Journal for ImmunoTherapy of Cancer

PurposeNovel therapies to prevent lethal castration resistant prostate cancer in response to standard-of-care androgen deprivation therapy (ADT) are required. Unfortunately, most prostate cancers are "immune cold" and fail to respond to checkpoint inhibitors (CPIs). To assess whether ADT induces changes that enable more effective CPI therapy, we examined the tumor immune micro-environment (TiME) following neoadjuvant ADT (nADT). DesignRadical prostatectomy specimens from 43 nADT-treated patients were stratified into three duration groups and compared to each other and matched controls. RNA sequencing and quantitative multispectral immunofluorescence (qmIF) staining were performed to analyze transcriptomic and TiME abundance and cellular spatial relationship differences after nADT. ResultsImmune and inflammatory pathways, particularly of antigen presentation and adaptive immune response, were increased, most notably in tumors receiving 3-5 months nADT. qmIF revealed a complex temporal response in the TiME, with a dramatic influx of CTLs and T-helper cells after 3-5 months of nADT. However, after 6 months nADT, M2-like tumor associated macrophages (TAMs) and Tregs were strikingly increased while CTLs decreased. Spatially, CTLs and T-helper cells, clustered near tumor cells at 3-5 months nADT, were replaced by M2-TAMs in tumors receiving [≥] 6 months of nADT. ConclusionThese data reveal the induction of a bi-phasic response in the TiME: robust CTL activation 3-5 months after nADT is initiated, followed by myeloid immunosuppression in tumors receiving prolonged nADT. This ADT-induced reprogramming of the TiME suggests a critical window of opportunity where short-duration ADT might augment CPI efficacy, converting cold into immunologically responsive tumors. Translational RelevanceImmune Checkpoint inhibitors (CPIs) have not been effective in treating most human prostate cancers. This study describes the temporal dynamics of the immune response of primary prostate cancers to neoadjuvant androgen deprivation therapy (nADT), and suggests a strategic approach to improve the efficacy of CPIs in prostate cancer. After several months of nADT, inflammation and immune-related pathways were activated, accompanied by a robust infiltration of both CD8+ and CD4+ T cell into prostate tumors, indicating effector T cell education and activation. In contrast, six or more months nADT leads to an immunosuppressive shift, evidenced by increased M2-like tumor associated macrophages and regulatory T cells. Thus, our findings suggest a critical window of opportunity following nADT for initiating CPIs. This provides a rationale for the precise sequencing of nADT and CPI regimens to maximize therapeutic benefit.

BackgroundImmune checkpoint inhibition has transformed cancer therapy; however, many patients fail to respond to single-agent blockade, and combination strategies are often limited by toxicity. Central nervous system tumors exploit multiple immunosuppressive pathways, including the CD200 and PD-1/PD-L1 axis to evade anti-tumor immunity and support tumor aggressiveness. MethodsWe investigated ARL200, a peptide ligand targeting the CD200 activation receptor (CD200AR) using in vitro immune assays, murine syngeneic tumor models, phosphoproteomics, and correlative studies from a first-in-human trial in recurrent glioblastoma. ResultsARL200 exposure activated DAP10/12-dependent signaling and downregulated multiple inhibitory immune checkpoint receptors, including CD200R1, PD-1, and CTLA-4, and checkpoint ligands, CD200 protein and PD-L1, through suppression of the JAK1/3-SHP-STAT-IKK/{beta}-NF{kappa}B pathway. Distinct ARL200 variant peptides elicited unique immune responses. In patients with recurrent glioblastoma, ARL200 treatment was associated with immune activation, reduced inhibitory checkpoint expression, and evidence of antigen-specific memory responses without treatment-related toxicity. ConclusionsTargeting CD200AR enables coordinated modulation of multiple immune checkpoints with a single agent, representing a next-generation immunotherapeutic strategy opening a new pathway for treating aggressive malignancies. Key PointsO_LIARL200 elicits an active immune response for the development of a potent and durable anti-tumor response C_LIO_LIARL200 abolishes the suppressive effects of multiple immune checkpoint blockades C_LIO_LIDifferent ARL200 sequences drive alternative immune responses. C_LI Importance of the StudyTumors exploit multiple immune checkpoint pathways to suppress antitumor immunity, particularly within the immunosuppressive microenvironment of the central nervous system. Current immune checkpoint inhibitors often require combination therapy to achieve clinical efficacy, frequently at the cost of increased toxicity. In this study, we demonstrate that targeting the CD200 activation receptor (CD200AR) with a peptide ligand provides a novel strategy to simultaneously downregulate multiple inhibitory immune checkpoints, including CD200R1, PD-1, PD-L1, and CTLA-4, through a shared intracellular signaling pathway. ARL200 engagement activates DAP10/12-dependent signaling while suppressing the JAK1/3-SHP-STAT-IKK/{beta}-NF{kappa}B axis, thereby overriding tumor-mediated immunosuppression. Importantly, this multi-checkpoint modulation is achieved with a single therapeutic agent and translates to immune activation and clinical responses in patients with recurrent glioblastoma, with minimal treatment-related toxicity. These findings establish CD200AR targeting as a next-generation immunotherapeutic approach with the potential to improve the safety and efficacy of immune-based therapies for aggressive CNS malignancies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/26345679v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@17a5010org.highwire.dtl.DTLVardef@11e67eborg.highwire.dtl.DTLVardef@1387c07org.highwire.dtl.DTLVardef@156d418_HPS_FORMAT_FIGEXP M_FIG C_FIG

In approximately one third of patients, resected head and neck squamous cell carcinoma will recur. We postulated that the induction of tumor neoantigen-specific T cell responses could prevent relapse. To this end, we developed TG4050, an individualized neoantigen therapeutic vaccine encoding up to 30 patient-specific predicted tumor neoantigens delivered by a Modified Vaccinia Ankara virus viral vector. We tested TG4050 as single agent in a randomized phase I trial. We found that of 16 evaluable patients randomized to immediate vaccination with TG4050, none relapsed after a median follow-up of 30 months, while 3 relapsed in the 16 control arm patients randomized to observation and treatment with TG4050 after recurrence. Polyepitopic responses to vaccine neoantigens were detected in the blood of patients from both arms after treatment initiation. These responses were maintained throughout treatment and persisted for over one year after the last dose. Vaccine neoantigen-specific CD8+ T cells had an effector phenotype and displayed high expression of cytotoxic and tissue-resident markers. TCR repertoire analysis showed that vaccine neoantigen-specific CD8+ T cell responses were polyclonal and comprised both de novo responses and amplification of pre-existing tumor-infiltrating T cell clones. Together, this translational data is consistent with the model in which single-agent delivery of TG4050 induces long-lasting tumor neoantigen-specific cytotoxic T cell responses that prevent tumor recurrence.

BackgroundGlioblastoma (GBM) represents an aggressive malignancy with limited therapeutic options. The immunosuppressive nature of GBM may be reversible with immune checkpoint inhibitor (ICI) treatment, however, initial studies have yet to demonstrate this. It is postulated that trafficking of peripherally activated lymphocytes may play a role in generating a robust intracranial immune response. Therefore, a blood-based assay to identify peripheral blood response may both predict response and better identify the ideal patient populations for future ICI clinical trials. MethodsThis was an open-label, Phase II, investigator-initiated exploratory study of patients with newly diagnosed GBM who completed maximal tumor resection and concurrent chemoradiation followed by standard adjuvant temozolomide and the combination of Nivolumab and Ipilimumab. The primary objective was to determine if the outcome, as measured by overall survival, is improved in patients when treatment with immune checkpoint inhibitors results in an immune response in peripheral blood T lymphocytes. The immune response is defined as changes in the CD4+/CD8+ precursor frequency and expansion index compared to the overall survival (OS) measured in months. ResultsThe study closed to enrollment early due to a shift in clinical priorities after the accrual of 40 patients. Twenty-three patients have died of their disease, and adequate samples for the primary analysis were available for 17 of these patients. The median OS for the 17 patients was 19 months (range 9-45months). For the four immune measurements, patients were categorized as reactive, indeterminate, or suppressed based on pre-defined protocol criteria. Only two patients were classified as reactive across all four measurements, and their median OS was 17.5 months, compared with 21 months for patients classified as suppressed. Across each of the 4 individual immune measurements, no statistical difference in OS were observed between reactive and suppressed groups. ConclusionIn this limited cohort, no detectable difference in the OS was observed between patients with a reactive immune signature and those with a suppressed immune signature.

The phase 1b TICIMEL clinical trial evaluated the safety, tolerability, and anti-tumor activity of combining the immune checkpoint inhibitors (ICI), ipilimumab and nivolumab, with tumor necrosis factor (TNF) blockers, certolizumab or infliximab, to treat advanced melanoma patients. A higher proportion of responses was observed in patients receiving ICI and certolizumab, while patients treated with ICI and infliximab demonstrated superior tolerability. Moreover, CITE-Seq analyses of circulating CD8 T cells showed that ICI plus certolizumab promoted an IFN signature, whereas ICI plus infliximab reduced the induction of genes associated with T cell activation. In preclinical models, ICI and TNF blockade with certolizumab increased IFN-{gamma}+ CD8 T cells and reduced regulatory T cells in tumors. The IgG1 Fc fragment of infliximab was identified as counteracting the benefits of TNF blockade. These findings underscore the importance of selecting the optimal TNF blocker to combine with ICI to enhance therapy efficacy in melanoma patients. ClinicalTrials.gov identifiers: NCT03293784; NCT05867004.

BackgroundTumor-associated macrophages (TAMs) display context-dependent functional polarization, but whether their prognostic impact is consistent across tumor types remains unclear. MethodsWe analyzed RNA-sequencing and clinical data from The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD; n=648), lung squamous carcinoma (LUSC; n=623), and melanoma (SKCM; n=466). Cox proportional hazards models adjusted for age and AJCC stage evaluated per-standard deviation (SD) expression of TAM markers (FOLR2, TREM2) and T-cell markers (CD8A, CXCL9). Cross-histology interaction terms tested divergence between LUAD and LUSC. ResultsIn melanoma, higher FOLR2 (HR 0.87), TREM2 (HR 0.83), CD8A (HR 0.69), and CXCL9 (HR 0.67) independently predicted improved survival. LUAD showed largely neutral macrophage effects. In contrast, LUSC demonstrated an adverse association for FOLR2 (HR 1.28). Interaction analysis confirmed significant divergence for FOLR2 and TREM2 between LUAD and LUSC. ConclusionsTAM-associated prognostic effects reverse by tumor histology, supporting tumor-context-dependent macrophage polarization and informing macrophage-targeted therapeutic strategies.

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.

Vitiligo is an autoimmune disorder characterized by melanocyte destruction. We performed a rank-based meta-analysis of six independent transcriptomic studies (115 samples) spanning microarray, bulk and single-cell RNA-seq platforms to identify consensus signatures of lesional skin. Robust Rank Aggregation identified 114 differentially expressed genes (FDR < 0.05) with striking asymmetry: 108 downregulated versus 6 upregulated. Downregulated genes were dominated by melanocyte markers (MLANA, TYRP1, DCT, PMEL, KIT). Upregulated genes included interferon-stimulated genes (OAS1, OAS2, EPSTI1). Pathway-level meta-analysis confirmed uniform suppression of melanogenesis, while immune activation was heterogeneous across datasets. Single-cell data from three included studies confirmed melanocyte depletion. The 108 downregulated genes showed exclusive expression in melanocytes. These include neural genes (PLP1, GPM6B, NRXN3), consistent with melanocytes neural crest origin. We also identified candidate melanocyte markers such as CYB561A3 and QPCT with high melanocyte specificity and consistent downregulation in vitiligo. These findings reveal a robust melanocyte loss signature in vitiligo detectable across all platforms, and study-dependent immune activation possibly influenced by sampling method and disease characteristics.

Backgroundfollowing a first cutaneous squamous cell carcinoma (CSCC), one-third of patients develop new primaries, escalating their risk of metastasis and poor outcomes. However, current follow-up strategies are not risk-stratified, representing a critical gap in patient management. Objectiveto develop and validate a prognostic model to quantify individualized absolute risk of a first metachronous CSCC after an index tumor, accurately accounting for the high competing risk of mortality in this typically elderly population. Methodswe conducted a nationwide, population-based cohort study of 11,737 patients with a first histologically confirmed CSCC (Netherlands Cancer Registry, 2007-2008) with up to 10 years of follow-up. Data on subsequent tumors was retrieved via linkage to the Automated National Pathological Anatomy Archive (Palga). A Fine-Gray competing-risk model was developed using routinely available clinical and pathological predictors (age, sex, hematologic malignancy, basal cell carcinoma (BCC) and actinic keratosis (AK) history, presence of synchronous CSCC, primary tumor location, and differentiation). Model performance was assessed 10-fold cross-validation, quantifying discrimination (time-dependent C-index) and calibration. Resultsduring follow-up, 3,288 (28%) developed a first metachronous CSCC. The model identified key predictors: markers of cumulative UV-exposure (included AK history, [&ge;]5 prior BCCs), and immunosuppression (chronic lymphocytic leukaemia/small lymphocytic leukaemia). Male sex, presence of synchronous CSCC at baseline were also associated with higher risk. While discrimination was modest (cross-validated 5-year C-index: 0.64), the model demonstrated excellent calibration. Conclusionsthis competing-risk model provides individualized, well-calibrated absolute risk estimates for a first metachronous CSCC. Based on routinely available clinical features, it offers insight into how established predictors shape risk in this high-susceptibility population. External validation and the identification of novel predictors are necessary to further refine the model and support personalized dermatologic care.

PurposeCirculating tumor DNA (ctDNA) analyses are informative as an early indicator of immunotherapy response in advanced non-small cell lung cancer (NSCLC); however, the clinical value of ctDNA molecular response requires further validation. Patients and MethodsAs part of a prospective clinical protocol (NCT05995821), we conducted targeted error-correction sequencing of ctDNA (n=328) and matched WBC DNA (n=109) from 109 patients with metastatic NSCLC who received anti-PD-(L)1 either as monotherapy or in combination. Following cellular origin resolution of 2,818 variants, landmark molecular response (mR) was defined as undetectable ctDNA within 3-9 weeks of treatment initiation. ResultsPre-treatment ctDNA burden, but not blood tumor mutation burden, predicted survival. Implementing a tumor-naive WBC DNA-informed approach increased the number of evaluable cases without compromising the overall accuracy of landmark ctDNA molecular responses. A direct comparison of single-timepoint on-therapy ctDNA assessment with ctDNA dynamics from baseline to the 3-9-week interval, along with an analysis of heterogeneity in molecular response within the 3-9-week window, showed that undetectable ctDNA at the landmark timepoint can effectively predict survival outcomes. A significant enrichment in landmark ctDNA mR was noted among patients with progression-free survival (PFS) [&ge;]6 months with immunotherapy (p=2.5e-05) and chemo-immunotherapy (p=0.02). Patients in the landmark mR group had longer progression-free (p=1.6e-06) and overall survival (p=2.5e-05) than those with molecular progression. ConclusionsLandmark ctDNA molecular response provides a real-time, accurate approach for monitoring immunotherapy clinical outcomes. Although not currently validated for regulatory use, these findings demonstrate the potential utility of ctDNA as an early endpoint in clinical trials. Translational RelevanceEmploying circulating tumor DNA (ctDNA) dynamics as an early indicator of immunotherapy response requires a roadmap for the next-generation sequencing approach, definition of molecular response and establishment of its clinical sensitivity. In this study, we introduce the concept of a landmark ctDNA molecular response, determined 3-9 weeks after initiation of immunotherapy, that maximizes the number of evaluable patients without sacrificing the specificity of the approach. Notably, when evaluating heterogeneity in ctDNA detection within the landmark 3-9-week window and assessing the impact of landmark interval dynamics on survival, we found that a single ctDNA assessment performed similarly to multiple ctDNA measurements within the landmark window (most notably, regardless of whether the timepoints were concordant or discordant). Our findings demonstrate that a single assessment of early on-therapy landmark ctDNA molecular response, can identify patients at risk of disease progression and enable future intervention and therapy optimization.

Aggressive variant prostate cancer (AVPC) is a lethal subtype of prostate cancer characterized by its androgen independence, resistance to chemotherapy, and display of neuroendocrine features which can emerge either de novo or via transformation after a prior diagnosis of adenocarcinoma. The poor clinical outcomes in patients with AVPC are associated with its profound molecular heterogeneity. In this study, we analyzed 23 consecutive AVPC cases treated at a dedicated small-cell clinic (2017-2025) using clinicogenomic and transcriptomic profiling. Transformed AVPC exhibited significantly shorter overall survival times than de novo AVPC (11.8 vs 26.0 months, P < 0.001). Integrative genomic analyses identified residual androgen signaling in subsets of cases harboring neuroendocrine lineage programs, highlighting a decoupling of lineage identity and morphology. To facilitate mechanistic and pharmacologic studies, we established NCI-LYM-1, a patient-derived organoid/PDX from an AR-negative, ASCL1+/SYP+ lymph node metastasis, which faithfully recapitulates the donor tumors molecular and phenotypic features. Short- and long-read whole-genome sequencing combined with optical genome mapping identified biallelic inactivation of PTEN, TP53, RB1 and BRCA2 as potential drivers, demonstrating clonal concordance with circulating tumor DNA from the original patient donor. Pathway and perturbation analyses suggested that NCI-LYM-1 harbored a strong dependency on apoptotic pathways, which was confirmed by in vitro organoid testing with the BCL-2/BCL-xL inhibitor navitoclax (IC50: 0.27 {micro}M) and the MCL-1 inhibitor AZD-5991 (IC50: 0.060 {micro}M). Overall, NCI-LYM-1 recapitulates the clinical aggressiveness and heterogeneity of AVPC, providing a tractable platform to identify novel precision therapies.

Regional lymph nodes (LNs) in the thoracic cavity serve as essential immunological hubs that coordinate humoral and cell-mediated responses against the development and progression of non-small cell lung cancer (NSCLC). To investigate immune dysregulation in the non-metastatic regional LNs of patients with aggressive NSCLC, we performed multimodal profiling on 36 LNs from 11 patients undergoing curative-intent resection including CITE-seq, scRNA-seq, and Imaging Mass Cytometry (IMC). Regional N1 LNs from patients with more aggressive disease (stage IB-IIIA) exhibited a significant enrichment of dysfunctional CD8 T cells and regulatory T cells (Tregs) compared to N2 LNs and LNs from patients with less aggressive disease (stage IA). These immune subsets were spatially co-localized with mature regulatory dendritic cells (mregDCs; CD1c, TIM3, LAMP3), forming an immunosuppressive niche uniquely enriched in the N1 LNs of higher-stage patients. Concurrently, higher-stage N1 LNs contained larger number of "decorticated" B-cell follicles characterized by decreased encapsulation of the mantle zone layer surrounding the germinal centers. This mantle zone disorganization was associated with increased spatial niches involving Tregs, CD68+ CD163 TIM3 Macrophages, CD163 TIM3dim Monocytic-Myeloid Derived Suppressor Cells (M-MDSC), plasma B cells, and a decrease in spatial niches involving CD4 T helper cells and fibroblastic reticular cells (FRCs). Together, our findings reveal parallel alterations in humoral and cell-mediated immunity within the regional LNs of patients with aggressive NSCLC.

PurposeTarlatamab is a DLL3-directed bispecific T-cell engager demonstrating clinically meaningful activity in relapsed small cell lung cancer (SCLC) in the phase II DeLLphi-301 trial. Determinants of tarlatamab sensitivity and resistance are incompletely understood, and thus we sought to identify genomic and transcriptional correlates of tarlatamab sensitivity using a clinical sequencing pipeline at a single comprehensive cancer center. Experimental DesignWe performed a retrospective, single-institution analysis of 12 patients with SCLC treated with tarlatamab. Whole-exome sequencing (WES) and exome-capture whole-transcriptome sequencing (WTS) were performed on 12 samples, and two matched samples after treatment with tarlatamab. Integrative analysis examined correlation between molecular features and clinical outcomes. ResultsThe overall response rate was 50%, which was consistent with outcomes reported in the DeLLphi-301 trial. Differences between SCLC driver alterations and tumor mutational burden were not significant between responders and non-responders, but homologous recombination deficiency scores were higher in responsive tumors. DLL3 expression was significantly greater in responders and demonstrated predictive discrimination for clinical response (AUC 0.83). Tumors responsive to tarlatamab were predominantly ASCL1-driven (SCLC-A) and demonstrated increased immune activation, such as enrichment of cytotoxic T-cell, NK-cell, and T cell transcriptional programs. Transcriptional subtype and a composite metric consisting of DLL3 expression and immune activity (DLI score) further discriminated between responders and non-responders (sensitivity 0.83, specificity 1). Paired post-treatment sample analysis identified loss of ASCL1 lineage and emergence of YAP1 expression and downregulation of DLL3, consistent with lineage plasticity as a mechanism of acquired resistance. ConclusionsSensitivity to tarlatamab is correlated with a combination of increased DLL3 expression, ASCL1-driven lineage, and an increased immune activation. Lineage state reprogramming and decrease in DLL3 expression accompany acquired resistance to tarlatamab. These findings highlight the utility of RNA based biomarkers which integrate target expression, lineage state, and immune context to guide tarlatamab therapy in SCLC. Prospective validation of the whole-transcriptome DLI score and transcriptional subtype will inform tarlatamab response prediction.

Abstract Background: Conventional MRI cannot reliably distinguish radiation necrosis (RN) from recurrent metastasis after cranial radiotherapy, as both can show similar enhancement despite different biology. We tested whether these entities are mechanically non-equivalent in vivo and separable by MRE-derived viscoelastic metrics and perilesional interface-instability features. Methods: In a prospective, histopathology-anchored cohort, 11 post-radiotherapy enhancing lesions were classified as RN (n=3) or recurrent/progressive tumor (n=8). MRE was acquired at 3.0 T with single-frequency 60-Hz excitation to derive storage modulus (G'), loss modulus (G''), and complex shear modulus magnitude (|G*|). Co-primary endpoints were median tumor G' and |G*|, each tested one-sided (RN > tumor) with Holm correction across the two co-primary tests. Median tumor G'' was tested two-sided. A prespecified secondary 6-endpoint family (absolute and tumor/NAWM-normalized G', G'', and |G*|) was analyzed with Benjamini-Hochberg FDR control. Exploratory instability mapping in a 0- 6 mm peritumoral shell generated interface-topology metrics, including convexity. Results: Absolute tumor-core medians were higher in RN than tumor for |G*| (1.79 vs 1.32 kPa; Cliff's {delta} = 0.67; q = 0.10), G' (1.62 vs 1.09 kPa; {delta} = 0.50; q = 0.14), and G'' (0.81 vs 0.46 kPa; {delta} = 0.75; q = 0.10). NAWM normalization improved separation: tumor/NAWM |G*| (2.26 vs 1.41; {delta} = 0.92; q = 0.04) and tumor/NAWM G'' (2.67 vs 0.87; {delta} = 1.00; q = 0.04) were FDR-significant. Convexity also differentiated RN from tumor (0.49 vs 0.36; {delta} = 1.00; MWU p = 0.01). Conclusions: Tumor/NAWM G'', tumor/NAWM |G*|, convexity, and tumor G'' emerged as the strongest candidate features, indicating that RN is mechanically harder and more dissipative than recurrent metastasis. Signal strength was high (Cliff's {delta} up to 1.00) but should be interpreted cautiously given sample size. Exploratory analyses further suggest that instability mapping captures biologically relevant interface behavior. These findings support a mechanics-based RN-versus-recurrence framework and justify prespecified, preregistered external validation.

BackgroundDistinguishing benign proliferative nodules (PNs) from melanoma arising within congenital melanocytic nevi remains a major diagnostic challenge. Copy number alteration (CNA) analysis is widely used to support classification, but current criteria were developed using array comparative genomic hybridization (aCGH). The performance of alternative platforms such as shallow whole-genome sequencing (sWGS) and methylation arrays in this setting is poorly defined. ObjectivesThe objective of this study is to compare CNA profiles obtained from aCGH, sWGS, and methylation arrays in atypical nodules arising within congenital nevi, and to correlate these molecular findings with clinical outcomes. MethodsSixteen samples from fourteen patients were retrospectively analyzed using all three platforms. CNAs were cataloged, concordance across methods was quantified using the Jaccard index, and molecular classifications were compared. Clinical follow-up was reviewed to provide clinical context. ResultsaCGH detected 39 CNAs, sWGS 60, and methylation profiling 66. Concordance was highest between sWGS and methylation (mean Jaccard 0.67), followed by aCGH versus sWGS (0.64) and aCGH versus methylation (0.49). Cases with high aneuploidy demonstrated strong cross-platform agreement, whereas low-burden lesions exhibited greater variability between methods. Divergent molecular classifications were observed in six cases. ConclusionsWhile all methods reliably detect broad chromosomal changes, sWGS and methylation arrays identify many additional focal CNAs that may not align with CGH-based diagnostic criteria. Until platform-specific thresholds are established, aCGH remains the most conservative and clinically validated approach for evaluating proliferative nodules in congenital nevi. SIGNIFICANCEAccurate molecular classification of melanocytic proliferations in congenital nevi is essential but challenging, particularly in patients with multiple proliferative nodules. This study provides the first systematic comparison of aCGH, sWGS, and methylation-based CNA profiling in this setting. We show that higher-resolution platforms detect substantially more focal aberrations, which can lead to discordant and potentially overcalled malignancy assessments when applying CGH-derived criteria. Our findings highlight the need for platform-adapted diagnostic frameworks and support continued use of CGH as the most conservative and clinically validated method for risk stratification. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=118 HEIGHT=200 SRC="FIGDIR/small/26347388v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1df3551org.highwire.dtl.DTLVardef@1256e50org.highwire.dtl.DTLVardef@6d8660org.highwire.dtl.DTLVardef@911b4f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Allogeneic hematopoietic cell transplantation (allo-HCT) hinges on a delicate trade-off between graft-versus-tumor control and graft-versus-host disease (GvHD), mediated by donor T-cell recognition of antigens presented by recipient human leukocyte antigen (HLA) molecules. We hypothesized that, beyond allele-level matching, sequence divergence at peptide-binding grooves across donor and recipient HLA loci shapes these responses. To this end, we evaluated the effect of HLA evolutionary divergence (HED), a metric quantifying amino acid variability at HLA peptide-binding sites, on selected hematological malignancies in 4,695 patients undergoing allo-HCT from a 9/10 mismatched unrelated donor (MMUD), reported to the EBMT database. We examined (i) locus-specific recipient HED (HED-R) and (ii) "HED-mismatch" (HED-MM), capturing immunopeptidome divergence at the mismatched locus. While dichotomous mismatch status explained differences in survival and acute GvHD risk (with overall greater detriment for class I loci), HED metrics uncovered substantial within-mismatch heterogeneity. In DRB1 mismatched subgroup, HED-MM at this locus, independently predicted inferior relapse-free survival (RFS) with an attenuating time-dependent association, further modulated by cross-locus HED-R. In this subgroup, higher HED-R at HLA-A and HLA-C associated with increased risks of acute GvHD and non-relapse mortality, respectively. Among HLA-B-mismatched pairs, higher DRB1 HED-R associated with worse overall survival (OS) and RFS and higher relapse risk. In the HLA-A-mismatched subgroup, higher HED-R at HLA-A increased chronic GvHD risk. Collectively, HED-derived metrics complement conventional mismatch classification by capturing qualitative differences in donor-recipient immunopeptidome interactions and reveal a complex, non-linear interplay among alleles across mismatch subgroups that modulates the clinical impact of mismatching. KeypointsO_LIIn mismatched unrelated HCT, baseline risk varies across mismatch constellations, with class I mismatches more detrimental than class II. C_LIO_LIHED complements conventional HLA mismatch classification by capturing qualitative donor-recipient immunopeptidome interactions. C_LI

BackgroundPATZ1 fusion-positive central nervous system (CNS) tumors frequently harbor MN1::PATZ1 fusions as driver mutations, provisionally classified as a rare DNA methylation class of low-grade neuroepithelial tumors. Radiographically, they resemble pilocytic astrocytomas with tumor and cystic components, but their supratentorial cortex location and higher recurrence rates are distinguishing features. An intermediate clinical course, despite focal high-grade histopathology, underscores the need for longitudinal molecular and immune analyses to refine classification and standard therapy. Case SummaryA female pediatric patient presented with neurological symptoms, including headache and right upper extremity weakness. MRI revealed a large cystic lesion in the left frontal lobe, leading to a differential diagnosis of low-grade glioma and ependymoma. Genomic analysis identified an MN1::PATZ1 fusion. The tumor recurred after gross total resection prompting a second resection. Transcriptomic and histopathologic assessments identified multiglial lineage, and high-grade features closely related to adult glioblastoma alongside pro-inflammatory activity in the primary tumor. The recurrent tumor showed reduced malignancy, and oligodendroglioma-like features. Increased MHC gene expression, immune checkpoint receptors (PDCD1, CTLA4, TIGIT,TIM3), T cell regulators (CXCR6), and elevated macrophage frequency, coupled with reduced PD-L1 in the recurrent tumor, suggest a complex anti-tumor immune response constrained by T cell dysregulation. This case, along with two other MN1::PATZ1 fusion-positive tumors, identifies a distinct transcriptomic subtype separate from circumscribed astrocytic glioma, highlighting upregulation of growth factor receptor pathways, like PI3K/AKT, and immune dysfunction linked to recurrence. ConclusionLongitudinal multi-omics analyses of recurrent MN1::PATZ1 fusion-positive CNS tumors revealed tumor maturation, immune dysfunction, and potential therapeutic targets. Introductory ParagraphPATZ1 fusion-positive central nervous system (CNS) tumors are rare, predominantly pediatric and frequently recurrent neoplasms provisionally classified as neuroepithelial tumors. Their pronounced histopathological and clinical heterogeneity, along with limited immunological characterization complicates their treatment standardization. We report a new case of an MN1::PATZ1 fusion-positive CNS tumor with recurrence, highlighting its radiographic similarities to low-to-intermediate grade pediatric glioma. Longitudinal multi-omics analyses of this case, along with additional MN1::PATZ1 fusion-positive CNS tumors, however, delineates a transcriptome subtype resembling adult high-grade glioma, with activated oncogenic and pro-inflammatory programs. The recurrent tumor exhibits features of decreased malignancy and enhanced glial differentiation, phenotypically shifting towards oligodendroglioma, suggesting tumor maturation. This was accompanied by increased antigen presentation programs, indicating immune engagement, while increased immune checkpoint expression and microglia/macrophage frequency indicate T cell exhaustion and immunomodulation, respectively. This longitudinal study highlights potential therapeutic strategies targeting both the tumor and its immune environment in MN1::PATZ1 fusion-positive CNS tumors.

Immune checkpoint inhibitors (ICIs) have transformed cancer treatment but commonly cause immune-related adverse events (irAEs), whether administered as monotherapy or in combination with other oncological agents. We present the first reported case of ICI-induced granulomatous sialadenitis in a male patient in his mid-fifties with BRAF-V600E-mutated papillary thyroid carcinoma who received sequential treatment with BRAF/MEK inhibitors followed by pembrolizumab. The patient experienced acute-onset severe xerostomia and salivary hypofunction, prompting ICI cessation and salivary gland biopsy. Integrative analysis using histology, single-cell RNA sequencing, and spatial transcriptomics revealed macrophage- and T-cell-mediated epithelial damage driven by epithelial senescence and Th1-polarized inflammation. Corticosteroid therapy reduced granuloma burden and improved salivary flow rates and tissue architecture; however, extensive fibrosis persisted despite treatment. These findings underscore the critical importance of early irAE recognition and intervention to preserve glandular function and enable continuation of cancer therapy.

IntroductionBRCA1/2 alterations are increasingly recognized as biologically and clinically relevant features in prostate cancer, yet the prognostic and therapeutic significance of zygosity status remains uncertain. Understanding differences between monoallelic and biallelic inactivation may refine risk stratification and guide therapeutic decision-making. Materials and MethodsA retrospective, desk-based observational analysis was performed using publicly accessible datasets from TCGA-PRAD (primary disease) and SU2C/PCF (metastatic disease). BRCA1/2 status was categorized as wild-type, monoallelic, or biallelic based on mutation, copy-number, and loss-of-heterozygosity profiles. Overall survival was evaluated using Kaplan-Meier estimates and Cox models. Systemic therapy outcomes were assessed by treatment class, incorporating exploratory interaction tests. ResultsIn TCGA-PRAD (n=300), OS did not significantly differ by zygosity (global log-rank p=0.45), with median OS of 80.0 months (wild-type), 78.0 months (monoallelic), and 55.0 months (biallelic). In SU2C/PCF (n=200), zygosity stratified outcomes significantly (global log-rank p=0.04): median OS was 22.0 months (wild-type), 14.0 months (monoallelic), and 16.0 months (biallelic). Treatment analyses showed ARSI exposure improved OS in wild-type disease (HR 0.60; 95% CI 0.38-0.95), while interaction testing suggested potential heterogeneity without statistical confirmation (interaction p=0.092). PARP inhibitor exposure showed directionally favorable HRs in wild-type and monoallelic groups but no significant interaction (interaction p=0.757). No therapy class demonstrated consistent effect modification by zygosity. ConclusionBRCA1/2 zygosity shows prognostic relevance in metastatic prostate cancer but not clearly in primary disease. While zygosity did not consistently modify systemic therapy associations in this dataset, findings support zygosity-aware reporting as a practical tool for molecular stratification and future research design.

Immune effector cell-associated neurotoxicity syndrome (ICANS) is a common and life-threatening complication of chimeric antigen receptor (CAR) T-cell therapy, with early detection being critical for timely intervention and improved outcomes. Cytokines such as interleukin-6 (IL-6) are key mediators of the inflammatory cascade underlying ICANS pathogenesis, but prospective clinical evidence for their predictive value is limited. Here we quantify IL-6 levels in a prospective cohort of 40 CAR-T patients (270 serum samples), using a simple in-house microfluidic bead immunoassay. IL-6 levels measured by our assay were significantly associated with ICANS onset. Specifically, each [~]3.4-fold increase in IL-6 levels was linked to a 74% increase in the odds of developing ICANS the following day, independent of other clinical variables. Overall, we show the prognostic value of IL-6 for next-day ICANS, demonstrate the potential of frequent cytokine measurement to guide CAR-T patient management, and develop a simple experimental method to perform such monitoring.