Immune checkpoint inhibitors (ICIs) are known to cause a wide spectrum of immune-related adverse events (irAEs). Among these, eosinophilic fasciitis (EF) is a rare, fibrosing disorder causing inflammatory infiltration of subcutaneous fat and fascia. It is characterized clinically by edema and subsequent induration and tightening of the skin and subcutaneous tissues. Several case reports have documented EF secondary to ICIs in patients on active treatment. Herein, we present two cases of delayed EF following treatment cessation with avelumab for metastatic urothelial carcinoma (Case 1) and following adjuvant nivolumab completion for stage IIIC melanoma (Case 2). Both patients had typical exam findings including erythema/edema of the extremities and trunk and diffuse thickening of subcutaneous fat and fascia, leading to severe respiratory restriction in Case 1. Both patients were diagnosed with EF by full-thickness skin biopsy showing sclerosis and lymphocytic infiltration of the subcutaneous fat and/or fascia. Only one of the two patients presented with definite eosinophilia. Both cases were treated with glucocorticoids and had early recurrence of symptoms post steroid taper, necessitating subsequent protracted steroid and steroid sparing agent use. Overall, we demonstrate the importance of considering delayed irAEs, specifically autoimmune fibrotic skin diseases even when ICI therapy has been discontinued. We underscore the need for collaboration between oncology and rheumatology as the scope of ICI treatments for cancer continues to expand.

Chimeric antigen receptor T-cell (CAR-T) therapy targeting for B-cell maturation antigen (BCMA) is used to treat patients with multiple myeloma (MM). To investigate gastrointestinal changes associated with CAR-T therapy, we performed a retrospective study. A total of 10 patients with 26 gastrointestinal biopsy specimens who underwent CAR-T therapy for MM were identified. Except for one patient with residual multiple myeloma, all specimens demonstrated markedly reduced or absent plasma cells. The most prominent biopsy findings occurred in the small intestine, primarily the duodenum, and included lamina propria lymphocytic infiltration, villous atrophy, foveolar metaplasia, an absence of plasma cells, and an increase in intraepithelial lymphocytes. There was an average of 7 apoptotic bodies per 10 high-power fields (hpf). The terminal ileum was also notable for increase in apoptotic bodies (average of 9.5 apoptotic bodies/10 hpf), villous atrophy, and lamina propria lymphocytic infiltration. The stomach biopsies overall typically showed mild inflammation with no increase in apoptotic bodies. A few colonic specimens demonstrated active colitis and prominent apoptotic bodies, while the majority of the colonic biopsies did not have significant findings other than melanosis coli, and an absence or reduction of plasma cells. The disproportionately greater injury in the duodenum versus the colon highlights the importance of upper endoscopic evaluation in symptomatic patients after CAR-T therapy. The mechanisms for these patterns of injury and differential anatomic findings are unknown; however, an immune-mediated injury associated with CAR-T therapy is suspected. Our study identifies a unique pattern of intestinal injury in patients with MM who received BCMA-targeted CAR-T therapy; this encompasses histologic findings more profound in the small intestine, which include absence of plasma cells, an increase in apoptotic bodies, lymphocytic infiltration, and villous atrophy.

This study presents a comparative analysis of sulfated polysaccharides extracted from Ulva lactuca collected in Tunisia (PSUT) and Morocco (PSUM). FTIR confirmed the presence of sulfate groups, while GC-MS identified diverse sugar components. Both polysaccharides showed strong antioxidant activity, with DPPH and ABTS radical scavenging rates exceeding 50% at the highest concentrations. In a rat model of gastric ulceration, PSUT and PSUM significantly reduced inflammation and lymphocytic infiltration and lowered the activities of 5-lipoxygenase and myeloperoxidase. Treatment also produced clear antioxidant effects, decreasing hydrogen peroxide by 48% and 61% and reducing TBARS by 65% and 71%, respectively. In addition, both polysaccharides inhibited gastric H+/K+-ATPase and pepsin, contributing to notable anti-acidic effects and improved protection of the gastric mucosa. Mucin levels increased by 82% and 100%, while gastric mucus content rose by 32% and 71% for PSUT and PSUM. Overall, PSUM displayed stronger in vivo anti-inflammatory and gastroprotective activities, whereas PSUT showed slightly higher in vitro antioxidant capacity. These findings confirm that the geographic origin of U. lactuca influences the structural features of its polysaccharides and their biological effects. Although based on a single animal model and dose, this work offers a solid basis for future mechanistic and clinical investigations.

Neoadjuvant PD1 blockade with laser interstitial thermal therapy for recurrent high-grade glioma.

Poikilodermatous skin changes represent a diagnostic challenge due to their non-specific clinical features and broad differential diagnoses. This report presents the case of a 29-year-old female with symmetrical, variably pigmented macules and patches over sun-exposed areas, including the neck and upper limbs. Histopathological examination revealed epidermal atrophy, pigment incontinence, basal layer pigmentation, dermal telangiectasia and a mild lymphocytic infiltrate - findings consistent with poikilodermatous change. No features suggestive of interface dermatitis, atypical lymphoid cells or basal vacuolar alteration were observed, effectively ruling out conditions such as cutaneous T-cell lymphoma, connective tissue disease and lichen planus pigmentosus. In the absence of systemic symptoms or alarming histological features, a diagnosis of benign acquired poikiloderma, likely actinic in origin, was favoured. This case underscores the critical role of histopathological correlation in accurately diagnosing poikilodermatous lesions and guiding appropriate clinical management.

Objective Although immune checkpoint inhibitors (ICIs) have improved survival in head and neck squamous cell carcinoma (HNSCC), associated adverse events, such as sialadenitis, remain poorly characterized. This study aimed to define the clinicopathological features, establish the causal pathogenic mechanism, and validate a therapeutic target for ICI-associated sialadenitis. Methods This study integrated three complementary approaches. First, a prospective cohort of 25 HNSCC patients underwent functional assessment of salivary and lacrimal glands before and after ICI therapy. Second, salivary gland tissues from separate cohorts of ICI-treated (n=30) and untreated control (n=30) patients were subjected to comprehensive analysis, including histology, multi-platform immunophenotyping (immunohistochemistry, multiplex immunofluorescence, flow cytometry), and cytokine quantification at both transcript and protein levels. Finally, a preclinical mouse model was established to confirm causality and validate the therapeutic efficacy of IL-17A blockade. Results Following ICI treatment, patients showed significantly reduced salivary and lacrimal secretion ( P < 0.05). Histopathological analysis revealed extensive lymphocytic infiltration, marked periductal fibrosis, and substantial loss of acinar structures. The immune infiltrate was dominated by CD4 + T cells, particularly the Th17 subset, with corresponding upregulation of IL-17A both at transcriptional and protein levels. Crucially, we established a mouse model of anti-PD-1-induced sialadenitis and demonstrated that therapeutic blockade of IL-17A restores salivary function. Conclusion This study establishes ICI-associated sialadenitis as a distinct pathological entity characterized by CD4 + T cell-driven inflammation mediated through the Th17/IL-17 axis, which differs from Sjögren syndrome, predominantly involving B cells and from IgG4 related sialadenitis. By demonstrating therapeutic efficacy in a preclinical model, our findings provide the first preclinical validation of the IL-17 axis as an actionable therapeutic target for this condition.

Background Immune checkpoint inhibitors (ICIs) are a frontline treatment for advanced non-small cell lung cancer (NSCLC), yet 80% of the patients exhibit resistance, creating an urgent need for novel therapeutic strategies. In this study, we investigated the synergistic efficacy of a triple-combination therapy comprising a PD-1 antibody, adoptive NK (natural killer) cells, and an oncolytic adenovirus Ad-anti-TGF-βRII (encoding a TGF-β inhibitor) in NSCLC xenograft mouse models. Methods We investigated the combined effect of Ad-anti-TGF-βRII and NK cells on PD-1 antibody monotherapy using both in vitro cell experiments and the mouse model of NSCLC. Cytotoxicity assays, quantitative real-time PCR, and western blot analysis demonstrated that Ad-anti-TGF-βRII exhibited stronger tumor-killing activity compared to the control oncolytic adenovirus Ad-null. Furthermore, cytotoxicity assays and flow cytometry were employed to explore how the combination of Ad-anti-TGF-βRII and NK cells with PD-1 antibody promotes NK cell proliferation and activation, as well as the potent tumor-killing effect of the combination therapy. In the mouse model of NSCLC, the anti-tumor efficacy of the combination therapy was evaluated by monitoring tumor volume changes, hematoxylin and eosin (H&E) staining. The underlying mechanisms were further investigated using immunofluorescence, immunohistochemistry, quantitative real-time PCR, western blot, and flow cytometry. Results The triple-combination therapy markedly inhibited tumor growth, augmented NK cell cytotoxicity and elevated the expression levels of perforin, granzyme B, and IFN-γ. Furthermore, it significantly increased lymphocyte recruitment and infiltration into tumor tissue. Comprehensive analysis demonstrated the favorable safety profile of this therapeutic regimen. Conclusions Our findings suggest that the combination of a PD-1 antibody, NK cells, and the oncolytic adenovirus Ad-anti-TGF-βRII represents a promising therapeutic strategy for NSCLC by remodeling the tumor microenvironment (TME) to overcome ICI resistance.

Autologous tumor-infiltrating lymphocyte (TIL) cell therapy is showing promising efficacy against immunologically "hot" tumors such as melanoma, cervical cancer, and renal cancer. However, generation of tumoricidal TIL from cold tumors with a low tumor mutational burden, such as many sarcoma types, poses a challenge due to limited infiltration of the tumor microenvironment (TME) with lymphocytes, low frequencies of tumor antigen-specific, high-affinity T cells, and incompletely understood mechanisms of immune-resistance prevailing in the TME. Here, we report the successful generation and expansion of TIL engineered with regulatable, membrane-bound IL15 (cytoTIL15™ cells) from immune-excluded, paucicellular chondrosarcoma biopsies largely consisting of collagenous matrix and demonstrate that these cells have potent tumor-killing capacity in cell culture and in tumor spheroid models in the absence of exogenous IL2. Comprehensive spatial profiling of the TME revealed ubiquitous collagen and myeloid infiltration as major resistance mechanisms, whereas lymphocytic infiltration was largely restricted to peripheral regions of the tumors, a relevant consideration when sampling these tumors for TIL harvest. Moreover, we demonstrate that IL15 reduced the signaling threshold of T-cell receptors isolated from TIL clonotypes, increasing their infiltration and cytotoxicity in autologous 3D tumor models. These results suggest the possibility of developing an effective IL2-free TIL therapy for patients with immune-excluded tumors.

Aging is a major risk factor for breast cancer, yet how it shapes tumor development, molecular phenotype, and immune evasion remains incompletely understood. Deciphering how aging influences cancer evolution is critical for improving risk assessment, prevention, and treatment. Here, using a N-nitroso-N-methylurea (NMU)-induced rat mammary tumor model that recapitulates key features of human breast cancer, we integrated bulk and single-cell transcriptomics, whole-exome sequencing, and histopathological analysis to dissect age-associated differences in mammary tumorigenesis. We found that the age at NMU exposure critically influences tumor incidence, mutational burden, molecular subtype, and the tumor immune microenvironment. Tumors arising in aged rats originated from aging luminal progenitor-like cells, exhibited increased genomic instability, reduced immune cell infiltration, and impaired antigen presentation linked to loss of heterozygosity at chromosome (Chr) 20p. The age-associated epithelial and immune changes we identified were conserved in human breast cancers, where the loss of the homologous Chr 6p region correlated with reduced lymphocyte infiltration and shorter relapse-free survival. These findings reveal that aging profoundly affects tumor-initiating cell populations and promotes immune evasion through chromosomal instability-driven defects in antigen presentation. Our work provides a molecular basis for understanding disease onset and progression that may impact efficacy of immunotherapy in older breast cancer patients.

Abstract Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, yet their long-term efficacy remains limited only to a subset of patients. A major barrier is the immunosuppressive tumor microenvironment (iTME), which hinders the infiltration and function of tumor-infiltrating lymphocytes (TILs). This challenge is particularly pronounced in neuroblastoma (NB), the most common extracranial solid tumor in infants and leading cause of childhood cancer deaths. We previously demonstrated that remodeling the NB iTME with a combination of anthracyclines and ICIs enhances anti-tumor immunity. Building on this, our current aim is to identify novel immunomodulatory agents (IAs) that selectively inhibit NB growth by boosting immune-mediated tumor control. To achieve this, we performed a high-throughput phenotypic screen of a chemical library containing clinical- and preclinical-stage compounds, including FDA-approved drugs with repurposing potential. The screen employed a co-culture system that mimics the iTME, combining PBMCs with MYCN-amplified and non-MYCN-amplified NB cell lines. Tumor growth and viability were measured through multiplexed imaging and biochemical assays. Compounds that reduced tumor viability while promoting immune activation were further evaluated in dose-response studies using NB spheroids, with immune activation further confirmed by flow cytometry. This strategy yielded 12 promising IAs capable of significantly inhibiting NB growth in the presence of immune cells. Some treatments reduced NB cell confluence by up to 40% and increased IFNγ and granzyme B production by cytotoxic CD8+ T cells, indicating potent immune activation. Six compounds were effective in both MYCN-amplified models, and two demonstrated broad activity across human and murine NB models. These two also demonstrated efficacy in the murine 9464D NB model by recruiting macrophage subsets capable of supporting an anti-tumor immunity. We propose that combining these IAs with existing immunotherapies could enhance NB treatment by reshaping the iTME to promote immune infiltration and tumor clearance. This strategy may also hold promise for other cancers characterized by a strongly immunosuppressive microenvironment. Citation Format: Gloria Bedini, Francesca Rita. Pellegrino, Martina Ardito, Fabio Pastorino, Doriana Fruci, Doriana Fruci. High-throughput phenotypic screening reveals novel immunomodulators that enhance immunotherapy in high-risk neuroblastoma [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C046.

Low immunogenicity and highly immunosuppressive tumor microenvironment (TME) present major challenges in immunotherapy, as they restrict T lymphocyte infiltration and activation. Although ferroptosis induction has emerged as a promising approach for enhancing tumor immunogenicity and promoting T cell recruitment, its efficacy is frequently compromised by cholesterol-driven T cell functional exhaustion. To address these limitations, we engineered tumor-targeting, TME-responsive, and size-switchable supramolecular nanoprodrugs that enable multidimensional immune activation. Nanoprodrugs orchestrate a sophisticated cascade of immune activation through four synergistic mechanisms: 1) size-switchable disassembly upon glutathione/ cholesterol exposure for deep tumor penetration; 2) redox imbalance driven by reactive nitrogen species accumulation and glutathione depletion via the synergistic action of oxaliplatin, ferrocene, and RRx-001 for ferroptosis augmentation; 3) immunogenic cell death induction via ferroptosis-apoptosis to initiate tumor immunity cycle, promoting T cell infiltration; and 4) T cell function reinvigoration with the downregulation of programmed cell death protein 1 and T-cell immunoglobulin 3 expression through cholesterol depletion in TME. This integrated approach achieved primary and distant tumor growth suppression, established durable immune memory against recurrence, and systemically enhanced the antitumor immunity. By concurrently targeting tumor immunogenicity, TME immunosuppression, and T cell exhaustion, this multidimensional strategy represents a transformative advancement in cancer immunotherapy.

Abstract Background: Bruton’s tyrosine kinase (BTK), a downstream mediator of the B-cell receptor (BCR) signaling pathway, is known to be essential in differentiation and proliferation of B-cell. Thus, BTK inhibitors are used to treat refractory mantle cell lymphoma and chronic lymphocytic leukemia. Recent discoveries have revealed the expression of BTK in myeloid-derived suppressor cells, known to worsen breast cancer (BC) outcomes. To this end, it was of interest to investigate the clinical relevance of BTK expression in BC. Methods: Total of 9297 BC patients from 23 independent cohorts with tumor transcriptome and clinical data were analyzed. Results: Based on two independent single-cell sequencing cohorts, approximately 15% of B-cells and 30% of myeloid cells within the tumor microenvironment expressed BTK, while no other cells exhibited BTK expression, suggesting that the BTK expression signal from bulk tumor samples primarily originates from these cells. Correlation with BTK expression was highest in macrophage regulation (r=0.94), and leukocyte fraction, lymphocyte infiltration, TCR Shannon and TCR Richness (all r>0.74), but not with BCR Shannon nor BCR Richness. Total macrophage, M1 and M2 macrophage, dendritic cells, total B-cells, memory B-cells, as well as CD8 and CD4 cells were all highly infiltrated in BTK high BCs. Moreover, BTK expression correlated with both PD1 & PD-L1 in all BC subtypes. Biologically, BTK high BC expression was linked to immune-related gene sets, with gene set enrichment analysis (GSEA) showing enrichment in pathways related to immune response such as Complement, Inflammatory response, Allograft rejection, IFN-γ, IFN-α, IL2, IL6, and TNFα signaling. At the same time, BTK high BC enriched tumor-aggravating signaling pathways such as PI3k/AKT/mTOR signaling and mTORC1 signaling. The cytolytic activity score, reflecting the global immune killing, was significantly higher in the BTK high BC. Given that BC with abundant tumor infiltrating lymphocytes respond better to chemotherapy, the relationship between BTK expression and response to neoadjuvant chemotherapy was assessed. Somewhat unexpectedly, BTK expression was associated with better response in only one out of ten neoadjuvant cohorts. Further, no change in BTK expression were observed before and after neoadjuvant chemotherapy in six cohorts. While BTK expression was higher in primary sites compared to metastatic sites, it was not associated with distant recurrence. Across the TCGA, METABRIC, and SCAN-B cohorts, no significant overall survival differences were observed between the BTK high and low expression groups. All findings were consistent regardless of the BC subtypes. Conclusions: Myeloid cells and B-cells were the source of BTK expression, and it was associated with macrophage regulation and infiltration of various types of immune cells and proteins including PD1 & PD-L1 and enriched immune related gene sets. However, BTK expression was not associated with response to neoadjuvant chemotherapy or Impact on survival, regardless of BC subtypes. Citation Format: T. ALRammah. Expression of Brutons tyrosine kinase (BTK) in breast cancer associates with tumor immune microenvironment but lacks predictive value for clinical outcome [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-13-11.

Abstract Introduction: Immune activation and lymphocyte infiltration into the tumor microenvironment are known to predict treatment response in HER2-positive (HER2+) breast cancer. Our work has shown that dynamic changes in peripheral absolute lymphocyte count (ALC) following initial neoadjuvant therapy (NAT) are also associated with a higher likelihood of achieving pathologic complete response (pCR). In this study, we aimed to develop and validate a predictive model for pCR based on clinically obtained pre-treatment ALC and ALC following a single dose of NAT, with the goal of creating a tool for early response prediction in HER2+ breast cancer. Methods: We assembled a retrospective discovery cohort of patients with HER2+ breast cancer treated with trastuzumab-based NAT from the Oncoshare database, which integrates electronic medical records and California Cancer Registry data from patients diagnosed between 2000 and 2020 at Stanford University. Our non-overlapping validation cohort consisted of patients with HER2+ breast cancer who underwent NAT on clinical trials of HER2-targeted therapy and/or who were enrolled in prospective institutional tissue collection studies. In the discovery cohort, we constructed a multivariable logistic regression model to identify predictors of pCR, including baseline ALC, early ALC change, days between baseline and on-treatment ALC draws, age at diagnosis, estrogen receptor (ER) status, stage, grade, and self-reported race/ethnicity. Early ALC change was defined as 1 minus the ratio of cycle 1 ALC to baseline ALC. To develop an ALC-based risk grouping strategy, we used logistic regression to create a composite risk score from baseline ALC and early ALC change, followed by receiver operating characteristic curve analysis. We then applied grid search optimization to test combinations of cutoff points across the 10th to 90th percentiles of the risk score distribution and identified the pair of cutoffs that maximized area under the curve (AUC) when patients were stratified into three risk categories. Model performance was evaluated using AUC analysis in both the discovery and validation cohorts. Results: In the discovery cohort (n = 154), higher baseline ALC and greater early ALC decline were independently associated with increased odds of pCR. The composite ALC risk score, using baseline ALC and early ALC change, stratified patients into three groups: low (25% of patients, n = 39), intermediate (45%, n = 69), and high (30%, n = 46) probability of pCR. Observed pCR rates in these groups were 15%, 51%, and 76%, respectively. When this ALC-based risk grouping was integrated with the other covariates (including time interval between ALC draws, age, ER status, clinical stage, tumor grade, and race/ethnicity), the full multivariable model demonstrated strong predictive performance with an area under the curve (AUC) of 0.81 (95% CI 0.74-0.88). In the separate validation cohort (n = 83), the composite ALC risk score stratified patients into low (30% of patients, n=25), intermediate (48% of patients, n=40), and high (22% of patients, n=18) probability of pCR groups, with observed pCR rates of 12%, 63%, and 78%, respectively. The model showed similar discriminative ability in the validation cohort, with an AUC of 0.81 (0.71-0.90). Conclusion: Early ALC dynamics, with no other covariates, effectively stratify patients with HER2+ breast cancer based on their likelihood to achieve pCR after NAT. We developed and validated a clincal model using routinely collected laboratory values, which achieved strong predictive performance in both discovery and validation cohorts. This ALC-based tool offers a cost-effective and scalable alternative for early response prediction, with potential to inform treatment adaptation strategies in real time. Citation Format: C. Bergstrom, I. Luo, E. Kotler, M. Satoyoshi, S. Philip, C. Curtis, A. Kurian, S. Han, J. Caswell-Jin. A clinical model to predict pathologic complete response using early peripheral absolute lymphocyte dynamics in HER2+ breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-10-30.

Biochemical recurrence (BCR) is a critical factor affecting the prognosis of prostate cancer (PCa) patients, while T cell exhaustion and metastatic prostate cancer (mPC)-related genes play significant roles in tumor progression. This study aims to identify key genes associated with BCR by integrating single-cell transcriptomics and deep learning techniques, and to validate their clinical significance. First, we identified highly expressed genes in CD8+ T cell exhaustion clusters from single-cell RNA sequencing data (scRNA-seq) of PCa and intersected them with mPC-related genes. Based on these genes, significant prognostic factors were screened using Cox regression analysis, and a deep learning neural network model was constructed to predict the risk of biochemical recurrence in prostate cancer patients. The tumor-infiltrating lymphocyte (TILs) infiltration was predicted by stratifying patients into high- and low-risk groups. ERBB2 was identified as the most predictive gene through model analysis. Subsequently, ERBB2 expression was validated in an independent PCa cohort using immunohistochemistry (IHC), and its association with biochemical recurrence and clinicopathological features was evaluated through survival analysis and statistical methods. The deep learning model demonstrated excellent performance in predicting BCR, with ERBB2 identified as the most important predictive factor. IHC results revealed that patients with high ERBB2 expression had significantly shorter biochemical recurrence-free survival (bRFS) (P < 0.05). Moreover, high ERBB2 expression was significantly associated with higher prostate-specific antigen (PSA) levels, Node-Metastasis (NM) stage, and International Society of Urological Pathology (ISUP) grade (P < 0.05). This study, for the first time, integrates single-cell transcriptomics, deep learning, and IHC to reveal the critical role of ERBB2 in biochemical recurrence of PCa. High ERBB2 expression is not only a potential biomarker for poor prognosis in PCa patients but may also provide a novel target for personalized therapy.

Abstract Background: Triple-negative breast cancer (TNBC) tumors are aggressive, highly immunogenic, and often associated with early recurrence. Studies suggest that high levels of tumor-infiltrating lymphocytes (TILs) are associated with improved clinical outcomes, including overall survival (OS) and progression-free survival (PFS). We evaluated the presence or absence of complete pathological response (pCR) according to the TILs found in the tumor analysis of patients included in this cohort. Patients and Methods: A total of 121 patients diagnosed with clinical stage (CS) II-III TNBC who received treatment based on the KeyNote-522 protocol (pembrolizumab combined with neoadjuvant chemotherapy) from April 2022 to January 2025 were evaluated. Patients were divided into two groups with a TILs cutoff of &amp;gt;30 and ≤30. Subsequently, clinical outcomes of pCR, overall survival (OS), and progression-free survival (PFS) were assessed. Results: A total of 121 patients, with a median age of 44 years, were analyzed, with the majority (69%) classified as CS II and 63% being premenopausal. TILs analysis was performed in 104 patients, with 74 (71%) having TILs ≤30 and 30 (28.8%) having TILs &amp;gt;30.Regarding tumor characteristics, the predominant histological subtype was ductal in both subgroups (81.8%). The majority of the population had histological grade 3 (67.7%). Regarding pCR, we observed that in the TILs ≤30 subgroup, 37 patients (50%) achieved a complete response, while in the TILs &amp;gt;30 subgroup, 25 patients (83.3%) achieved a complete response. Patients with TILs greater than 30 had a higher likelihood of pCR (OR 0.24; p &amp;lt; 0.01). Eight patients experienced tumor recurrence, with 7 in the TILs ≤30 subgroup.Regarding PFS and OS analysis, no statistically significant difference was observed (p = 0.58 and p = 0.88, respectively). In the TILs ≤30 subgroup, with a 40-month follow-up, the median OS was 23.5 months, and the median PFS was 21.5 months. In the TILs &amp;gt;30 subgroup, the median OS was 23 months, and the median PFS was 19 months. However, when analyzing the relationship between pCR and TILs, it was found that patients with TILs greater than 30 had a higher likelihood of pCR (OR 0.24; p &amp;lt; 0.01). Conclusion: The presence of TILs greater than 30 was associated with a significant increase in pCR. The data obtained in this study suggest that tumor lymphocytic infiltrate may serve as a prognostic biomarker for pCR in patients with TNBC. However, it should be considered that the sample size was small, and further studies in this area are needed. Citation Format: F. BALINT, G. de Almeida, N. Pandolfi, M. Cesca, L. Leite, S. Sanches, E. Santos, V. Cordeiro, M. Tavares. Tils as a predictor of response in triple-negative breast cancer: analysis of clinical and pathological outcomes in patients undergoing neoadjuvant chemotherapy based on the keynote-522 protocol in a brazilian cancer center [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-10-18.

Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cell therapy, where the lymphocytes of a cancer patient's tumor are harvested, expanded in vitro using IL-2 stimulation, and then infused back into the patient[1], [2]. However, even with the use of TIL therapy, cancer cells can survive for various reasons, such as poor lymphocyte infiltration into tumors, chronic activation of the T cell receptor and the immunosuppressive tumor microenvironment [3].Cytokine-inducible SH2-containing (CISH) protein is a negative regulator of T cell activation, and in a recent clinical trial was knocked out in TILs to improve TIL therapy efficacy [4]. In this study, we developed a mechanistic signaling pathway model to theoretically evaluate the efficacy of CISH knockout (CISH KO) in T cell activation and examine potential alternative target genes that can theoretically be targeted using multiplex gene-editing or drugs to further improve T cell activation and function [5]. Our modeling results demonstrate that CISH knockout increases the transcription of activation biomarkers IL-2 and TNF-α, but also inhibitory biomarkers such PD1 and FasL. Using global sensitivity analysis, we also found that GSK3B, which is responsible for the deactivation of the NFAT, is also predicted to further increase T cell activation when knocked out. In addition, we predict that PDCD1, FAS and CTLA4 can be knocked-out in combination with CISH to further enhance T cell activation and prevent exhaustion and apoptosis.

Abstract Background: Sacituzumab Govitecan (SG) is an antibody-drug conjugate (ADC) containing a TROP2-directed antibody bound to a topoisomerase I inhibitor payload. SG is approved for the treatment of patients (pts) with metastatic triple-negative breast cancer who have received at least two prior systemic therapies, including one for advanced disease, and pts with hormone receptor-positive (HR+)/HER2-negative metastatic breast cancer (mBC) after endocrine therapy and ≥2 systemic treatments in the advanced setting. SG efficacy does not seem to correlate to TROP2 protein expression, and no additional efficacy biomarkers have been identified. Methods: SOLTI ACROSS-TROP2 (NCT06236269) is a phase II, open-label, single-arm trial investigating biomarkers of efficacy and mechanisms of resistance to SG in 100 pts with HR+/HER2- mBC who progressed during or after treatment with CDK4/6 inhibitors and received up to one prior chemotherapy or ADC regimen for metastatic disease. Pts received SG at 10 mg/kg via IV infusion on Days 1 and 8 of each 21-day cycle. Mandatory tumor biopsies were obtained at baseline and after 2-3 weeks of treatment (C2D1). The primary objective is to evaluate the change in CelTIL score (a composite score of tumor cellularity and tumor infiltrating lymphocytes) between baseline and C2D1 tumor samples (Nuciforo et al, Ann Oncol 2019). Key secondary endpoints include progression-free survival (PFS), overall response rate (ORR), and safety. Here we present the planned interim analysis results from the first 50 enrolled pts. Results: Between April and December 2024, 50 pts were enrolled. Median follow-up was 8.6 months (m). Median age was 58 (range 30-79), all pts were female, most were postmenopausal (78%), had no visceral metastases (64%), and received SG as second line for mBC after chemotherapy or ADC (70%). At data cut-off (May-25), 18/50 (36%) remained on treatment and 43/50 (86%) were evaluable for the primary endpoint. The mean increase in CelTIL score from baseline to C2D1 was 4.33 points (95% CI 0.99-7.65), indicating a significant overall change, with a greater and significant increase in patients that achieved radiological response (6.33 vs 3.13 points). PFS events occurred in 28/50 pts (maturity 56%). Median PFS was 6m (4.2-NR) both overall and among pts treated in second line for mBC. ORR was 37% and 38%, respectively. Grade ≥3 treatment-related adverse events (TRAEs) occurred in 48.0% of pts. Most common TRAEs were fatigue (58%), anemia (58%), neutropenia (54%), nausea (46%) and diarrhea (38%). Treatment discontinuation rate due to TRAEs was 8%. Conclusions: In this interim analysis of SOLTI ACROSS-TROP2, one cycle of SG led to an increase in CelTIL score, particularly among patients who achieved a radiological response. Consistent efficacy was observed in terms of PFS and ORR. The final results from the full cohort of 100 patients, along with a comprehensive translational analysis of paired pre- and on-treatment biopsies, are expected to provide further insights into biomarkers of response and mechanisms of resistance to SG in HR+/HER2- mBC. Citation Format: E. Ciruelos, T. Pascual, B. Adamo, M. Alva Bianchi, I. Blancas, L. Carnerero, R. Villanueva-Vázquez, B. Fullana, M. Melé, J. Salvador Bofill, J. M. Cejalvo, A. Ferrando-Díez, Y. Izarzugaza, M. Borrell, X. González Farré, G. Villacampa, M. Paes Dias, F. M. Juan, M. Oliveira. Biomarkers of response and resistance to Sacituzumab Govitecan in HR+/HER2− advanced breast cancer: interim results from the phase II SOLTI ACROSS-TROP2 Trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-02-23.

Abstract Background: Neoadjuvant immunotherapy with chemotherapy is widely used for management of localized triple negative breast cancer (TNBC). While tumor infiltrating lymphocyte (TILs) have been associated with therapy response, the impact of absolute lymphocyte count (ALC) and neutrophil-to-lymphocyte ratios (NLR) in modulating response is not known. In this study, we evaluated the association between ALC, NLR, and therapeutic response to chemo-immunotherapy in patients with TNBC. Methods: In this multi-institutional retrospective study, patients with stage I-III TNBC diagnosed between 2019 to 2023 were included who were treated with at least one cycle of neoadjuvant pembrolizumab and chemotherapy at two comprehensive cancer centers. Pre-treatment laboratory values were collected for all patients. An NLR cut-off value of 5 was used based on previous studies. Univariate and multivariate logistic regression and Cox proportional hazards models were used to assess the impact of pretreatment NLR on pCR. Results: A total of 372 patients with early-stage TNBC were included with an overall pCR rate of 61.3%. The median baseline NLR was 2.21 (95% CI, 2.10-2.30), and 9.4% of patients had a baseline NLR &amp;gt;5. Patients with a baseline NLR &amp;gt;5 had significantly lower pCR rates compared to those with NLR &amp;lt;5 (45.7% vs. 62.9%; p=0.047). Similarly, baseline lymphopenia was also associated with lower pCR rates (45.9% vs. 63.0%; p=0.043). Multivariate logistic regression demonstrated that baseline NLR &amp;gt;5 was significantly associated with inferior pCR rates (odds ratio [OR], 0.49; 95% CI 0.24-0.997; p=0.049), adjusting for age, race and stage. Conclusions: This is the first study to demonstrate that absolute (and relative) lymphopenia is associated with lower pCR rates in patients with early-stage TNBC treated with neoadjuvant chemo-immunotherapy. Further research is needed to further evaluate lymphocyte count as a biomarker of neoadjuvant chemo-immunotherapy response and develop potential therapeutic strategies to improve lymphopenia in patients with early-stage TNBC. Citation Format: A. LeVee, E. Yang, K. Tsai, N. Baclig, A. Soliman, S. Zhang, A. Kordic, J. Mortimer, M. Lechner, S. Alkassis, N. McAndrew, M. Lipsyc-Sharf, M. Sedrak, R. Callahan, A. Master, D. Prager, K. McCann, A. Bardia. Pretreatment neutrophil-to-lymphocyte ratio is associated with pathologic complete response following neoadjuvant chemo-immunotherapy in early triple-negative breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-10-26.

Abstract Background The Neo-N trial previously showed a pathological complete response (pCR) rate of 53% with 12 weeks of carboplatin/paclitaxel plus nivolumab. We report 3-year EFS and associations of an ultrasensitive tumor-informed structural-variant (SV)–based circulating tumor DNA (ctDNA) assay with EFS. Methods Neo-N is an investigator-initiated, non-comparative, open-label, randomized phase 2 trial across 14 sites. Adults with operable stage I–II TNBC received carboplatin AUC5 q3w + weekly paclitaxel for 12 weeks with nivolumab per assigned schedule- A: lead in monotherapy or B: concurrent Nivolumab, followed by surgery and adjuvant therapy per investigator. A cohort underwent tumor-informed ctDNA SV assay derived from whole genome sequencing and plasma was collected at: T0 (baseline) and T1 (5+/-1 weeks) and T2 &amp;lt;28 days post-surgery. Primary objectives of this report are 3-year EFS and association of ctDNA status and dynamics, as well as tumor infiltrating lymphocyte (TIL) changes with EFS. Results As of the planned data cutoff median follow-up is 36 months (range 20-43mo). Among all randomized participants (n=108), the 36mo EFS rate is 92.4% (90% CI 83.5-96.6%) for cohort A and for B 83.1% (90% CI: 72.4% to 89.9%). 59% (64/108) had ≥1 ctDNA assay timepoint. Baseline T0 ctDNA was detectable in 91% with median ctDNA tumor fraction = 0.3526% (P25 = 0.0481, P75 = 1.856; range 0–28.61). At T1 and T2, ctDNA was detectable in 18% (11/62) and 4% (2/56), respectively. There was comparable early ctDNA clearance at T1 across the two cohorts (A 76% vs B 77%) despite less chemotherapy exposure in Cohort A. T1 ctDNA + had no pCRs and 36mo EFS of 45.5% (90% CI: 20.8% to 67.3%); T1 ctDNA- had a pCR rate of 58.8% and EFS of 91.5% (90% CI: 81.5% to 96.2%). Across ctDNA dynamics: T0-T1 74% (46/62) went from Pos to Neg i.e. cleared ctDNA and had a pCR rate 63.0% (90% CI: 49.9% to 74.9%), EFS 90.6% (90% CI: 79.6% to 95.8%) vs Pos-Pos had 0 pCRs (n=11), EFS 45.5% (90% CI: 20.8% to 67.3%)and Neg-neg (n=5) had a 100% EFS. No new safety signals were observed; immune-related adverse events were consistent with prior reporting. Full efficacy (EFS/Overall survival), TIL and ctDNA data will be presented. Conclusions A short-course, non-anthracycline carboplatin/paclitaxel plus nivolumab achieves durable 3-year disease control in early TNBC. An on treatment tumor-informed SV-ctDNA measurement is associated with EFS, complementing surgical endpoints. These findings support ultrasensitive ctDNA for risk-stratification tool and provide a rationale for early on treatment ctDNA-guided escalation/de-escalation strategies to refine neoadjuvant chemo-immunotherapy for early TNBC patients. Citation Format: S. Loi, S. M. Niman, N. Zdenkowski, N. Segui, P. A. Francis, S. Baron Hay, W. Fox, K. Punie, A. M. Menzies, R. Angus, S. Zardawi, B. Mitchell, C. Mavin, S. Dawson, K. Howarth, M. J. J. Kuper-Hommel, M. M. Regan. Neo-n (neon): three-year event-free survival and ultrasensitive ctdna dynamics in early triple-negative breast cancer (tnbc) treated with neoadjuvant carboplatin/paclitaxel and nivolumab [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD7-10.

Advanced cell therapies often face high parameter uncertainty at launch, prompting calls to collect further data about long-term effectiveness and safety. However, the value of collecting these data to support resource allocation decision-making is not known. Therefore, this study aimed to appraise all published value of information analyses for advanced cell therapies. A systematic review (PROSPERO: CRD42023446874) identified value of information analyses for advanced cell therapies between inception and 14 May 2025 (databases: Medline; Embase). Included studies reported the expected value of perfect information (EVPI), expected value of partial perfect information (EVPPI), expected value of sample information (EVSI) or expected net benefit of sampling (ENBS). Study design and value of information results were summarised in a narrative synthesis. Quality of reporting was assessed using the Consolidated Health Economic Evaluation Reporting Standards Value of Information (CHEERS-VOI) checklist. Three published value of information analyses were identified: tisagenlecleucel for relapsed/refractory acute lymphoblastic leukemia; tisagenlecleucel for relapsed/refractory diffuse large B-cell lymphoma, and tumor infiltrating lymphocyte cell therapy for advanced melanoma. The beneficiary populations were 6, 36, and 400 individuals per year, respectively. All studies reported EVPI; two studies reported EVPPI. Estimated base case population EVPI was: €314,455, €0, and €2,250,000, respectively. Estimated EVPPI indicated that input parameters for survival extrapolations were the most valuable targets for further research specifically during scenario analyses that explored a lower cost of treatment acquisition. Value of information analyses will help decision-makers, analysts, and manufacturers understand whether long-term data collection is worthwhile to reduce decision uncertainty for advanced cell therapies at launch. Current estimates indicated that the value of further research is likely to be low. The value of collecting additional data will likely increase if future advanced cell therapies are priced such that their corresponding incremental cost-effectiveness ratio aligns with a relevant cost-effectiveness threshold and if they are indicated for larger beneficiary populations.