BACKGROUND & AIMS:Liver transplantation (LT) is a curative treatment option in early and intermediate hepatocellular carcinoma (HCC) following downstaging with locoregional therapies (LRT). Tumor response to immune checkpoint inhibitors may extend LT eligibility to intermediate and advanced stages. METHODS:In this prospective phase II study, patients with intermediate and advanced HCCs beyond extended transplant criteria, not amenable to further LRTs, were downstaged with atezolizumab-bevacizumab (Atezo-Bev) prior to LT. The primary endpoint was recurrence-free survival with safety and efficacy as additional outcomes. Spectral quantitative pathology and immune signatures in tumor tissue and peripheral blood were studied longitudinally. RESULTS:Sixteen patients with HCC beyond expanded transplant criteria (median tumor size 6.5 cm [IQR 3-8], median AFP 283 ng/ml [IQR 6-1,080], portal vein thrombosis 50%) were downstaged to LT after a median of 4.7 months (IQR 2.4-7.6). Prior LRTs were used in 15 (94%) patients. The washout period from the last Atezo-Bev dose to transplant was 57.5 (IQR 29-87) days. Median follow-up was 16 months (95% CI 4-22). Pre-transplant immune-related adverse events occurred in 3 (19%) patients and post-transplant acute rejection in 4 (25%). Post-LT 90-day morbidity and mortality were 62.5% (95% CI 35-85%) and 6.3% (95% CI 0.2-30%) respectively. Explant pathology revealed 10 complete and 6 partial responses. Responding patients harboured a tumor microenvironment with features suggestive of immune activation/extinguishment, correlated with duration of Atezo-Bev treatment and length of pre-LT washout. One (6.2%) HCC post-LT recurrence occurred during follow-up. Recurrence-free and post-transplant overall survival were 90% and 94% after 2 years, respectively. CONCLUSIONS:LT following Atezo-Bev downstaging achieves promising recurrence-free survival in intermediate and advanced HCC beyond conventional transplant criteria. Acute rejection seemed to be increased but was clinically manageable. LT should be considered in patients with HCC who respond to immunotherapy. IMPACT AND IMPLICATIONS:Immune checkpoint inhibitors are now standard treatment for advanced hepatocellular carcinoma (HCC) and some intermediate-stage cases. Emerging evidence suggests that immunotherapy may also have a role in the neoadjuvant setting before liver resection or transplantation. By inducing tumor responses, immunotherapy may downstage patients initially ineligible for curative therapies, enabling liver transplantation (LT) and favorable post-transplant outcomes. However, treatment-related adverse events, particularly graft rejection, must be balanced against potential survival benefits. In this first prospective study evaluating LT after atezolizumab-bevacizumab (Atezo-Bev) in patients with intermediate or advanced HCC not amenable to other therapies, 26% achieved transplant eligibility. Two-year post-LT outcomes were comparable to those observed with conventional transplant criteria. Immune monitoring indicated that tumor response and graft rejection are closely linked through the PD-1/PD-L1 pathway. Although rejection risk appeared increased, it was manageable with standard steroids and intensified early immunosuppression. These findings support considering expanded transplant criteria in selected patients with sustained responses to immunotherapy. REGISTRATION NUMBER:NCT05879328.

BackgroundImmune checkpoint inhibitors (ICIs) provide durable benefit for a subset of patients with advanced non-small cell lung cancer (NSCLC), however predictive biomarkers remain limited. Peripheral blood offers accessible immune and inflammatory signals that may reflect the onset of systemic mechanisms of resistance. This study aimed to identify circulating immune features associated with clinical outcome and to develop a clinically applicable blood-based score for risk stratification in patients receiving ICIs.MethodsTwo independent real-world cohorts of advanced NSCLC patients treated with anti-PD-1 therapy were analyzed. Plasma cytokines and chemokines, soluble immune checkpoint molecules and blood cell counts were measured before and during ICI-therapy. Baseline biomarkers associated with progression-free survival (PFS) and overall survival (OS) were incorporated into an Immune-Suppressive Blood Index Score (ISBIS). The ISBIS discriminative performance was first determined in the discovery cohort (n=57) and then validated in an independent cohort (n=56), and nomogram integrating ISBIS with clinical variables was generated.ResultsISBIS score predicts poor outcome to be associated with an immunosuppressive blood profile composed of baseline IL-6, IL-8, CCL2, CXCL10, neutrophils, PLR, IFN-γ and lymphocytes. On-treatment increase in CXCL10, neutrophils, and NLR, as well as decreases in lymphocytes and soluble PD-L2, were associated with early disease progression. ISBIS stratified patients into three risk groups with significantly different survival in both cohorts and remained independently associated to OS. A nomogram combining ISBIS with ECOG-PS (Eastern Cooperative Oncology Group - Performance Status), disease burden, sex, and NLR (neutrophil-to-lymphocyte ratio) demonstrated strong discrimination and validated accuracy.ConclusionsA systemic immune-suppressive profile identifies patients with poor outcomes to ICIs. ISBIS integrates critical immune and inflammatory signals into a clinically applicable tool, supporting personalized immunotherapy strategies in advanced NSCLC.

Abstract Desmoid fibromatosis (DF) is a rare mesenchymal neoplasm with an unpredictable clinical course, where spontaneous regression or progression occurs in a significant subset of patients through largely undefined mechanisms. The use of active surveillance (AS) offers the opportunity to investigate whether tumor- or host-driven systemic and local immune features may explain these divergent outcomes, improving patient management. A prospective observational study enrolled 55 patients with primary sporadic DF managed with AS. Clinical evolution was categorized as progression, regression, or stable disease according to RECIST 1.1. Immunomonitoring with multicolor flow cytometry identified distinct systemic T-helper polarization states stratifying clinical trajectories: regressors showed a Th2-skewed profile, while progressors displayed activated T-helper cells and Th1/Th9/Th17 subsets. Higher baseline Th2 levels associated with regression and longer progression-free survival. Plasma proteomic and whole-blood transcriptomic analyses confirmed coordinated IL-4/IL-13–linked pro-resolving programs in regressors and inflammatory, early T-cell activation signatures in progressors. Tumor transcriptomics revealed adaptive, antigen-presentation and restrained immune programs in regressing lesions versus innate inflammatory, interferon and TGF-β–driven fibrotic pathways in progressing tumors. These findings identify systemic T-helper polarization as a biomarker of DF behavior and highlight coordinated systemic–tumoral immune programs underlying clinical outcomes, supporting more precise clinical management.

Genomic, non-genomic, and immune alterations contribute to melanoma resistance to BRAF and MEK inhibitors. Here, we investigated the role of the SPACA6-hosted miR-99b~125a~let-7e cluster in modulating inflammatory processes and therapy resistance. We found that miR-99b, miR-125a, and let-7e were upregulated in progressing tumors from treated melanoma patients compared with untreated lesions, and in patients with short response duration compared with long-term responders. Similarly, miR-99b~125a~let-7e expression levels were high in melanoma cell lines with acquired resistance to BRAF/MEK inhibitors, showing upregulation of immunosuppressive cytokines. Combined inhibition of miR-99b, miR-125a and let-7e during drug treatment reduced proliferation of resistant cells and decreased the expression of pro-inflammatory cytokines such as CCL2, IL6, and IL8. Conversely, enforced overexpression of these miRNAs in drug sensitive cells promoted resistance and enhanced cytokine transcripts. In silico miR-99b, miR-125a and let-7e target gene analysis uncovered GNAI1, ADCY1 and NR6A1 genes in lipid metabolism pathways linked to BRAF/MEK inhibitor resistance, which converge on the activation of the mTOR signaling, and show down-regulation in resistant cells and tumors. RNA-seq and proteomic profiling of 3D cultures of patient-derived melanoma explants demonstrated that inhibition of the miR-99b~125a~let-7e cluster reprogrammed the tumor microenvironment, enhancing immune activation and suppressing mTOR signaling. Together, these findings identify the SPACA6-hosted miR-99b~125a~let-7e cluster as a regulator of BRAF/MEK inhibitor resistance through promotion of tumor survival and of an immunosuppressive microenvironment. Targeting this miRNA cluster may provide novel therapeutic opportunities to overcome drug resistance in metastatic melanoma.

BackgroundAntiangiogenics promote immune activation by reducing myeloid-derived suppressor cells (MDSCs) and enhancing natural killer (NK) and T cell functions in metastatic RCC patients. However, these effects are transient, leading to compensatory immunosuppression. Platelets (PLT) and their extracellular vesicles (PLT-EVs) modulate immune and angiogenic pathways, suggesting a role in immune reprogramming during therapy.MethodsCirculating EVs were longitudinally profiled in metastatic RCC patients (n=8) undergoing Pazopanib therapy. EVs, isolated by differential ultracentrifugation from baseline, 3- and 6-month plasma samples, were characterized by bead-based multiplex assay and nanoparticle tracking analysis. Results were correlated with blood counts, RNA-seq and flow cytometry immune profiles.ResultsPazopanib induced temporally structured EV compartment alterations. After three months, EVs were enriched in immune markers (CD8, CD56, CD19, CD1c, HLA-DR), consistent with immune activation, whereas PLT-derived markers (CD41b, CD42a, CD29) were diminished. By six months, PLT-EV markers recovered, with CD62P+ and CD29+ EVs co-expressing immunoregulatory and angiogenic molecules (CD209, CD105). PLT-EV abundance correlated with the expansion of regulatory T cells (Tregs), PD-L1+ monocytes and MDSCs, together with suppression of NK cells. PLT activation and PDGF signaling pathways decreased in PBMC from patients with clinical benefit.ConclusionsDespite the small sample size and absence of functional experiments, our results suggest that Pazopanib promotes cytotoxic immune programs but, by six months, reprograms PLT-EVs towards different adhesion characteristics contributing to Treg and MDSC expansion while suppressing NK activity. PLT-EVs may influence the balance between immune activation and suppression during anti-angiogenic therapy, suggesting PLT-EVs as biomarkers and therapeutic targets in mRCC.

586 Background: Immune checkpoint blockade is a standard-of-care treatment for mRCC patients, but the immune mechanisms driving clinical benefit remain underexplored. In the I-RENE trial (NCT04891055), we conducted a comprehensive evaluation of myeloid-derived suppressor cell (MDSC) and lymphoid dynamics and their impact on the efficacy of nivolumab. Methods: The I-RENE study is a prospective, translational, real-world multicenter trial involving mRCC patients treated with nivolumab after failure of previous VEGFR-targeted therapies. Sixty patients were enrolled between December 2018 and August 2022. Peripheral blood (PB) samples were collected at baseline and at 2, 4, and 12 weeks, as well as at disease progression. PBMCs were analyzed by high-resolution flow cytometry (profiling 144 lymphoid and myeloid cell subsets), and plasma was assessed using multiplex analysis (69 soluble factors). CD14+ monocytes from PB and tumor tissue were subjected to RNA sequencing. Results: Significant immune modulations in PB were detectable as early as 2 weeks into treatment across all patients, regardless of clinical response, and persisted throughout the 3-month observation period. These changes included substantial alterations in the profiles of immune cells and plasma soluble factors. Responders to nivolumab exhibited a marked reduction in monocyte subsets associated with immune suppression, such as CD14+HLA-DR- cells, CD14+PD-L1+ cells, and intermediate monocytes. In contrast, non-responders showed a progressive increase in suppressive monocytes and a concomitant decrease in CD14+ cells involved in anti-tumor immunity, including non-classical monocytes and CD14+ cells expressing HLA-DR and CX3CR1. Lymphoid compartment analysis revealed that responders experienced an increase in CD8+ T cells and CD4+ effector memory T cells, along with a reduction in CD38+ T cells. Conversely, non-responders showed significant upregulation of senescent CD8+ T cells (KLRG1+CD28-CD57+) and CD38+ T cells. All progressing patients showed a notable resurgence of suppressive myeloid cells (HLA-DR-, PD-L1+), confirmed by an enriched MDSC gene signature, as well as an increase in PB CD38+ and senescent T cells. Notably, the detrimental role of myeloid-driven immune suppression in non-responders was further confirmed by an enriched MDSC gene signature in matched tumor samples. Conclusions: The pervasive and detrimental impact of myeloid cells committed to immune suppression is evident even before radiological progression in mRCC patients and persists despite immunotherapy. These findings highlight the need for early identification of immune-suppressive infiltrates and the development of strategies to overcome or reprogram these cells, both at the systemic and tumor level, following diagnosis.

Supplementary Data from Development of a Molecular Blood-Based Immune Signature Classifier as Biomarker for Risks Assessment in Lung Cancer Screening

Supplementary Tables S1-S6_S17-S18

587 Background: Collecting Duct Carcinoma (CDC) represents 1% of all renal cell carcinomas (RCC) and is characterized by aggressive clinical behavior and a particularly dismal prognosis. Cabozantinib and platinum-based chemotherapy are active therapeutic options, but survival remains poor with no novel agents approved for the treatment of metastatic disease. Antibody-Drug Conjugate (ADC) targeting Nectin-4 and TROP-2 are dramatically changing the therapeutic landscape of Urothelial Carcinoma (UC). Given the biological and clinical similarities between CDC and UC, we evaluated Nectin-4 and TROP-2 expression in CDC. Methods: CDC tissue samples were collected from patients enrolled in the CICERONE study (NCT05372302), a multicentric trial that aims to define the transcriptomic profile of CDC with the goal of changing the paradigm of CDC management by using biology-driven treatments. Nectin-4 and TROP-2 are surface proteins involved in cell adhesion and proliferation. Their expression was assessed by immunohistochemistry (IHC) using a validated assay with a Nectin-4 antibody (clone M22-321b41.1) and a TROP-2 antibody (Ab227689 by Abcam).Staining area was quantified as a percentage (0–100%) and considered positive if >10% of the tumor surface was stained. Staining intensity was dichotomized into positive and weakly positive, based on a clear distinction from the negative control. Results: 59 tissue samples from patients with diagnosis of CDC were considered eligible after centralized histological review. Fresh or archival tumor tissue was collected from either primary lesion or metastatic sites biopsies at baseline before the start of systemic therapy. Preliminary results demonstrated that Nectin-4 was expressed in 24 out of 59 samples (41%), while TROP-2 positivity was observed in 58/59 cases (98%). Conclusions: CDC appears to be a unique kidney tumor, which lies between UC and RCC clinically and biologically. Here we report for the first time the expression of two potential therapeutic targets in metastatic CDC, Nectin-4 and TROP-2, confirming the similarities between CDC and UC.Based on this encouraging data, along with the interesting results from EV-302 trial, we hypothesize that pembrolizumab in combination with Antibody-Drug Conjugate (ADC) could be active in mCDC and this combo will be assessed in the phase II RePRINT trial (NCT06302569). Clinical trial information: NCT05372302 .

Proton pump inhibitors have been explored for potentiating cancer therapies via reverting the tumor acidity and promoting the activation of anti-tumor immune responses. To regulate the intracellular pH of melanoma and immunosuppressive myeloid cells, we developed poly(L-lactide-co-glycolide) nanoparticles loaded with esomeprazole (ESO-NPs). The effect of ESO-NPs on melanoma cells was observed as alkalinization and reduction of melanin content accompanied by a decrease of microphthalmia-associated transcription factor (MITF), poliovirus receptor (PVR), and programmed death ligand 1 (PD-L1) immune checkpoint expression. ESO-NP treatment of melanoma-patient-derived and in vitro-induced myeloid-derived suppressor cells (MDSCs) reduced the expression of immunosuppression-associated molecules PD-L1, CD206, and CD163 on patient-derived myeloid cells while inducing the expression of co-stimulatory molecule CD86 and HLADR in the in vitro model. Our findings suggest that reprogramming the intracellular pH of melanoma and immune-suppression-associated myeloid cells with ESO-NPs can modulate the expression of proteins involved in resistance to cancer therapy and immunosuppression, thus potentially improving the response to immunotherapies.

Immunotherapy (IT) with immune-checkpoint inhibitors is becoming a universal strategy for cancer treatment. However, most solid malignancies require the combination of IT with standard therapies to achieve consistent clinical benefit. Nevertheless, the synergistic mechanisms underlying the efficacy of these different combinatorial regimens, particularly in term of immunological outcomes, are still poorly defined. IT should be combined with other oncological treatments based on a scientifically-sounded rationale to possibly potentiate, and not jeopardize, the IT-mediated priming of anti-tumor immunity. An extreme example of this concept is represented by hepatocellular carcinoma (HCC), in which IT is starting to be administered with liver transplantation (LT). Indeed, such a combination has been considered in recent clinical studies with rather promising results in terms of efficacy. Notwithstanding, the immunosuppressive regimens required for avoiding graft rejection could heavily penalize T cell responses induced by IT. Hence, how could such a combination therapy be clinically effective? This is the topic we have been recently addressing with the ImmunoXXL study (NCT05879328), a prospective clinical trial in which multimodal immunomonitoring has been applied to HCC patients undergoing IT+LT or receiving the two regimens alone. Data obtained so far suggest the occurrence of an unexpected immune synergism based on the ability of LT to sustain IT-primed T cell memory through the engagement of trained immunity, thus shedding light on the rationale of transplant oncology in solid tumors.

Colorectal cancer (CRC) remains challenging to diagnose, necessitating the identification of a noninvasive biomarker that can differentiate it from other conditions such as inflammatory bowel diseases (IBD) and diverticular disease (DD). Raman spectroscopy (RS) stands out as a promising technique for monitoring blood biochemical profiles, with the potential to identify distinct signatures identifying CRC subjects. We performed RS analysis on dried plasma from 120 subjects: 32 CRC patients, 37 IBD patients, 20 DD patients, and 31 healthy controls. We also conducted longitudinal studies of CRC patient's postsurgery to monitor the spectral changes over time. We identified six spectral features that showed significant differences between CRC and non-CRC patients, corresponding to tryptophan, tyrosine, phenylalanine, lipids, carotenoids, and disulfide bridges. These features enabled the classification of CRC patients with an accuracy of 87.5%. Moreover, longitudinal analysis revealed that the spectral differences normalized over 6 months after surgery, indicating their association with the presence of the disease. Our study demonstrates the potential of RS to identify specific biomolecular signatures related to CRC. These results suggest that RS could be a novel screening and monitoring tool, providing valuable insights for the development of noninvasive and accurate diagnostic methods for CRC.

Immune checkpoint inhibitors (ICIs) have revolutionized the management of advanced non-small cell lung cancer (NSCLC), although patient survival is still unsatisfactory. Accurate predictive markers capable of personalizing the treatment of patients with NSCLC are still lacking. Circulating extracellular vesicles involved in cell-to-cell communications through miRNAs (EV-miRs) transfer are promising markers. Plasma from 245 patients with advanced NSCLC who received nivolumab as second-line therapy was collected and analyzed. EV-miRnome was profiled on 174/245 patients by microarray platform, and selected EV-miRs were validated by qPCR. A prognostic model combining EV-miR and clinical variables was built using stepwise Cox regression analysis and tested on an independent patient cohort (71/245). EV-PD-L1 gene copy number was assessed by digital PCR. For 54 patients with disease control, EV-miR changes at best response versus baseline were investigated by microarray and validated by qPCR. EV-miRNome profiling at baseline identified two EV-miRs (miR-181a-5p and miR-574-5p) that, combined with performance status, are capable of discriminating patients unlikely from those that are likely to benefit from immunotherapy (median overall survival of 4 months or higher than 9 months, respectively). EV-PD-L1 digital evaluation reported higher baseline copy number in patients at increased risk of mortality, without improving the prognostic score. Best response EV-miRNome profiling selected six deregulated EV-miRs (miR19a-3p, miR-20a-5p, miR-142-3p, miR-1260a, miR-1260b, and miR-5100) in responding patients. Their longitudinal monitoring highlighted a significant downmodulation already in the first treatment cycles, which lasted more than 6 months. Our results demonstrate that EV-miRs are promising prognostic markers for NSCLC patients treated with nivolumab.