Molecular Oncology

BackgroundThis study investigates the role of the pioneer transcription factor FOXA1 as a master gene in sustaining epithelial cell polarization in early-stage lung adenocarcinoma. The partial loss of FOXA1 is explored to determine if it will affect plasticity and progression of lung adenocarcinoma. The study also addresses the transcriptional circuitry that links polarity defects to lysosome homeostasis. MethodsA multiomics approach was used to define the status of the chromatin in epithelial and mesenchymal states of A549 adenocarcinoma cells obtained with a newly synthetized TGF-{beta} receptor inhibitor or TGF-{beta} respectively. The study leveraged ATAC-seq, RNA sequencing, Cut&Tag sequencing of FOXA1 and histone marks profiling. The functional impact of FOXA1 was examined by partial silencing in vitro and by heterozygous FOXA1 deletion in a KrasG12D mouse model. Three-dimensional organoid culture, high-resolution electron microscopy, spatial transcriptomics and multiplex immunohistochemistry assessed carcinoma cell polarity, proliferation, the tumor microenvironment and organelle content. Group differences were evaluated with two-tailed t tests or one-way analysis of variance. ResultsFOXA1 binding and expression were highest in cells harboring an epithelial phenotype. In mouse KrasG12D LUAD tumors FOXA1 marked polarized, CDH1-positive cells; heterozygous loss diminished CDH1, disrupted apical-basal architecture, lowered organoid-forming efficiency and remodeled the immune microenvironment. Spatial transcriptomics and ultrastructural analyses showed that FOXA1-deficient carcinoma cells accumulated lysosomes, down-regulated vesicle fusion genes of the SNARE family and activated the lysosomal CLEAR gene network. FOXA1 occupied enhancers of lysosome-associated genes and competed with the transcription factor TFE3, thereby suppressing transcription of cathepsin B and cathepsin C and restricting lysosome biogenesis. ConclusionsFOXA1 is a central regulator that preserves epithelial cell polarity and limits lysosome formation in lung adenocarcinoma. Targeting the FOXA1-TFE3-lysosome axis may affect tumor plasticity and provide new therapeutic opportunities.

BackgroundDespite extensive characterization of key oncogenic drivers, pancreatic ductal adenocarcinoma (PDAC) continues to exhibit profound molecular heterogeneity and inconsistent responses to standard therapies, including gemcitabine. The role of pathway-level alterations, particularly in the context of age at onset and therapeutic exposure, remains insufficiently defined. MethodsIn this study, we leveraged a conversational artificial intelligence framework (AI-HOPE-TP53 and AI-HOPE-PI3K) to enable precision oncology, driven interrogation of clinical and genomic data from 184 PDAC tumors, stratified by age at diagnosis and gemcitabine exposure. Using AI-enabled cohort construction and pathway-centric analyses, we evaluated alterations in TP53 and PI3K signaling networks, with findings validated through conventional statistical methods. ResultsTP53 pathway analysis revealed a significantly higher frequency of TP53 mutations in early-onset compared to late-onset PDAC among gemcitabine-treated patients (86.7% vs. 57.1%, p = 0.04), with a similar trend observed between treated and untreated early-onset cases (86.7% vs. 40%, p = 0.07). Notably, in late-onset PDAC patients not treated with gemcitabine, absence of TP53 pathway alterations was associated with improved overall survival (p = 0.011). Complementary analyses of the PI3K pathway demonstrated a higher prevalence of pathway alterations in late-onset gemcitabine-treated tumors compared to untreated counterparts (13.2% vs. 2.7%, p = 0.02). Importantly, among late-onset patients not receiving gemcitabine, those without PI3K pathway alterations exhibited significantly improved overall survival (p < 0.0001). ConclusionTogether, these findings identify distinct TP53 and PI3K pathway dependencies that are modulated by both age-of-onset and treatment exposure in PDAC. This work highlights the utility of conversational artificial intelligence in enabling rapid, integrative, and hypothesis-generating analyses within a precision oncology framework, supporting the identification of clinically relevant molecular stratification strategies for this aggressive disease.

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.

Background: Gastric cancer surveillance in CDH1 pathogenic variant carriers is challenging, as predictors of localized (stage T1a) and advanced (stage >T1a) signet ring cell carcinoma (SRCC) are not well defined. We established the Group of investigAtors STriving toward Research In CDH1 (GASTRIC) consortium to identify clinicopathological factors associated with localized and advanced SRCC. Methods: A retrospective observational study (1998-2025) of CDH1 carriers across twelve academic centers was performed. Clinical, endoscopic, and pathological data were compared between carriers with and without SRCC on endoscopy, and between those with advanced versus localized or no cancer on gastrectomy specimens. Results: Overall, 390 CDH1 carriers from 235 families were included. Presence of SRCCs on endoscopy was significantly associated with thickened folds, nodularity, masses, and intestinal metaplasia, while gastritis was negatively associated. Of 196 carriers (52.4%) undergoing gastrectomy, 11 (5.6%) had advanced cancers, 10(90.9%) of which showed endoscopic abnormalities. Identification of SRCC on baseline endoscopy was the most sensitive feature for advanced disease (0.81) but had moderate specificity (0.74), whereas masses and thickened folds were highly specific (0.99 and 0.96, respectively) but less sensitive. Negative predictive values were high (0.94-1.0), while positive predictive values were modest (0.13-0.66). On multivariate analysis, masses and SRCC foci on baseline endoscopy were independent predictors of advanced disease. Conclusion: Among CDH1 carriers, absence of endoscopic findings was reassuring, whereas significance of detected endoscopic and pathological abnormalities was less certain. Advanced cancer occurred in a small number of carriers, with endoscopic abnormalities in nearly all cases. Endoscopic surveillance might be an alternative to surgery in carriers without worrisome mucosal findings.

BackgroundChemo-resistance remains a major clinical challenge in Oral Squamous Cell Carcinoma (OSCC), attributed to the intrinsically resistant cells. Although tumour-derived extracellular vesicles (EVs) have been implicated in cell-cell communication, their role in propagating chemo-resistance remains poorly defined. This study aims to identify salivary EV-associated miRNAs capable of predicting chemoresistance and to delineate the role of miR-1307-5p in modulating CSC-driven therapeutic refractoriness. MethodsSalivary EV-derived expression profile of miR-1307-5p was assessed by qPCR in chemo resistant OSCC patients and further validated in TCGA small RNA sequencing datasets. Expression was validated by qPCR and correlated with clinicopathological outcomes. Functional assays including cell-cycle analysis, apoptosis, migration/invasion, 3D spheroids, angiogenesis, and CAM assays were performed in miR-1307-5p inhibited CD44 CSC subpopulation compared to its vehicular control. Transcriptomic profiling cross-referencing with TCGA was conducted to identify potential novel targets of miR-1307-5p. Chemo-sensitisation was assessed by treating the knockdown chemo resistant cells with low dose cisplatin and validating it using in-vitro functional assays and orthotopic xenograft model. ResultsmiR-1307-5p was significantly elevated in salivary EVs of chemo resistant OSCC patients and correlated with poor overall survival (p = 0.03). The miRNA was markedly enriched in endogenously resistant CD44 CSCs. Silencing of miR-1307-5p induced G2/M arrest, triggered apoptosis, impaired invasion, and reduced angiogenesis both in-vitro and in ex-vivo assays. Transcriptomic profiling, TCGA validation, and integrative pathway analysis identified key oncogenic hubs which converge on PI3K-AKT, MAPK/ERK, and YAP signalling pathways governing EMT. Inhibition of miR-1307-5p restored cisplatin sensitivity in resistant CSCs, with low-dose cisplatin producing substantial tumour suppression in-vitro and in-vivo. Reduced CD44 expression in xenograft models confirmed CSC reprogramming. EVs from anti-miR-treated cells confer chemo sensitisation upon uptake by resistant CSCs. Xenograft models substantiated that EVs can initiate tumour formation and that EV-mediated delivery of anti-miR-1307-5p drives significant tumour regression. ConclusionThis study identifies salivary EV-derived miR-1307-5p as a clinically relevant biomarker of chemoresistance in OSCC and reveals its mechanistic role in sustaining CSC-driven therapeutic failure. Targeting miR-1307-5p offers a promising avenue for restoring cisplatin sensitivity and developing exosome-based therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/709730v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@19f88e0org.highwire.dtl.DTLVardef@d36b95org.highwire.dtl.DTLVardef@3c2579org.highwire.dtl.DTLVardef@c04ef5_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundHead and neck squamous cell carcinoma (HNSCC) comprises a heterogeneous group of epithelial malignancies associated with poor survival ({approx}50%), limited therapeutic options, and a lack of predictive biomarkers. Concurrent chemoradiotherapy (CRT) remains the standard treatment for advanced disease; however, many patients fail to respond, develop resistance, or eventually relapse. The development of three-dimensional organoid technology has enabled the generation of patient-derived organoids (PDOs), offering a promising platform for personalized therapeutic testing. MethodsWe established a biobank of HNSCC PDOs from fresh laryngeal and pharyngeal tumor samples, including human papillomavirus-positive (HPV+) cases. Organoid formation and expansion rates were analyzed in relation to clinical parameters. Selected representative PDOs were histologically and molecularly characterized. Additionally, several models were exposed to cisplatin and radiation to evaluate treatment response, and a subset was assessed for tumorigenicity in subcutaneous mouse models. ResultsFifty-seven PDO models were successfully established, long-term expanded, and cryopreserved. Prior chemotherapy and/or radiotherapy was identified as an independent negative predictor of organoid outgrowth and expansion capacity compared with treatment-naive samples. Histological features, including differentiation grade and immunohistochemical markers, were largely preserved and strongly correlated with the original tumors. PDOs displayed heterogeneous responses to cisplatin and radiotherapy, with HPV-positive models showing greater sensitivity, consistent with clinical observations. Global transcriptomic profiling revealed molecular subtypes concordant with established HNSCC classifications and suggested an additional subtype characterized by low MYC and mTORC1 transcriptional activity. ConclusionHNSCC PDOs faithfully recapitulate tumor histology and molecular diversity, providing a robust platform to investigate tumor biology and therapeutic response.

PurposeTreatment options of advanced oral squamous cell carcinomas (OSCC) are limited, and cisplatin toxicity and drug resistance are major clinical issues. Src is a central kinase that integrates multiple oncogenic pathways and a promising therapeutic target. However, Src inhibitors have shown suboptimal efficacy as monotherapies and their sensitivity in OSCC remains elusive. Experimental DesignWe examined the activation of major oncogenic signaling pathways and the antitumor effects of six Src inhibitors (dasatinib, ponatinib, vandetanib, saracatinib, PP2, bosutinib) in seven human OSCC cell lines (HSC-2, HSC-3, HSC-4, SAS, HO-1-u-1, CAL27, SCC-25). BALB/cAJcl nu/nu mice bearing CAL27 xenografts received dasatinib (30 mg/kg, intraperitoneally, daily), bosutinib (50 mg/kg, intraperitoneally, daily), cisplatin (2 mg/kg or 4 mg/kg, intraperitoneally, weekly), or combinations. Tumor volume, bioluminescence imaging, and body weight were monitored for 17 or 21 days, followed by histopathological assessment. ResultsThe activation of the key pathways, including Src and MAPK, considerably differed among the cell lines and was linked to heterogeneous sensitivity to Src inhibitors. Effective growth suppression required Src dephosphorylation and downstream MAPK pathway inhibition, which vary depending on the cell line. Additionally, combination treatment with a Src inhibitor and cisplatin showed additive antitumor effects, allowing the reduction of cisplatin doses by half without efficacy loss. Notably, dasatinib alone and in combination with cisplatin decreased tumor burden with characteristic internal tumor death in vivo. ConclusionsThese findings elucidate Src signaling dependency on OSCC and the potential of Src inhibition to decrease cisplatin toxicity, paving way for Src targeted therapeutic strategies.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer characterized by a high abundance of cancer-associated fibroblasts (CAFs), which influence therapy response, tumor biology and tumor aggressiveness. CAFs are a heterogeneous cell type and previous single-cell RNA sequencing (scRNAseq) of PDAC tumors identified three main CAF subtypes: myofibroblastic, inflammatory and antigen-presenting CAFs (myCAF, iCAF, apCAF, respectively). However, scRNAseq on large patient cohorts is often not feasible due to costs and technical constraints. Therefore, bulk RNAseq deconvolution can be used to identify cell types within the heterogeneous tumor microenvironment. Here, Statescope deconvolution was used to identify different cell types of the tumor microenvironment within an early onset PDAC cohort, comprising 74 patients aged under 60. Three CAF populations were identified (iCAFs, myCAFs and desmoplastic CAFs), and their correlations with tumor microenvironment components, mutational signatures and survival were examined. iCAFs were associated with classical-like tumor cells, whereas myCAFs and desmoplastic CAFs correlated with basal-like tumor cells. Desmoplastic CAFs are associated with inflammatory granulocytes/neutrophils, while negatively associating with monocyte-derived macrophages and immature/transitional B cells. No associations were observed between mutational signatures and the abundance of CAF and epithelial tumor subtypes. Interestingly, a high abundance of CAFs, and specifically increased iCAF abundance, was associated with improved survival. This iCAF-mediated survival effect was predominantly apparent in female patients. All in all, deconvolution of bulk RNA sequencing data, followed by its integration with clinical and biological parameters, reveals the heterogeneity and prognostic implications of CAF subpopulations in the tumor microenvironment of early onset PDAC patients.

Background: The tumor suppressor gene TP53 and the oncogene KRAS are among the most frequently altered core drivers in human malignancies. Although they cooperatively regulate critical biological processes, the prognostic impact of their co alterations remains poorly defined and exhibits striking inconsistency across different cancer types. Methods: We comprehensively analyzed genomic and clinical data from multi-cancer cohorts sourced from the cBioPortal database and The Cancer Genome Atlas (TCGA). Genetic alterations, including sequence variations and copy number alterations (CNAs), were classified for TP53 and KRAS. Patients were stratified into four subgroups based on individual or combined alteration status. Survival analyses were performed using Kaplan-Meier methods. Integrated multi-omics analyses were conducted to assess the relationship between genetic alterations and mRNA/protein expression, and to characterize co-occurring genetic events and their prognostic implications. Results: Patients harboring concurrent TP53 and KRAS alterations exhibited significantly shorter overall survival in pancreatic cancer, colorectal cancer, and ampullary carcinoma, but surprisingly demonstrated the longest survival in gastric cancer. Distinct KRAS mutation subtype distributions were observed across cancer types: G12D/G12V predominated in pancreatic and colorectal cancers, G12C in non small cell lung cancer, and G13D in gastric cancer, with copy number alterations representing a substantial proportion of KRAS alterations in gastric and lung cancers. Multi-omics analysis revealed a lack of concordance between genetic alterations and mRNA/protein expression, indicating that mutation status alone does not reliably reflect downstream molecular changes. Concurrent genetic events displayed striking cancer-type specificity: CDKN2A alterations frequently co-occurred with TP53/KRAS double alterations in pancreatic cancer and were associated with worse prognosis, whereas APC mutations co-occurred in colorectal cancer and correlated with improved survival. Integrated analysis further demonstrated that KRASaltered/TP53altered patients were highly enriched in pancreatic, colorectal, and lung cancers, each exhibiting unique background genomic landscapes. Conclusions: The prognostic significance of TP53 and KRAS alterations is profoundly cancer-type specific, driven by differences in mutation subtype distribution, copy number alteration patterns, co-occurring genetic events, and the discordance between genotype and functional expression. These findings challenge the simplistic view of dual-gene alterations as universal markers of poor prognosis and underscore the necessity of incorporating cancer-specific molecular contexts into prognostic models and precision oncology strategies.

BackgroundWilms tumour (WT) relapse occurs more frequently in patients with blastemal-type WTs. The presence of cancer stem cells (CSCs) is linked to tumour survival and relapse, and CSCs may be found in greater numbers in blastemal cell foci. CSC-associated phenotypes have been described in untreated WT, but their persistence, organisation and relevance after neoadjuvant chemotherapy is unknown. MethodsWe analysed 23 formalin-fixed paraffin-embedded blocks from 18 chemotherapy-treated patients where WTs were enriched for viable blastema, using human fetal kidney as developmental control. Immunohistochemistry and -fluorescence analysis determined progenitor (PAX2, SIX2, CITED1) and CSC-associated (NCAM, ALDH1, CD133) marker expression. We qualitatively and semi-quantitatively evaluated spatial expression patterns and co-localisation across tumour compartments. ResultsPAX2 and SIX2 were co-expressed in blastema in most cases (15/18), with PAX2 expression higher at the periphery of blastemal foci and SIX2 expression found uniformly in central aspects. CITED1 expression was also associated with SIX2 in blastema tissues (14/18). NCAM was blastema-enriched (15/18) with higher central intensity, frequently adjacent to PAX2-expressing peripheral zones. ALDH1 expression was present across blastema and epithelium while NCAM-, ALDH1-double-positive cells were rarely observed (4/18). CD133 expression was less commonly seen (2/18), localising near epithelial/nephrogenic structures. ConclusionsAfter neoadjuvant chemotherapy, WT blastema retained overlapping but non-identical progenitor/CSC-associated marker landscapes with reproducible peripheral-centre gradients. These spatial arrangements suggest a blastemal niche for CSCs that may sustain a therapy-resistant state. Our analysis provides the foundation for future functional validation and molecular profiling to define key lineage relationships and therapeutic vulnerabilities in post-chemotherapy WT. [250/250 words]

IntroductionNovel therapeutic options are urgently required to improve outcomes and survival for patients with lung squamous cell carcinoma (LUSC). In particular, understanding the unique histological features that define LUSC is essential to improving lung cancer mortality. Many pre-clinical models fail to accurately represent intratumour heterogeneity and recapitulate the tumour microenvironment. This is partly responsible for the poor translation of clinical findings to approved therapies. Our objective was to investigate whether patient-derived organoids, replicate the histological morphological, and structural features of keratinizing LUSC, a poor prognostic subtype of lung cancer. MethodsOrganoid cultures were established and maintained from two patients presenting with keratinizing lung squamous cell carcinomas. Immunofluorescent staining of individual organoids and confocal microscopy was performed to confirm expression of tumour markers. Whole organoid domes were fixed, and immunofluorescent staining and imaging was performed to investigate the structural features of the organoid cultures. Findings were compared with histopathological features of the original tumour tissue. ResultsPatient-derived organoids expressed tumour markers specific to the squamous cell carcinoma subtype of non-small cell lung cancer, which were confirmed to be expressed in the parent tissue. Within organoid cultures, keratin pearl structures spontaneously developed, matching the keratinizing pattern demonstrated by hematoxylin and eosin staining of the original tumour. ConclusionsPatient-derived organoids have the capability to replicate key histological features of their parent tumour. This high degree of fidelity makes these 3D models an important and valuable tool for understanding complex tumour biology and as a platform for preclinical drug testing to advance novel therapies into the clinic.

Introduction: Glioblastoma multiforme (GBM) remains the most lethal primary brain tumor with median survival of 14-15 months. Current prognostic markers inadequately stratify patient outcomes. PINK1 (PTEN-induced putative kinase 1), a mitochondrial kinase regulating mitophagy and cellular stress responses, has emerged as a promising prognostic candidate. Our preliminary analysis of 20 GBM cases demonstrated significant PINK1 expression with correlation to aggressive phenotypes (Turizo Smith et al., 2025). This multicenter study aims to prospectively validate PINK1 as a prognostic biomarker for survival and functional outcomes in a Latin American cohort. Methods and analysis: PINK1-GBM Colombia is a multicenter, observational cohort study across four tertiary hospitals in Bogota, Colombia (Hospital de Kennedy, Hospital El Tunal, Hospital Santa Clara and Hospital Universitario de la Samaritana). We will enroll at least 26-50 adults (18+ years) with newly diagnosed IDH-wild type GBM undergoing surgical resection. PINK1 expression will be quantified by immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue using standardized protocols. Primary outcomes: overall survival (OS) and progression-free survival (PFS). Secondary outcomes: functional status trajectories (KPS/ECOG). Follow-up extends 24 months with clinical, imaging (RANO 2.0), and telephone assessments. Survival analyses will employ Kaplan-Meier methods, log-rank tests, and Cox proportional hazards models adjusted for established prognostic factors. Ethics and dissemination: Approved by Universidad Nacional de Colombia Ethics Committee (Acta 001, February 5, 2026; Ref: 2.FM.1.002-CE-002-26), Subred Sur Occidente (P-AP-19-2025, July 11, 2025), and Subred Centro Oriente (CEI 067/2025, October 24, 2025). Conducted per Declaration of Helsinki and Colombian Resolution 8430/1993. Results will be disseminated via peer-reviewed publication, international conferences, and thesis submission.

Lung adenocarcinoma (LUAD) is classified internationally into six histological subtypes that predict clinical outcomes. Mutation analyses identify targets but provide less prognostic information than histological appearances. Immunotherapy in LUAD is constrained by the unpredictable immune environment within tumours. We therefore characterised relationships between WHO histological classification, common mutations, and underlying transcriptomic and immune profiles in 89 LUAD cases. Mutation profiles poorly correlated with histology or survival. Global gene expression was structured into 12 modules, identifying different tumour cells and pathways within WHO subtypes. Tumour classes also held distinctive immune cell profiles. Transcripts within high-risk solid tumours indicated enrichment of CD8+ and activated CD4+ T-cells, suggesting responsivity to immunotherapy. Independently from histologic classification, 31 transcripts were strongly associated with survival and were enriched in macrophage and fibroblast derived networks. The results suggest histological subtype stratification and typing for survival-associated markers have the potential to inform clinical trials of LUAD.

BackgroundThe tumor immune microenvironment likely plays a central role in progression of thyroid cancer. As for most other solid tumors, it is unknown if immune dysregulation contributes to earlier, subclinical stages of thyroid tumor development, or whether thyroid tumor heterogeneity might involve differential expression of pro-inflammatory mediators. MethodsThe time course of tumor-associated inflammation was studied in Tg-CreERT2;Braf CA/+ mice representing a model of BRAFV600E-driven papillary thyroid carcinoma (PTC). Tumor growth was estimated by histological examination and magnetic resonance imaging. Cytokine expression was monitored by quantitative RT-PCR, RNAScope and Western blot analyses. ResultsBased on spontaneous BrafCA activation due to leaky Cre activity in a minority of targeted cells tumors developed within a preserved thyroid tissue architecture to multifocal papillary thyroid carcinoma (PTC) over a period of 12 months. Tumorigenesis was accompanied by a gradually increased mRNA and protein expression of interleukin-1beta (IL-1{beta}), interleukin-6 and tumor necrosis factor-alpha (TNF-) starting already before Braf mutant cells commenced neoplastic growth. RNAScope revealed that both follicular cells and stromal cells expressed Il1b whereas Il6 and Tnfa transcripts were mostly confined to neoplastic epithelia. Early cytokine expression was associated with oncogene-induced senescence, whereas during tumor development (3-6 months) and in advanced tumor stages (at 12 months) the cytokine expression pattern differed among glands and tumor foci of the same gland accompanied by a highly variable locoregional lymphocytic infiltration. Oral treatment of mutant mice for 1 month with PLX4720, a vemurafenib prodrug, partially reduced cytokine expression along with inhibited tumor growth and redifferentiation of thyroid function. The magnitude of reduced cytokine expression differed much between glands and among mice of both sexes. ConclusionsThese findings indicate that oncogenic BRAFV600E targeted to the thyroid both stimulates endogenous production of IL-1{beta}, IL-6 and TNF- and recruits inflammatory cells to foci of early tumor development. PTCs of different clonal origin are distinguished by differential expression of pro-inflammatory cytokines. The anti-inflammatory effect of mutant Braf kinase inhibition varies presumably related to heterogeneous tumor development, which evolves from stochastic BrafCA activation suggesting there are clonally different probabilities of acquiring drug resistance among Braf mutant thyroid follicular cells.

Current clinical management of multiple myeloma (MM) relies on bone marrow (BM) biopsies for minimal residual disease (MRD) assessment. While BM biopsies are the gold standard, their invasive nature and potential to miss extramedullary or patchy disease necessitate sensitive, non-invasive liquid biopsy platforms. In this study, we evaluated the analytical performance of the CellSearch CMMC assay to determine its utility for deep-MRD monitoring. Using a standard 4 mL whole blood input, the assay achieves a WBC-normalized sensitivity of 2.45 x 10-7, supported by a limit of quantitation of 5 cells per run. Given this high analytical sensitivity, the assay provides a robust negative predictive value, rendering false-negative findings highly unlikely in populations with detectable peripheral disease. These findings characterize the CellSearch CMMC assay as a highly sensitive, analytically validated platform for non-invasive deep-MRD level longitudinal surveillance monitoring. When integrated into a clinical workflow that accounts for its specificity profile, the platform offers a patient-friendly complement to serial BM biopsies, with the potential to reduce their frequency in appropriate clinical contexts.

The central nervous system (CNS) is a common site of metastatic spread for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases remain limited. Tyrosine kinase inhibitors have shown promising results in increasing the overall response in brain metastases, owing to their brain penetrance and increased effectiveness; however, their use is limited to the small group of tumors carrying specific oncogenic drivers. Among these, inhibitors with activity against neurotrophic tyrosine receptor kinases (NTRKs) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the brain metastatic niche and can, in a paracrine manner, activate NTRK function in cancer cells. Here we show that NTRKs are expressed in primary tumors, brain metastases, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous BDNF and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented brain metastatic colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for brain metastases of WT-NTRK+ lung cancers, and therefore, NTRK inhibitors can be used to target non-fusion NTRK function to prevent or decrease brain metastases. SIGNIFICANCEThese studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

The RUNX1 transcription factor is a critical regulator of hematopoiesis and frequently mutated in myeloid malignancies. In the myeloproliferative neoplasm, chronic myeloid leukemia (CML), secondary somatic RUNX1 mutations and RUNX1::MECOM/EVI1, are associated with tyrosine kinase inhibitor (TKI) resistance and progression to the blast-phase (BP-CML). Research has predominantly focussed on transcriptional dysregulation mediated by RUNX1 mutations in myeloid malignancies, whilst post-transcriptional dysregulation remains comparatively unexplored. To address this, we used orthogonal organic phase separation (OOPS), to characterise the RNA-binding proteome of RUNX1 deficient BP-CML cells. RUNX1 depleted BP-CML cells exhibited significant alterations to RBP abundance involved in stress response pathways and translation/ribosome-biogenesis (RiBi). Furthermore, RUNX1 depletion or expression of RUNX1::EVI1 in BP-CML cells induced expression and RNA binding activity of SPATS2L, a component of stress granules (SG); membraneless cytoplasmic condensates protecting mRNAs from degradation, promoting survival under stress. Whilst RUNX1 depletion increased SG-assembly, SPATS2L depletion reduced SG-assembly in BP-CML cells and inhibited the growth and survival of multiple BP-CML cell lines. The translation inhibitor homoharringtonine (HHT), used historically in TKI-resistant CML, ablated SG-assembly in BP-CML cells with RUNX1 depletion, and, primary BP-CML cells with LOF/hypomorphic RUNX1 mutations (characterised by defective DNA-binding/CBF{beta}-interaction) were preferentially sensitised to HHT. Finally, suppressing SPATS2L expression induced by RUNX1 depletion, increased the HHT-sensitivity of RUNX1 depleted BP-CML cells, suggesting SPATS2L contributes to therapeutic resistance in CML with RUNX1 mutations. This study suggests that SPATS2L and SG induction could be critical to RUNX1-mutant leukemias, and, provides preliminary evidence for a mutationally-targeted approach in CML with RUNX1 aberrations.

BackgroundNeoadjuvant chemoimmunotherapy (nCIT) has become a standard treatment for locally advanced resectable non-small cell lung cancer (NSCLC), yet the spatial biology underlying treatment resistance remains poorly understood. We used spatial transcriptomics to define the microenvironmental architecture of residual cancers in patients who did not achieve major pathologic response (non-MPR) compared with those who did (MPR). MethodsSpatial transcriptomics was performed on 10 formalin-fixed paraffin-embedded (FFPE) tumor blocks (5 MPR, 5 non-MPR) obtained from 8 patients treated with nCIT. A deep learning algorithm was applied to detect viable residual cancer spots from treatment-induced fibrosis and necrosis. Spatial deconvolution, distance modeling, ligand-receptor analysis, and functional pathway scoring were integrated to characterize niche-specific programs. ResultsMPR cancer core displayed an immune-permissive remodeling environment with deep infiltration of cytotoxic CD8+ T cells, mature dendritic cells (LAMP3+, CCR7+), and active efferocytosis signaling (APOE-TREM2), alongside robust MHC class II expression. Non-MPR cancer core, by contrast, exhibited spatial immune exclusion: a dense fibroblast barrier reinforced by TIMP1-CD63 signaling and Treg-enriched boundaries physically restricted effector T cell access to the cancer core. Residual cancer cells in non-MPR samples maintained active cell cycling and independently upregulated cytochrome P450-mediated drug detoxification and DNA damage response pathways without inducing MHC class II expression -- effectively decoupling intrinsic survival from immune recognition. The non-MPR core also showed a hyper-metabolic profile, including elevated glutathione metabolism consistent with antioxidant buffering against chemotherapy-induced oxidative stress. TROP2 was broadly expressed across the non-MPR cancer core and co-localized with DNA damage response and nuclear factor erythroid 2-related factor 2 resistance signatures. ConclusionsResidual cancer cores in non-MPR tumors appear to represent evolved resistant niches sustained by structural immune exclusion, metabolic rewiring, and DNA repair proficiency. These findings highlight the spatial co-localization of epithelial anchors, such as TROP2, with intrinsic resistance pathways, providing a structural rationale for developing novel precision therapeutic strategies to bypass stromal barriers and overcome the cancer cores intrinsic repair capacity.

BackgroundTriple-negative breast cancer (TNBC) presents significant therapeutic limitations due to its aggressive heterogeneity and the rapid emergence of adaptive resistance to apoptosis-based regimens. Addressing these challenges requires polypharmacological strategies capable of modulating multiple signalling networks simultaneously. While the Cannabis sativa phytocomplex offers a vast chemical space for multi-target intervention, the quantitative pharmacological basis of its synergistic interactions remains largely uncharacterised. PurposeThis study aimed to deconstruct the synergistic landscape of high-purity phytocannabinoids (CBD, CBG, CBD-A) in combination with the sesquiterpene {beta}-caryophyllene (BCP) against TNBC, using MDA-MB-231 as a primary model and Hs578T as a validation line. MethodsGrowth Rate (GR) inhibition metrics and the SynergyFinder+ framework were used to map pharmacological interactions across four reference models. Subcellular dynamics and phenotypic transitions were characterised by high-resolution label-free holotomographic microscopy combined with live-cell kinetic imaging and single-cell fate mapping. ResultsTwo highly potent synergistic clusters were identified for CBD-CBG-BCP combinations, with ZIP, HSA, and Bliss synergy scores exceeding 65. CBD-A exhibited minimal interaction potential and was excluded from ternary studies. GR-based quantification further revealed that these combinations produced net cytotoxicity (GR < 0) at sub-IC concentrations of each component. Single-cell fate mapping by holotomographic microscopy identified a temporally ordered death programme: an initial phase of extensive cytoplasmic vacuolisation associated with focal perinuclear space swelling and progressive nuclear compression, morphological hallmarks of autosis, which is followed by a transition to apoptotic execution. The autotic nature of the primary death phase was confirmed by pharmacological rescue with digoxin, a selective inhibitor of the Na,K-ATPase. To the best of our knowledge, this sequential engagement of autosis followed by apoptotic execution represents the first documented instance of such a two-stage death programme in any cellular model. ConclusionThese findings provide robust evidence that specific phytocannabinoid-terpene ratios engage a Na,K-ATPase-regulated autotic programme as an upstream commitment step, followed by apoptotic execution, effectively circumventing the caspase-independent resistance mechanisms characteristic of TNBC. This study establishes a rational, quantitatively validated framework for transitioning from empirical botanical use to evidence-based, multi-target cannabinoid polypharmacology in aggressive breast cancer.

Introduction: Solitary plasmacytomas (SP) are rare neoplasm of localised proliferation of clonal plasma cells. It can be classified based on site of involvement and bone marrow involvement. It is an indolent disease in the majority of patients. Primary modality of treatment is radiotherapy and surgical excision. Materials and methods: This was a retrospective audit of SP who were treated and followed up at a tertiary care center in eastern India from January 2012 to December 2025. Patients who has solitary plasma cytoma with more than 10% plasma cells, POEMS syndrome, have been excluded from analysis. Results: We identified 46 patients of SP. The median age of the studied population was 53 years (23-75 years). Males were more commonly affected than females (M:F=2.2:1). Most common chief complaints were bony pain (67.4%). SBP was seen in 39 (84.8%) cases whereas SEP was seen in 7 (15.2%) cases. Vertebra was the most common site of involvement (61.4%). Median M band concentration 0.24 g/dL (0.1 to 1.95 gm/dL). IgG was the most common isotype accounting for 60.6% cases. Six cases (13%) had minimal bone marrow involvement. The majority of the patients received local radiotherapy (89.1%). With a median follow up of 5.4 years (95% CI: 1.8 - 9.0), median OS was not reached, median PFS was 9.22 years (95% CI: 5.8-12.6), median time to next treatment (TTNT) was 9.86 years (95% CI: 6.8 - 12.9). Conclusion: Solitary plasmacytoma commonly affects young males. Bones are more commonly affected than extramedullary sites. SP has a lower rate of progression and excellent prognosis when treated with local radiotherapy.