it drives tumorigenesis in malignant cells, sustains breast epithelial cell proliferation, and bolsters T cell anti-tumor functionality--while also acting as a highly accurate biomarker for predicting immunotherapy response. This indicates that JSP targeting demands a nuanced approach: broad inhibition may impair anti-tumor immunity, and optimized therapeutic strategies paired with precise biomarkers are critical to maximize JSPs utility in breast cancer immunotherapy. Our findings highlight JSPs functional heterogeneity in epithelial, tumor, and T cells, with high JSP activity correlating with enhanced immunotherapy efficacy in breast cancer. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/703506v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@17f041corg.highwire.dtl.DTLVardef@1e6f724org.highwire.dtl.DTLVardef@6b8784org.highwire.dtl.DTLVardef@18e1c90_HPS_FORMAT_FIGEXP M_FIG C_FIG

Testicular germ cell tumours (TGCTs) are the most common cancer in young males and are considered curable if they respond to platinum-based therapy. However, a significant number of refractory patients develop metastatic disease and the lack targeted therapy remains an unmet clinical need. To identify novel therapeutic targets, we investigated the epigenetic instability of TGCTs and characterised novel oncogenic gene networks regulated by transposable elements (TEs)-derived long noncoding RNAs (lncRNAs) which are controlled by PIWI-interacting RNAs (piRNAs). A TGCT-specific piRNA signature was identified by bioinformatics analysis of the The Cancer Genome Atlas (TCGA) TGCT dataset and analysis of piRNAs mapped to active LINE1 sequences identified piR-hsa-7221 as a transcriptional regulator of the lncRNA CASC9 in seminoma tumours. We show that piR-hsa-7221 binds to a complementary LINE1 LIPA5 sequence and regulates the expression of CASC9 driven by the LINE1 antisense promoter. Therefore, loss of piR-hsa-7221 drives the upregulation and oncogenic activity of CASC9, which as is impaired after silencing, leading to reduced cancer cell proliferation and invasion, as well as increased sensitivity to cisplatin treatment. These effects are associated with the regulation of the cell cycle, developmental pathways, extracellular matrix, hormone metabolism and immune responses, highlighting WNT signalling as a significant downstream target. Therefore, this novel epigenetic mechanism provides new insights into the role of piRNA-mediated regulation of oncogenic lncRNAs derived from active transposable elements. Importantly, the identification of piR-hsa-7221 and the lncRNA CASC9, together with the associated gene networks highlights novel therapeutic targets for the treatment of seminoma TGCTs.

ObjectivesHomologous recombination repair (HRR) deficiency is associated with improved immunotherapy responses in non-small cell lung cancer (NSCLC) patients. The HRR genes BRCA1/2 are key regulators of DNA repair, yet their impact on the tumor microenvironment (TME) in lung adenocarcinoma (LUAD) remains unclear. MethodsUsing single-cell sequencing and multi-omics data, we characterized BRCA1/2 mutation-associated transcriptional programs, immune cell composition, and functional alterations in T cells, investigating the molecular and immune architecture of BRCA-mutant LUAD patients. ResultsBRCA1/2 mutations were associated with increased genomic instability and poor prognosis in LUAD patients, but predicted better clinical outcomes following immune checkpoint blockade (ICB) treatment. BRCA1 mutations correlated with an upregulated type I IFN/IFN-{gamma} signature and CD8+ T cell activation. BRCA2 mutations were associated with alveolar/stress/inflammatory responses and enhanced MHC-II antigen presentation, linked to CD4+ T cell differentiation. Both alterations coincided with reduced CD28 co-stimulation and CTL activity, hinting of immune evasion. We identified two tissue-resident memory T cell (Trm) subsets as predictors of clinical outcomes and ICB response. BRCA1 mutations were associated with CD8+ Trm expansion, whereas BRCA2 mutations linked to tumor CD4+ Trm expansion and peripheral T/NK cell cytotoxicity. Furthermore, a cancer-promoting program activated by BRCA1 mutation was vulnerable to histone deacetylase inhibitors, which inhibited LUAD tumor growth. ConclusionsThis study provides a preliminary characterization of the BRCA-mutant TME in LUAD patients, revealing distinct transcriptional and immune patterns that highlight differences in BRCA1/2-associated molecular architecture and offer a framework for improving therapy efficacy in LUAD.

Despite vaccination, cervical tumors remain a health issue and require treatment improvement. In 2023, Pembrolizumab, an anti-PD-1 immune checkpoint blockade (ICB), was introduced in the case of advanced or metastatic cervical tumors. This treatment significantly increased progression free survival from 20% to 50%. However, some patients remain resistant to anti-PD-1 treatment, which calls for new targets. In our study, we highlighted poliovirus receptor (PVR) and Nectin-2 as potential ICB targets. Indeed, PVR and Nectin-2 are TIGIT ligands, an immunomodulatory checkpoint expressed by regulatory T cells or exhausted T cells. The binding of PVR or Nectin-2 to TIGIT maintains the immunosuppressive signal in the immune cells allowing tumor progression. Furthermore, we observed that PVR and Nectin-2 were highly expressed on tumor cells and tumor associated macrophages (TAMs) accross the different histological subsets of cervical tumors. Therefore, we hypothesized that using anti-PVR and anti-Nectin-2 anti-bodies would lift immunosuppression in cervical tumors. To that end, we used CyTOF to asses precise immunophenotyping of the targets ex vivo, high throughput confocal microscopy to assess phagocytosis, monocyte derived macrophages (MDM) coupled with 3D cell culture models to assess the impact of our treatment on MDM and TAM repolarization and tumor growth. We could demonstrate that our treatment repolarized macrophages towards an inflammatory profile and that this was followed by a reactivation of macrophage cytotoxic function such as phagocytosis. We also demonstrated that anti-PVR and Nectin-2 treatment allowed the control of tumor growth in 2D and 3D cell culture models. We could also develop a pre-clinical model of autologous cell culture from cervical cancer patients. Using the MIVO technology, which combine organotypic culture and fluidics, we could assess peripheral blood mononuclear cells recruitment towards tumor cells in the presence or absence of anti-PVR and anti-Nectin-2. In conclusion we could demonstrate that targeting macrophages via the PVR/Nectin-2 couple reactivates cervical tumor growth control and improves immune cell recruitment.

The pivotal role of tumor infiltrating myeloid cells in lung cancer composition and response to therapy is universally recognized. Nevertheless, their main cradle being the bone marrow (BM), remains vastly understudied owing to the spatiotemporal complexity of hematopoiesis and its hard to access anatomical location. Therefore, the BM niche of lung cancer subjects remains understudied which is why we integrated transcriptional and translational single-cell profiling, ELISA and two-photon microscopy to characterize the medullary hematopoietic compartment in orthotopic lung cancer-bearing mice with validation in human non-small cell lung cancer (NSCLC) samples. In brief we found that lung cancer remotely alters the entire hematopoietic process resulting in higher levels of hematopoietic stem cells (HSCs), myeloid and lymphoid multipotent progenitors (MPPs) and downstream predominance of Granulocyte Monocyte Progenitors (GMP), early Granulocyte Progenitors (GP) and Common Monocyte Progenitors (cMoP) at the expense of mature neutrophils and B cells. Furthermore, a significant increase in the expression and secretion of S100A9 and Lipocalin-2 (LCN2), was characteristic across the entire hematopoietic trajectory in lung cancer-bearing mice and patients. In vivo inhibition of S100A9 with Tasquinimod reduced tumor growth, irrespective of its combination with immunotherapy. In addition, it altered the secretion profile of S100A9 but also LCN2 in the BM, suggesting that S100A9 serves as an upstream regulator of LCN2 and holds therapeutic premise to treat immunotherapy refractory lung cancer.

Background&AimsPatients with microsatellite-instable (MSI) colorectal cancer (CRC) benefit from immune checkpoint therapy. For patients with microsatellite-stable (MSS) non-MSI tumors targeting alternative immune checkpoints, such as the Poliovirus receptor (PVR/CD155), may extend response to checkpoint inhibitors and thereby improve outcomes. The drugable transcription factor REVERB (NR1D1) is a master repressor of macrophage function. We hypothesized that regulation of PVR allows elimination of tumor cells by macrophage-directed precision therapy. MethodsHuman CRC cell lines (MSS+: HT29, SW480; MSI+: HCT116), patient-derived organoids (MSS+ PDOs) and tissues were assessed by PCR, Western blot, immunohistochemistry and flow cytometry. 3D co-cultures of CRC cells with macrophages derived from THP1 monocytic leukemia cells or peripheral blood of healthy donors were analysed by microscopy and viability assays. Functional perturbation of PVR and REVERB was achieved by CRISPR/Cas9-sgRNA gene modification, synthetic ligands or blocking antibodies (Abs). ResultsREVERB bound a cognate DNA-element in the -1 kb human PVR gene promoter, and its agonist (SR9009) super-repressed, whereas its antagonist (SR8278) de-repressed transcription of PVR mRNA in macrophages. Macrophages with CRISPR-modified REVERB were unresponsive to ligand, devoid of PVR protein, showed more phagocytosis, tumor cell efferocytosis and expression of genes related to host immunity (PDL1, TLR4 e.a.) than clones with the wild-type receptor. Macrophages lowered the viability of tumor cells, potentiated by PVR blocking Ab or PVR knock-out in tumor cells. Consistently, REVERB antagonist augmented tumor cell death in co-cultures with macrophages and PVR blocking Ab. ConclusionCo-addressing the checkpoint axis REVERB-PVR may represent a novel intervention strategy for patients with MSS+ CRC.

BackgroundTreatment optimization in HPV-associated oropharyngeal cancer (OPSCC) remains challenging, as recent de-escalation trials have shown limited success. Current patient selection strategies based on smoking history and TNM classification are insufficient, highlighting the need for robust, standardized prognostic biomarkers. We report the first validation of the Immunoscore (IS) for prognostic stratification in HPV-associated OPSCC. Patients and methodsWe analyzed 191 HPV-associated (p16+ and HPV DNA/RNA+) OPSCC patients from an international multicenter cohort (2015-2024), comprising a French monocentric retrospective training cohort (N = 48) and three validation cohorts: French monocentric retrospective (N = 48), French multicenter prospective (N = 50), and US multicenter retrospective (N = 45). IS is a standardized digital pathology assay quantifying CD3lJ and CD8lJ densities in tumor cores and invasive margins, with cut-offs defined in the training cohort and validated across cohorts. Associations with disease-free survival (DFS), time to recurrence (TTR) and overall survival (OS) were assessed, alongside 3RNA-seq and sequential immunofluorescence profiling of immune composition. ResultsMedian age 65; 80% male; 74% smokers; 66% T1-2; 82% N0-1 (AJCC8th). IS-High patients demonstrated superior 3-year DFS in the training and validation cohorts 1-3 (all log-rank P < 0.05). Multivariable analysis identified IS-Low as the strongest independent risk factor for DFS (HR 9.03; 95% CI: 4.02-20.31; P < 0.001). The model combining IS with clinical factors showed higher predictive accuracy for DFS (C-index 0.82) than clinical variables alone (0.7; P < 0.0001). Similar findings were observed for TTR and OS. IS-High tumors showed markedly higher enrichment of lymphoid and myeloid immune cell populations, contrasting with immune-poor signatures in IS-Low tumors. ConclusionsIS is a robust biomarker that outperforms standard clinical variables in both prognostic and predictive accuracy. The enriched cytotoxic immune infiltrate in IS-High tumors explains favorable outcomes and supports their suitability for treatment de-escalation. Prospective validation is warranted.

Autophagy, a key lysosomal degradation pathway regulating metabolic adaptation in cancer, plays fundamental roles in both the tumor and host stromal compartments during cancer progression. An important unanswered question is whether and how autophagy in specific host stromal elements, such as endothelial cells, influences metastasis. Here, we scrutinize how the genetic loss of autophagy in endothelial cells impacts primary tumor progression and metastasis in the Polyoma Middle T (PyMT) model of luminal B breast cancer. In both autochthonous and orthotopic mammary transplant models, PyMT primary tumor growth is significantly delayed upon endothelial cell Atg12 or Atg5 genetic deletion (Atg12 or 5 ECKO), which correlates with increased tumor cell apoptosis and HIF1 activation. In contrast, PyMT-bearing Atg12 ECKO mice exhibit increased metastasis, as well as higher rates of primary tumor and lung metastatic recurrence following surgical resection of PyMT primary tumors. Experimental metastasis assays further corroborate that loss of endothelial cell autophagy in Atg12 ECKO host animals promotes PyMT metastatic colonization and outgrowth, resulting in increased lung metastases compared to controls. Similarly, in the Rat Insulin Promoter T antigen pancreatic neuroendocrine tumor (RT2-PNET) model, endothelial cell deletion of Atg12 promotes liver micro-metastases. Taken together, these results from distinct preclinical cancer models reveal that endothelial cell autophagy suppresses metastatic seeding and progression and broach that autophagy inhibition in host endothelial cells may adversely influence the efficacy of systemic autophagy-lysosomal pathway inhibition in the clinical oncology setting.

Alternative lengthening of telomeres (ALT) is a telomere elongation mechanism activated during oncogenesis and primarily acting in tumors of mesenchymal origin. Although the proteins involved in the machinery enabling ALT to elongate telomeres are becoming better understood, the underlying biology of this mechanism remains unclear. In the present study, we took advantage of a fully characterized cohort of 98 leiomyosarcomas (LMS) from the French Sarcoma Group to further our understanding of the ALT mechanism. We first compared the transcriptomic profiles of ALT+ and TERT+ LMS and demonstrated a strong enrichment of the CINSARC signature in ALT+ tumors. The establishment of an ALT+-related signature in these LMS confirmed the close association between CINSARC and ALT in two additional cohorts of non-translocation-related sarcomas. In vitro mesenchymal models of spontaneous ALT induction showed increased CINSARC expression following acquisition of the ALT mechanism. Conversely, ALT inactivation, through BLM inhibition, led to decreased CINSARC expression. These results establish CINSARC as a new hallmark of the ALT mechanism in non-translocation-related sarcomas and demonstrate the association of a cellular biological process with the CINSARC prognostic signature, namely the ALT mechanism.

BackgroundCutaneous melanoma (CM) is an aggressive skin cancer with rising incidence, representing a growing public health concern. Despite the remarkable success of immune-checkpoint inhibitors (ICIs) in the management of advanced disease, mortality remains high due to therapy resistance. Identifying reliable prognostic and predictive biomarkers is therefore essential to improve patient stratification, optimize treatment selection, and minimize unnecessary toxicity. MethodsWe comprehensively profiled the circulating immune landscape of 54 treatment-naive CM patients by integrating flow cytometry immunophenotyping with clinicopathological data, and performed tumor gene expression analysis in a subset of 26 patients. ResultsElevated HLA-DR and CD69 expression on circulating CD4+ T cells, together with reduced circulating CD8+ T cell frequency, emerged as candidate prognostic biomarkers associated with improved survival. Prognostic models combining these immune variables with clinical covariates accurately stratified patients by overall survival (89.5% sensitivity, 72.7% specificity; AUC = 0.872, p < 0.0001) and progression/recurrence risk (75% sensitivity and 71.4% specificity; AUC = 0.763, p = 0.001). In a subset of 43 patients subsequently treated with ICIs, elevated baseline HLA-DR and CD69 expression on circulating CD4+ T cells was also associated with therapeutic benefit. A predictive model integrating these markers with clinical covariates achieved good discriminatory performance (65.2% sensitivity, 88.9% specificity; AUC = 0.775, p = 0.0027). Tumor gene expression profiling supported the role of IFN-{gamma}-related signatures, previously linked to ICI response, as complementary prognostic and predictive tools. ConclusionThese findings highlight systemic CD4+ T cell activation status as a promising, easily measurable biomarker in CM, laying the foundation for future strategies to refine patient stratification and guiding immunotherapy decisions.

BackgroundSequential alterations in APC, KRAS, TP53, and SMAD4 have been proposed as a framework for colorectal cancer progression. Human colorectal cancer datasets have not revealed the biological transitions associated with these mutations. When examining a cohort of TCGA-colorectal tumors grouped as AK (APC/KRAS), AKP (APC/KRAS/TP53), and AKPS (APC/KRAS/TP53/SMAD4), we observed no significant differences in immune-cell composition, four previously defined Consensus Molecular Subtypes (CMS1/2/3/4), or transcriptomic clustering between these genomic groups. Therefore, these canonical alterations do not sufficiently characterize the known properties of metastatic progression in human colorectal cancer. MethodsTo overcome these limitations, we developed a genetically defined, organoid-based, orthotopic mouse model whereby mouse colon organoids modeling sequential APC, KRAS, TP53, and SMAD4 alterations were orthotopically injected into the colon. This was followed by RNA-sequence processing, normalization with DESeq2, differential expression, pathway enrichment, and immune/stromal inference. Gene co-expression modules were identified from variance-stabilized mouse expression data, mapped to 1:1 human orthologs, and summarized as eigengenes. A multinomial logistic regression model trained on mouse eigengenes was applied to TCGA-COAD human tumors to assign them to mouse-informed transcriptomic states (AK-like, AKP-like, AKPS-like), which were then used for downstream visualization and comparative analyses. ResultsWhole-transcriptome analysis revealed discrete transcriptional states and immune-cell differences between the organoid AK/AKP/AKPS groups. Early TP53 loss led to strong activation of immune pathways, accompanied by increased infiltration of NK and T cells. As tumors progressed with SMAD4 loss and metastasis, this immune activity collapsed, giving rise to broad immune suppression. CMS classifications also shifted, with AK tumors resembling epithelial CMS2, AKP tumors displaying immune-rich CMS1 features, and AKPS and metastatic lesions adopting mesenchymal CMS4 characteristics. We then applied a progression-based transcriptomic classifier to 460 human colorectal tumors. This reclassification revealed conserved immune remodeling, CMS transitions, pathway-level differences, and significant differences in patient survival. ConclusionWe show that organoid-derived progression profiles reveal hidden evolutionary structure in human colorectal cancer and provide a transcriptional framework for interpreting metastatic potential and clinical outcomes.

Background: Prostate cancer (PCa) presents a formidable clinical paradox. It is immunologically cold and resistant to immune checkpoint blockade (ICB), yet bulk genomic analyses consistently reveal low and non-prognostic expression of CD274 (PD-L1), the primary molecular target of such therapies. We hypothesised that this paradox arises from a failure of current methodologies to account for two critical, interacting dimensions: the granular heterogeneity of basal gene expression (the static engine) and the spatiotemporal dynamics of adaptive resistance mediated by interferon-gamma (the adaptive engine). Methods: We developed a rigorous, multi-phase computational framework integrating clinical genomics with hybrid agent-based modelling. In Phase I, we extracted and normalized CD274 mRNA expression from the TCGA-PRAD cohort (n = 554) to define the empirical landscape of basal resistance. In Phase II, we developed a spatial Agent-Based Model (ABM) parameterized by this distribution to simulate clonal selection. In Phase III, we extended this into a Hybrid Discrete-Continuum model, coupling discrete agents with a reaction-diffusion Partial Differential Equation (PDE) representing the IFN-{gamma} field. We simulated 50 stochastic replicates per arm across four experimental arms, including Diffusion and Induction knockouts. Results: Bulk TCGA analysis confirmed low average PD-L1 expression (Median Transcripts Per Million (TPM) = 1.48; Interquartile Range (IQR): 0.91-2.14) with no prognostic value (Hazard Ratio (HR) = 1.15; 95% Confidence Interval (CI): 0.67-1.97; log-rank p = 0.605). However, the static ABM revealed that rare, high-expressing genomic outliers (>9.0 TPM) drive persistence through Darwinian immunoediting, enriching the surviving population's resistance by 3.86-fold. The hybrid adaptive model demonstrated a far superior survival strategy: the IFN-{gamma}/PD-L1 feedback loop facilitated the emergence of "protective sanctuaries"-localised regions of high resistance at the tumour-immune interface. This mechanism increased final tumour burden by ~4.5-fold compared to static selection alone (p<0.001). Spatiotemporal analysis confirmed that resistance is not a fixed trait but a dynamic state induced by immune pressure. Diffusion knockout (D = 0) abolished sanctuary formation, reducing final burden by 65% (p<0.001), while induction knockout (Pmax = 0) reverted to static outcomes. Conclusions: This study resolves the cold tumour paradox by demonstrating that PCa resistance is driven by a twin engine of rare genomic outliers and adaptive spatial dynamics. The failure of biomarkers in PCa is due to their inability to capture the dynamic mirage of adaptive sanctuaries. Our validated framework offers a platform for testing synchronised therapeutic disruptions targeting both the static genomic landscape and the dynamic cytokine signalling axis.

Cancer remains a major therapeutic challenge despite substantial advances in diagnosis and treatment, including immune checkpoint blockade. Among emerging immunotherapeutic approaches, adoptive cell transfer (ACT) has attracted growing interest. Human peripheral V{gamma}9V{delta}2 T cells are promising candidates for ACT because they combine rapid and potent antitumor functions with major histocompatibility complex (MHC)-independent tumor recognition, enabling allogeneic use with limited risk of graft-versus-host disease. This raises the possibility of generating standardized V{gamma}9V{delta}2 T-cell banks from healthy donors for off-the-shelf immunotherapy. Here, we provide preclinical evidence supporting the suitability of allogeneic human V{gamma}9V{delta}2 T cells for ACT. We characterized peripheral blood V{gamma}9V{delta}2 T cells from healthy donors after successive antigen-specific and non-specific amplification steps, assessing their phenotype, effector functions, and metabolic state. Amplified cells maintained a strong pro-inflammatory Th1-like profile, preserved cytotoxic activity, and did not produce immunoregulatory cytokines. They also displayed high purity, a predominant effector memory phenotype, reduced expression of several inhibitory immune checkpoints, and sustained antitumor reactivity. Altogether, these findings support the development of allogeneic V{gamma}9V{delta}2 T-cell products as a scalable platform for next-generation cancer immunotherapies.

Thoracic malignancies, including lung adenocarcinoma (ADC) and malignant pleural mesothelioma (MPM), remain associated with poor prognosis and limited durable therapeutic responses in advanced stages. Although targeted therapies and immunotherapy have improved outcomes in selected patients, systemic chemotherapy continues to play a central role in routine clinical practice. However, treatment response is highly heterogeneous, and reliable predictive biomarkers of chemotherapy sensitivity are lacking. Both ADC and MPM frequently involve the pleural cavity and are commonly associated with malignant pleural effusion (MPE), which contributes to symptoms such as dyspnea and chest pain and requires therapeutic drainage. Importantly, MPE represents a clinically accessible source of viable tumor cells obtained through minimally invasive procedures. In this study, we established patient-derived organoids (PDOs) from malignant pleural effusion samples obtained from five patients with advanced lung adenocarcinoma and, as an exploratory extension, from one patient with malignant pleural mesothelioma. Organoids were characterized by immunohistochemistry and subjected to systematic chemotherapy drug screening. Inter-model variability in treatment response was assessed, and selected drug sensitivities were further validated through dose-response assays. Pleural effusion-derived organoids successfully recapitulated tumor-specific phenotypic features and revealed marked heterogeneity in chemotherapy sensitivity across models. Secondary validation confirmed the reproducibility of selected responses. Our findings support the feasibility of generating functional organoid models from malignant pleural effusions and highlight their potential as translational platforms for individualized chemotherapy profiling in advanced thoracic malignancies.

Anal squamous cell carcinoma (ASCC) is a rare malignancy associated with high-risk HPV, with rising incidence among younger adults. While immunotherapy has improved outcomes in metastatic ASCC, treatment for localized disease remains largely unchanged, with high recurrence rates. This study provides comprehensive exome and transcriptome profiling of 40 stage I-III non-metastatic ASCC patients treated with curative chemoradiotherapy (CRT) to identify predictors of treatment response and progression-free survival. Transcriptomic analysis revealed 350 differentially expressed genes between complete responders (CR) and non-complete responders (NCR) (p-value<0.01; FC>2). CR was associated with modulation of immune-related pathways, cytokine production, epidermis development, cell differentiation, and signaling pathways associated with TNFA/NFkB and epithelial to mesenchymal transition. Immune infiltrate analysis showed significant enrichment of CD8+ central memory T cells (p=0.008) in CR cases, correlating with increased tertiary lymphoid structure and improved overall (p=0.0026) and disease-free survival (p=0.0098). Exome-seq identified alterations in novel and known cancer driver genes without association to CRT response, despite high tumor mutational burden (TMB) was significantly associated with shorter overall (p=0.03) and disease-free survival (p=0.027) compared with low TMB cases. These findings highlight the potential of incorporating gene expression signatures (e.g., FDCSP, ALDOB, ADGRB1, SPINK7) alongside immune-related markers into clinical practice to enhance the prediction of treatment response and guide personalized therapies in ASCC. A robust and functionally active immune microenvironment, characterized by specific T and B cell populations and the presence of tertiary lymphoid structures, emerges as a hallmark of complete response and improved survival in ASCC patients undergoing chemoradiotherapy.

Malignant peripheral nerve sheath tumours (MPNSTs) are high grade soft-tissue sarcomas with an unmet need for novel therapies. Tumour antigen-based approaches, including neoantigen and tumour-associated antigen (TAA) directed therapies, offer potential opportunities for immunotherapy. Here, we integrated public domain tumour DNA and RNA sequencing data with in-silico prediction to systematically characterise the (neo)antigenic landscape of MPNST. We stratified the predictions across the two known sub-groups of MPNST, those associated without and with Polycomb Repressor Complex 2 (PRC2) loss of function variants (PRC2-Loss). Using computational pipelines including pVACtools, we identified high-confidence neoantigens based on pMHC affinity derived from somatic mutations and gene fusions, as well as recurrently overexpressed cell-surface TAAs. All predicted neoantigens were private to individual MPNST cases, with different neoantigens across both tumour subtypes. PRC2-Loss tumours showed reduced immune infiltration with downregulation of antigen processing and presentation pathways compared to PRC2-WT, confirming intrinsic constraints to effective neoantigen-directed immune priming. Moreover, PRC2-Loss MPNSTs demonstrated recurrent copy number driven overexpression of cell surface TAAs (chromosome 8), providing alternative immunotherapeutic targets that are pMHC independent. These findings confirm a PRC2-independent private immuno-antigenic peptide repertoire with an immune resistant MPNST microenvironment in PRC-Loss. These data provide further impetus for rational development of complementary immune based treatment strategies, including personalised neoantigen vaccines and cell surface protein TAA-directed therapies dependent on PRC2 status.

Cancer heterogeneity is traditionally attributed to multiple parallel signaling pathways. This belief is challenged here by proposing the ER/PR axis as the dominant pathway underlying the full spectrum of breast cancer. Absolutely quantitated ER, PR, Her2 and Ki67 protein levels were accumulated over 8 years from 1652 specimens collected non-selectively and measured with Quantitative Dot Blot (QDB) method over time. Cox analysis showed ER and Ki67 as independent adverse prognostic factors while PR was an independent favorable factor statistically. Their optimized stratification framework demonstrated that prognosis across all clinical subtypes was predominantly aligned along the ER/PR axis rather than being subtype-specific, including repeated identification of a subgroup with near-perfect 10-year survival probability from three independent cohorts to be proposed as the biological basis of the ultra-safe group in MINDACT trial. A parsimonious model is proposed where the ER/PR signaling hierarchy supersedes current prevailing clinical subtyping, with its balance essential for survival until ER levels become uncontrollable. This concept of pathway hierarchy may also exist in other major cancer types, and cannot be addressed without clinical epidemiology.

Head and neck cancer (HNC) is highly prevalent in South-Asia, driven by additional region-specific exposures such as chewing tobacco and betel nut. Despite therapeutic advances, five-year survival rate remains around 50-60%, underscoring urgent need to identify novel therapeutic targets and improve disease-free survival. This study was designed to identify both somatic and germline drivers contributing to HNC pathogenesis. Through whole-exome sequencing of 103 patients, we detected mutations in known HNC drivers (TP53, CDKN2A, NOTCH1) as well as novel hotspots in several genes, including TRIM48, MAP3K19, and CDC20. A recurrent hotspot mutation (p.A187T) in POLQ gene was identified in patients with high tumor mutation burden and was absent in both TCGA and ICGC cohorts. Among known hotspots, the MYC p.T73A mutation was highly prevalent, occurring in over 50% of patients. As MYC is considered an "undruggable" target, alternative strategies targeting upstream regulators such as BRD4 with specific inhibitors may hold promise for South-Asian HNSCC patients harboring the p.T73A mutation. Copy-number variation analysis further revealed EGFR amplification and TP73 deletion in the majority of patients, highlighting additional layers of genomic dysregulation. Comparative genomic analyses showed no recurrent mutations in epigenetic regulators (ARID2, EP300, KMT2B/MLL2, KMT2D/MLL4, NSD1, and TET1). We report p.S456L germline variant in SDHA consistently among South-Asian cohorts. Patients with p.S456L mutation were younger than those without it, reflecting typical epidemiological signature of a genetic variant that increases susceptibility. Systematic molecular characterization of recurrent mutations is required to elucidate mechanism of action of these variants and to find actionable therapeutic targets.