OncoImmunology

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.

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.

BackgroundInterferon-gamma (IFN-{gamma}) is the primary effector cytokine of adaptive anti-tumor immunity, yet it paradoxically induces a potent immunosuppressive tumor microenvironment (TME). The full mechanistic scope of this paradox in head and neck squamous cell carcinoma (HNSC) has not been characterized at the transcriptomic scale. MethodsUsing TCGA HNSC RNA-seq data (n = 522), we applied an integrated computational pipeline: Spearman correlation analysis, principal component analysis (PCA), UMAP, K-means clustering (k = 4), Random Forest regression, deep neural networks, permutation importance, JAK-STAT cascade mapping, and DNN-based transcriptome-wide mediation analysis across 57 IFN-{gamma} pathway and 78 immunosuppressive genes. ResultsIFN-{gamma} pathway activity was universally and positively correlated with six immunosuppressive axes, including checkpoints (CD274; LAG3; IDO1), Tregs, myeloid suppression, and tryptophan catabolism. K-means clustering identified four immunologically distinct tumor subgroups. DNN models predicted suppressive TME. Permutation importance identified IRF8 as the dominant mediator linking IFN-{gamma} signaling to immunosuppression. DNN mediation analysis identified PDCD1LG2 (PD-L2) as the strongest intermediary between IFNG and PD-L1 regulation, followed by JAK2 and GBP5. ConclusionsIFN-{gamma} orchestrates coordinated immunosuppression in HNSC through JAK-STAT-IRF8 signaling. PDCD1LG2 and JAK2 are actionable mediators of this paradox, supporting combination strategies co-targeting IFN-{gamma}-induced checkpoint induction and direct checkpoint blockade in HNSC immunotherapy. GRAPHICAL ABSTRACT

Diffuse large B-cell lymphoma (DLBCL), the most common aggressive lymphoma, encompasses histologically similar but genetically distinct cancers. Recent genetic studies have defined at least six molecular subtypes, yet none account for Epstein-Barr virus (EBV), despite 5-15% of DLBCLs being EBV-associated. By reanalyzing published whole-exome and RNA-sequencing data from 481 tumors, we identified 19 EBV-positive cases. These were significantly enriched in the BN2 subtype (6/19), while most (11/19) remained unclassified. In BN2 tumors, several subtype-defining mutations were reduced in frequency among EBV-positive cases, supporting the hypothesis that EBV oncogenes substitute for specific cellular alterations and may confound DLBCL classification algorithms. Extending our analysis to cell lines, we found that the widely used Val cell line harbors the B95-8 laboratory EBV strain; other EBV-positive lines appeared authentic but modeled only non-BN2 subtypes and expressed an atypical viral latency III program, whereas some DLBCL tumors expressed the atypical latency III program and others latency I or II. Together, these findings demonstrate that EBV-positive DLBCL, like DLBCL itself, is not a single disease, and that current in vitro models only partially capture its biological heterogeneity. Key pointsO_LIEBV-positive DLBCL is not a single disease and EBV status can impact genetic-based classifications. C_LIO_LICurrent EBV-positive DLBCL cell lines do not adequately capture tumor complexity; we determined that Val is a problematic cell line. C_LI

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: Although thin, T1 melanomas have an excellent cure rate with surgery alone, >25% of melanoma deaths originate from thin melanomas (TMs). There is, therefore, an urgent need to improve the identification and management of patients with TMs at high risk of recurrence. Methods: Patients with T1 melanoma and recurrence [&le;] 2 years of diagnosis (T1 rapid group) were compared to patients with T1 melanoma and recurrence [&ge;]10 years after diagnosis (T1 late group). Results: 442 patients from 14 sites were included: 310 and 132 patients in the T1 rapid and late groups, respectively. Median age at primary melanoma diagnosis was 51 years [15-85], 272 (62%) male, 254 (58%) superficial spreading and 101 (23%) head/neck primary. The majority (73%) of recurrences in the T1 rapid group were locoregional. Using univariable logistic regression analysis, age >65 years (p<0.0001), lentigo maligna (LM) melanoma subtype (p=0.025), head/neck primary site (p=0.0065), mitoses [&ge;]1/mm2 (p=0.0181) and ulceration (p=0.0087) were significantly associated with T1 rapid recurrence compared to T1 late recurrence. Using multivariable analysis, age >65 years (p=0.0010), mitoses [&ge;]1/mm2 (p=0.049) and ulceration (p=0.037) remained significant. Conclusions: Rapid recurrence of TM is associated with age >65 years, LM subtype, head/neck primary site, mitoses [&ge;]1/mm2 and ulceration.

Immune checkpoint inhibitors such as anti-PD1 antibodies are essential in cancer therapy. Emerging data suggest that lower doses may be effective and more economical, though further evidence is needed. We conducted a retrospective study at Centre Leon Berard to assess the efficacy and safety of low-dose nivolumab (20 mg every three weeks) in patients with advanced cancer, mainly squamous cell carcinomas (SCC). Between 2023 and 2024, 53 patients were treated, with a median age of 74 years; 39.6% were over 80. Most were male (64%) and had ECOG >1 (69.9%). Primary tumor sites included cutaneous SCC (34%), head and neck SCC (32%), and soft tissue sarcoma (15%). After a median follow-up of 8.3 months, median overall survival was 7.5 months. The objective response rate (ORR) was 20.8% overall, rising to 35.3% in cutaneous SCC and 23.5% in head and neck SCC-comparable to standard-dose nivolumab. Toxicity was manageable: 18.7% experienced immune-related adverse events, with only 3.7% grade 3. Low-dose nivolumab demonstrates encouraging efficacy and tolerability in a frail population, supporting its potential role in resource-limited settings. Prospective trials are warranted to confirm these findings in broader populations.

Chimeric antigen receptor (CAR) T-cell therapy has transformed treatment for relapsed /refractory(r/r) lymphoid malignancies. Yet, these cellular immunotherapies are often associated with immune-related adverse events (irAEs), namely cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), that pose significant risks to patient safety and limit broader clinical implementation of CAR T-cell therapies. In the current study, we used proteomics technology to establish circulating protein signatures that would predict severe CRS and ICANS in r/r lymphoma patients that subsequently received CAR T-cell therapy. Initial discovery was performed using plasma samples collected preceding CAR T-cell infusion from 39 r/r lymphoma patients at MD Anderson Cancer Center. A 5-marker and 8-marker protein panel was developed for predicting Grade [&ge;] 2 CRS and ICANS respectively, yielding respective AUCs of 0.85 [95% CI: 0.72-0.98] and 0.91 [95% CI: 0.81-1.00]. Independent testing of the CRS and ICANS panel was performed in a cohort of 59 r/r lymphoma patients from the Moffitt Cancer Center, with resultant AUCs of 0.76 [95% CI: 0.63-0.89] and 0.67 [95% CI: 0.51-0.84] for the CRS and ICANS panel, respectively. Patients were further classified into low-, intermediate-, and high-risk groups based on panel score tertiles. In the combined dataset (MDACC + Moffitt), compared to patients in the low-risk group (reference), patients in the intermediate- and high-risk groups were 3.15 [95% CI: 0.92-12.71] and 13.84 [95% CI: 4.21-56.26] more likely to have Grade [&ge;] 2 CRS, and 1.21 [95% CI: 0.36-4.23] and 8.59 [95% CI: 2.87-29.09] more likely to have Grade [&ge;]2 ICANS. The protein biomarker panels provide a means to risk stratify patients who are at high risk for developing severe CRS and ICANS, to inform on the need for prophylactic interventions and improve patient outcomes.

BackgroundCompanion canines need advances in therapeutic options for solid tumor malignancies. Prior studies established feasibility of autologous natural killer (NK) cell infusions in canines with solid tumors; however, autologous products are limited by dysfunctional immunity and a manufacturing process that delays care. Allogeneic NK cells offer the possibility of "off-the-shelf" therapy to be administered from healthy donors. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from healthy canine donors via density gradient separation. NK cells were expanded with recombinant human IL-2 and canine IL-21 with the addition of K562 feeder cells transfected with CD137 ligand and membrane bound human IL-15. Additional experiments included IL-12 in the expansions. In vitro potency was assessed via co-culture with the D17-mKate2 canine osteosarcoma cell line. Three canines were enrolled in a phase 1 trial infusing ex vivo expanded allogeneic NK cells after lymphodepletion. ResultsFlow cytometric analysis confirmed successful expansion of canine NK cells with up to 50% of cells demonstrating NKp46+ after 14 days of expansion. Residual T cell numbers varied based on donor. The addition of IL-12 led to increased NK cell expansion. Incucyte demonstrated potency with increasing osteosarcoma cell death at higher effector to target ratios. Three canines with metastatic/refractory solid tumors were successfully lymphodepleted and infused with allogeneic NK cell products. The canines tolerated the infusions well. ConclusionsCanine allogeneic NK cells were successfully expanded and activated ex vivo, demonstrated potency in vitro, and safety in vivo. Further studies will optimize the NK cell product and escalate dosing to reach the maximal tolerable dose.

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

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.

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.

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.

Engineering macrophages with chimeric antigen receptors is emerging as a promising cancer therapeutic. Chimeric antigen receptor-expressing macrophages (CAR-Ms) engineered to recognize tumor-specific antigens have been shown to inhibit tumor growth and activate adaptive immune responses, leading to robust tumor control in animal studies. Based on this work, clinical trials have been initiated. While the trials have shown promise, challenges remain. The dynamic interactions between CAR-Ms and cancer cells and the exact mechanisms driving anti-tumor effects remain poorly defined. Defining the dynamic interactions between CAR-Ms and cancer cells will provide critical insights for optimizing future CAR-M design and improving therapeutic efficacy. We sought to directly visualize CAR-M interactions with glioblastoma cells at high-resolution and in real-time using CAR-Ms engineered to recognize Neural-Glial Antigen 2 (NG2), an antigen expressed on glioblastoma cells. Using patient-derived glioblastoma cells, we formed glioblastoma spheroids and embedded them in a 3D matrix together with CAR-Ms. Using time-lapse microscopy, as expected, we found that NG2-targeting CAR-Ms engulfed glioblastoma cells. However, excitingly, we found that NG2-targeting CAR-Ms blocked >85% of glioblastoma cell invasion in 3D. This inhibition of glioblastoma invasion was not due to a significant change in CAR-M polarization states. Together, these data suggest that NG2-targeting CAR-Ms both engulf glioblastoma cells and block glioblastoma invasive behavior.

Atypical teratoid rhabdoid tumor (ATRT) is a malignant brain tumor of children that has an overall survival of less than 40 percent even with aggressive therapy. We identified upregulation of the mitogen activated protein (MAP) kinase pathway in ATRT. The novel, brain-penetrant MEK inhibitor mirdametinib inhibited the growth of ATRT cell lines in culture at nanomolar concentrations. Mirdametinib suppressed proliferation as measured by BrdU incorporation and induced apoptosis as measured by cPARP and Annexin V staining. Monotherapy with mirdametinib extended the life of mice bearing orthotopic xenografts. Combination therapy with the brain-penetrant cyclin dependent kinase 4/6 inhibitor abemaciclib further suppressed growth and BrdU incorporation in ATRT cell lines representing all molecular subgroups. Mirdametinib and abemaciclib combined to extend survival of mice bearing orthotopic ATRT xenografts. In conclusion, mirdametinib has single agent activity against ATRT and combines with abemaciclib to decrease proliferation and extend survival in orthotopic xenograft models of ATRT.

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.

PURPOSE: Newly-diagnosed glioblastoma (nGBM) is a devastating tumor with median survival of only 14-18 months despite aggressive standard of care (SOC). Dendritic cell (DC) homologous antigenic double-loading provides a powerful pattern-based signal that initiates cDC1-like skewing of monocytic precursors, inducing downstream development of CD8+ memory effectors. Here we report phase I results for DOC1021 (dubodencel), a novel DC vaccine regimen integrated with SOC. METHODS: In this dose-escalating study, DC prepared from mobilized peripheral blood were doubly loaded with autologous tumor lysate and amplified tumor mRNA and administered bilaterally near the deep cervical node chains in three biweekly courses given with weekly peg-IFN after conclusion of chemoradiation. Four dose levels from 3.5x106 to 3.6x107 total cells were tested. Patients with subtotal resection or tumor progression prior to vaccination were not excluded. RESULTS: Eighteen patients (median age 61 years (range 47-73), 94% MGMT unmethylated, 25% subtotal/partial resected) completed vaccination (16 nGBM, 2 recurrent) with no dose-limiting toxicities. Attributable AE were mostly mild and flu-like or injection-site reactions. Twelve-month OS among the newly-diagnosed cohort was 88% compared to an expected ~60% for SOC alone. Patients who received observation rather than reoperation in response to worsening MRI contrast-enhancement demonstrated gradual lesional resolution and improved OS. Immunophenotyping revealed post-vaccination elevations in CD4 and CD8 memory T-cells in peripheral blood, and spatial transcriptomic analysis revealed foci of activated inflammatory complexes at the primary tumor site. CONCLUSIONS: DOC1021 was safe, feasibly integrated within SOC, and associated with more favorable outcomes in this challenging patient population. Patients who received observation rather than reoperation for worsening MRI contrast-enhancement exhibited superior survival, suggesting an immune-reactive tumor microenvironment manifesting as pseudo-progression. These data supported initiation of a randomized Phase II trial (NCT06805305) for nGBM.

CAR-T immunotherapy has achieved remarkable efficacy in hematologic malignancies. However, the widespread clinical adoption of autologous CAR-T products remains constrained by high costs, lengthy manufacturing process, and limited accessibility. Universal or off the shelf CAR-T (UCAR-T) cells derived from healthy donors offer a promising alternative, enabling immediate treatment at a lower cost. However, the allogeneic nature of UCAR-T cells triggers immune rejection by the host immune system after infusion, thereby compromising their persistence and therapeutic efficacy. Current strategies to circumvent this rejection focus on disrupting HLA class I expression. Although this modification allows UCAR-T cells to successfully evade T cell mediated elimination, the loss of HLA class I molecules renders them vulnerable to attack by host natural killer (NK) cells. In contrast to previous approaches that attempt to retain certain non-classical HLA molecules (such as HLA-E or HLA-G) to inhibit NK cells, we directly focused on editing the ligands that mediate NK cell rejection. Through transcriptomic and in vitro validation analyses, we found that UL16 binding proteins (ULBP) 2/5/6 were substantially upregulated in UCAR-T cells compared with nontransduced donor T cells. Elevated ULBP expression effectively activates the NKG2D receptor on allogeneic NK cells and leads to killing of UCAR-T cells, thereby impairing UCAR-T function. To test whether abrogating this NK activating signal could improve UCAR-T persistence and antitumor efficacy, we generated ULBP knockout UCAR-T cells using CRISPR-Cas9 editing. Deletion of ULBP2/5/6 significantly reduced NK cell mediated killing in vitro without affecting CAR expression or T cell effector function. Compared with wild type UCAR-T cells, ULBP deficient UCAR-T cells exhibited enhanced tumor killing efficacy in the presence of NK cells. Collectively, our findings identify ULBP upregulation as one of the mechanisms underlying NK cell mediated rejection of HLA deficient UCAR-T cells. Targeted ablation of ULBP molecules provides a novel strategy to confer resistance to host NK cells, thereby improving the therapeutic potential of universal CAR T products.

Long-term follow-up of the CASSIOPEIA trial (NCT02541383) demonstrated superior progression-free survival (PFS) with daratumumab, both in combination with bortezomib, thalidomide, and dexamethasone during induction and consolidation, and during maintenance therapy, in transplant- eligible patients newly diagnosed with multiple myeloma (MM). However, outcomes among CASSIOPEIA patients remain heterogeneous across treatment groups. Measurable residual disease (MRD) is a strong indicator of the depth and duration of therapeutic response and is independently associated with both PFS and overall survival (OS), but it does not fully capture the biological diversity of MM. We performed a risk prediction analysis based on transcriptomic subgroups in CASSIOPEIA patients. A subset of 628 patients was characterized using RNA sequencing and consensus clustering identified five transcriptomic subtypes of MM. Long-term follow-up allowed the definition of three transcriptomic risk categories, with estimated 72-month PFS rates of 70%, 51%, and 27% for low, intermediate, and high-risk groups, respectively, among patients who received daratumumab in at least one treatment phase. In these patients, MRD negativity rates after consolidation and six months later were significantly higher in the low and high-risk groups compared with the intermediate-risk group. In the high-risk group, MRD status was not associated with PFS or OS. This suggests that, although daratumumab administered during both the induction/consolidation and maintenance phases improves the clinical outcomes of patients with activation of NSD2 or overexpressing members of the MAF family, highly aggressive minor clones may rapidly expand. These findings emphasize the need for novel therapeutic strategies in this high-risk population.

Immune mobilizing monoclonal TCR against cancer (ImmTAC) are cancer therapeutics that activate T cells through recognition of a tumor-associated antigenic MHC/peptide complex. A first-in-class ImmTAC, Tebentafusp, is approved for the treatment of metastatic uveal melanoma. While clinical efficacy is thus established, the cellular mechanisms underpinning ImmTAC action are not fully resolved. Using a recently established experimental strategy to generate suppressed human primary cytotoxic T lymphocytes (CTL), we have investigated an ImmTAC that recognizes a peptide derived from the tumor associated antigen NY-ESO-1 in comparison to direct engagement of a TCR recognizing the same MHC/peptide complex. In response to endogenous antigen presentation, ImmTACs could elicit tumor cell cytolysis by suppressed CTL, but not IFN{gamma} secretion, in a manner dependent on the engager affinity for CD3{varepsilon}. ImmTACs enhanced the efficient execution of subcellular CTL polarization steps required for effective cytolysis and could trigger calcium signaling. These data establish that ImmTACs activate CTL similarly to direct engagement of a TCR by MHC/peptide and are likely to retain this capability under suppressive conditions such as in the tumor microenvironment.