Citations

Abstract Background: The association of TILs with pathologic complete response (pCR) and survival in HER2+ breast cancer is not well established with several prior studies showing inconsistent results. In this secondary analysis of EA1181, we evaluated the association between stromal TILs (sTILs) and pCR rates. Methods: EA1181 (NCT04266249) enrolled patients (pts) with anatomic clinical stage II/III HER2+ breast cancer who preoperatively received 4 cycles of trastuzumab and pertuzumab with 12 weeks of paclitaxel or docetaxel q3w x 4 (THP), followed by surgery. sTILs density was assessed on full-face hematoxylin and eosin (H&E)-stained tumor biopsy sections. sTILs scores were analyzed per protocol as a continuous variable (every 10% increment) and as a categorical variable (<10% vs 10-60% vs >60% as per Denkert et al, 2018), and also with an exploratory 30% cutoff commonly used in TNBC. Cox proportional hazards models were used to examine the association between sTILs and pCR, adjusting for available baseline factors. The associations between sTILs (by category) and clinicopathologic characteristics were examined using Fishers’ exact test. Results: Among 2141 pts with HER2+ BC treated on EA1181, H&E slides were available for 1328 (62%). The study population evaluated for sTILs was comparable to the overall population. pCR rates were 44.5% overall, 64% for HER2+/ER- and 33% for HER2+/ER+ disease. sTILs distribution was: 623 (47%) <10%, 698 (52%) 10-60%, and 7 (0.5%) >60%, which led us to merge the latter two groups (705 [53%] had ≥10% sTILs). In univariable and multivariable analyses, increasing sTILs (as a continuous variable) were associated with increasing pCR rates in both HER2+/ER+ and HER2+/ER- disease (p<0.001; Table). sTILs analyzed as a categorical variable (<10% vs ≥10%) were significantly associated with pCR for all patients (combined) and for those with HER2+/ER+ disease. In exploratory analysis using a cutoff of <30% vs ≥30% sTILs, an association with pCR was also seen in univariable analysis for both HER2+/ER+ disease (p<0.001) and HER2+/ER- disease (p=0.045), though in multivariable analysis with clinical factors, sTILs >30% remained a significant predictor for pCR only in HER2+/ER+ disease. Additional exploratory analysis with other molecular biomarkers, intrinsic subtypes, and immune signatures will be presented. Conclusion: sTILs were associated with pCR after THP, further supporting the important role of immune mechanisms in HER2+ breast cancer, and highlighting a potentially robust predictive tool to assess pathologic response. Baseline sTILs could potentially inform the preoperative design of future trials of therapy optimalization. Association of baseline sTILs with recurrence free survival in EA1181 will be reported in the future. Citation Format: S. S. Badve, F. Zhao, N. Tung, Y. Gokmen-Polar, C. C. O'Sullivan, A. Prat, E. P. Winer, J. L. Wright, A. Recht, A. C. Weiss, J. A. Tjoe, S. M. Feldman, G. B. Rocque, M. Smith, N. Unni, S. Sardesai, S. Tang, S. Modi, W. J. Irvin, P. Villagrassa, C. Battelli, A. K. Krie, N. Bagegni, M. A. George, M. L. Telli, V. F. Borges, N. D’Abreo, P. Shah, K. D. Miller, A. H. Partridge, L. A. Carey, A. M. DeMichele, A. C. Wolff, ECOG-ACRIN.Tumor infiltrating lymphocytes (TILs) and pathologic complete response (pCR) in stage II/III HER2+ breast cancer treated with taxane, trastuzumab, and pertuzumab (THP): secondary results from the ECOG-ACRIN EA1181/CompassHER2 pCR 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 GS1-04.

Abstract Recurrence scores based on a 21-gene assay are clinically useful for predicting prognosis and chemotherapy benefit in postmenopausal node-positive breast cancer patients, but its performance in premenopausal patients is inconsistent. Here, we evaluated Ataraxis Breast RISK (ATX), an AI test that predicts recurrence risk, and compared it with the genomic assay. ATX identified high risk patients misclassified as low risk by the genomic assay and therefore may refine selection of patients for adjuvant chemotherapy.

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, and despite advances in treatment, immune suppression remains an obstacle to effective therapy. Effector CD4+ T cells (CD4+ Teff) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Treg), which accumulate in lung tumors and mediate suppressive functions through multiple mechanisms. This suppression leads to tumor progression and poor patient outcomes. However, the mechanisms underlying Treg-mediated suppression are not fully understood. Herein, we identify the extracellular adenosine 5-triphosphate receptor P2RX7 as a key regulator of Treg function in lung tumors. In a murine lung cancer model induced by Lewis lung carcinoma cells, we found that P2RX7 enhanced the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell–specific P2RX7-knockout (P2RX7-KO) mice, reduced Treg infiltration was accompanied by increased CD4+ Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibited reduced tumor growth, confirming a Treg-intrinsic role of P2RX7. Suppression assays revealed that tumor-infiltrating wild-type Tregs had greater suppressive activity compared with P2RX7-KO Tregs, which failed to inhibit type 1 and follicular helper T–like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that wild-type Tregs expressed higher levels of the immunosuppressive molecule CTLA-4 when compared with P2RX7-KO Tregs. Thus, we conclude that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer and targeting P2RX7 may constitute a strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

Abstract Background The incidence of early onset breast cancers (BCs) has increased, paralleling rising trends in delayed childbearing. We hypothesize that a distinct postpartum BC (PPBC) subtype, identifiable by time since last birth (TSLB) and biomarker expression, contributes to this trend. Methods We applied GeoMx Digital Spatial Profiling (DSP) to measure associations between TSLB and 71 proteins in 640 BCs from women aged ≤40 years included in the Young Women’s Breast Cancer Study. We analyzed data using univariable linear regression and multivariable sliced inverse regression to account for higher order interactions among biomarkers. Results In keratin-rich segments, progesterone receptor (PR) (P = 1.00 × 10−4) and PTEN (P = 1.00 × 10−3) were associated with longer TSLB; multivariable analyses revealed positive associations for GZMB (P = 4.00 × 10−4), SMA (P = 1.00 × 10−4), and NF-1 (P = 1.00 × 10−3). In keratin-poor segments, univariable significant positive associations were found for PR (P = 2.00 × 10−4) and PTEN (P = 1.00 × 10−3), whereas CD20 (P = 3.00 × 10−4) and CTLA4 (P = 4.00 × 10−5) were negatively associated; multivariable significant associations were found for fibronectin (P = 3.00 × 10−5) and pan-Akt (P = 1.00 × 10−3). Associations persisted after adjustment for multiple comparisons and BC molecular subtypes. Associations including for PR, PTEN, and CD20 were strongest among women with the shortest TSLB. OPAL multiplex immunofluorescence assays for PR, PTEN, CD20, SMA, and CTLA4 replicated several DSP findings, particularly when stratified by subtype and with compartment matching. In TCGA, RNA species linked to proteins associated with TSLB correlated strongly with a T-cell exhaustion signature previously linked to poor prognosis among premenopausal women. Conclusion These data support PPBC as a biologically coherent phenotype defined by TSLB and biomarker profile, with potential implications for prevention and therapy.

Importance The choice of induction agent during rapid sequence intubation (RSI) of critically ill adults may affect clinical outcomes. Although ketamine and etomidate are frequently used for RSI, their comparative effectiveness remains controversial. Objective To compare the safety of etomidate vs ketamine for emergency RSI in critically ill adults. Design, Setting, and Participants This cohort study used a target trial emulation with observational data collected between March 1, 2022, and April 30, 2024, from 18 emergency departments across Brazil, with data from the Brazilian Airway Registry Cooperation. Adults who underwent RSI and received either etomidate or ketamine as a sole hypnotic agent were included; those with preintubation cardiac arrest or immediate postintubation transfer were excluded. Exposure Administration of etomidate or ketamine as the induction agent for RSI. Main Outcomes and Measures The primary outcome was 28-day in-hospital mortality. Secondary outcomes included 7-day in-hospital mortality, first-attempt intubation success, and major adverse events (new hemodynamic instability, severe hypoxemia, and cardiac arrest) within 30 minutes after intubation. Inverse probability of treatment weighting was used to adjust for confounding. Risk ratios (RRs) and risk differences (RDs) with 95% CIs were used to compare outcomes between groups. Results Among 1810 patients (median age, 64 years [IQR, 50-74 years]; 1048 men [57.9%]), 514 received ketamine and 1296 received etomidate. The median shock index was higher in the ketamine group than the etomidate group (0.81 [IQR, 0.65-1.01] vs 0.76 [IQR, 0.59-0.99]), and preintubation vasopressor use was more common in the ketamine group than the etomidate group (191 of 514 [37.2%] vs 391 of 1296 [30.2%]). Weighted 28-day mortality was higher with etomidate than ketamine (60.5% [95% CI, 57.2%-63.8%] vs 54.4% [95% CI, 45.0%-63.9%]; RR, 1.14 [95% CI, 1.03-1.27]; RD, 7.6% [95% CI, 2.0%-13.3%]). Seven-day mortality was also higher with etomidate than ketamine (35.2% [95% CI, 32.0%-38.3%] vs 30.1% [95% CI, 23.5%-36.7%]; RR, 1.19 [95% CI, 1.04-1.35]). New hemodynamic instability within 30 minutes after intubation was more frequent with ketamine (24.2% [95% CI, 20.4%-28.0%] vs 18.9% [95% CI, 16.7%-21.0%]; RR, 0.78 [0.64-0.95]). There were no statistically significant differences in the other secondary outcomes. Conclusions and Relevance In this cohort study of critically ill adults undergoing RSI, etomidate use was associated with higher in-hospital mortality at 7 and 28 days compared with ketamine. These findings highlight the need for definitive randomized clinical trials to compare both agents.

Abstract Introduction: Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy, in which nearly all patients are expected to relapse and undergo multiple lines of treatment over the course of their disease. By the time patients receive CAR-T cell therapy, they have typically been exposed to numerous prior therapies, including high-dose melphalan (HDM). Chemotherapeutic agents that cause genotoxic stress, such as HDM, can induce cellular senescence in normal cells. More recently, it has been demonstrated that tumor cells can also exhibit features of therapy-induced senescence (TIS). TIS in tumor cells is associated with distinct gene expression patterns that have been implicated in resistance to therapy and relapse. To investigate whether TIS is activated in MM cells following exposure to chemotherapies, particularly in the context of late-stage disease, we performed an integrated analysis of single-cell RNA sequencing (scRNA-seq) datasets. Methods: We integrated scRNA-seq data from bone marrow plasma cells and MM cells using the following three sources: 1) Boiarsky, et al. (GSE193531, dbGaP: phs001323.v3.p1), which includes healthy donors, MGUS, SMM, and newly diagnosed MM (NDMM) patients; 2) Dhodapkar, et al. (GSE210079), which includes late-stage MM patients pre- and post-CAR-T cell therapy; and 3) newly generated scRNA-seq data from healthy donor (N=3) and late-stage MM patients pre- (N=10) and post- (N=2) CAR-T cell therapy. Non-plasma cells were excluded based on canonical plasma cell markers. We used Seurat v4.3 for data integration, employing canonical correlation analysis (CCA) to define anchors using the FindIntegrationAnchors and IntegrateData functions. Integration quality across datasets was validated by the consistent clustering of healthy plasma cells and expected expression patterns of chromosomal translocation markers CCND1 in t(11;14) cases and FGFR3 in t(4;14) cases, as previously described (PMID: 36396631). To evaluate senescence, we applied single-cell gene set enrichment analysis (scGSEA) using previously published gene sets (PMID: 40164720): SenUp (Senescence Upregulated), SenGA (Senescence Growth Arrest), and SCAPs (Senescent Cell Anti-apoptosis Pathways). We also curated a Plasma Cell Senescence (PCSen) gene set, composed of senescence-related genes that were differentially upregulated in pre-malignant plasma cells across two independent datasets (GSE5900, GSE47552).A normal plasma cell gene set was also generated, comprising genes differentially expressed between healthy and abnormal plasma cells in GSE193531. Results:In the integrated dataset, scGSEA revealed significantly higher enrichment of PCSen, SenUp, SenGA, and SCAPs in late-stage MM cells (both pre- and post-CAR-T cell therapy) compared to NDMM and healthy donors (p<0.05, Kruskal-Wallis with Dunn's posttest). Conversely, the Normal gene set was significantly enriched in healthy plasma cells relative to MM (p<0.05). Expression of CDKN2A, which encodes the senescence marker p16, was also increased in late-stage MM relative to NDMM or healthy plasma cells. Notably, in our newly generated scRNA-seq samples collected within 6 months before CAR-T therapy, we observed a significant inverse correlation between senescence marker expression and progression free survival (PFS) post-CAR-T, suggesting that MM cells exhibiting TIS may be more resistant to T cell mediated cytotoxicity. Supporting this, preliminary in vitro experiments showed that MM cells treated with HDM displayed reduced sensitivity to T cell-mediated cytotoxicity, consistent with a senescence-associated resistant phenotype. Conclusion: Our findings demonstrate that late-stage MM cells exhibit transcriptional signatures consistent with therapy-induced senescence, potentially driven by prior exposure to chemotherapy. This senescent phenotype may contribute to resistance to immunotherapies, including CAR-T cells. These data highlight senescence as a mechanism of resistance and a potential biomarker for predicting CAR-T outcomes. Further studies are needed to explore senescence-targeted strategies to enhance immunotherapeutic efficacy in MM.

Abstract Description Vaccination is essential for preventing viral infections and promoting public health; however, its efficacy may vary due to factors such as the nature of the pathogen, vaccine type, and individual immune response. It is known that intranasal immunizations can enhance the immune response by stimulating tissue-resident memory T cells (TRMs), which promote early viral clearance at the infection site. Our study aims to optimize the generation and maintenance of TRMs in the lungs using purinergic signaling components as adjuvants. For this, we crossed B knockout mice with hACE2 mice, generating a B cell-deficient lineage capable of internalizing active SARS-CoV-2 (BKO-hACE2). C57BL/6 and BKO-hACE2 mice were immunized with inactivated SARS-CoV-2 receiving one intravenous dose followed by three intranasal booster doses. After 30 days, lungs were analyzed by flow cytometry and histopathology. Additionally, we evaluated the effects of ATP (0.5 mM to 10 mM) during immunizations, finding that lower doses (0.5 mM) increased the frequency of TRMs (CD4+ and CD8+), while higher doses reduced this population. This suggests that ATP may interact with the P2X7 receptor, promoting either proliferation or cell death. Although infection data did not provide conclusive evidence of T cell-mediated protection in the absence of antibodies, the potential of ATP as a possible adjuvant opens new avenues for enhancing immune responses in experimental models. Funding Sources FAPESP - 2023/15373-9 Topic Categories Viral Immunology (VIR)

Abstract Description The presence of Foxp3+CD4+ regulatory T cells (Tregs) hinders the protective antitumor T cell responses to lung cancer. The accumulation of Tregs is linked to lung cancer progression, resulting in poorer survival rates. However, the mechanisms behind this accumulation are not fully understood. Tregs express elevated levels of the extracellular ATP (eATP) receptor P2RX7 in both human and mouse lung cancer. Here, we show that P2RX7 is crucial for the accumulation of Tregs in lung tumor, promoting tumor growth in a murine lung cancer model. We observed, in T cell-specific P2RX7-KO mice (CD4-cre P2rx7fl/fl), reduced Treg infiltration and a concomitant increase in tumor CD4+ effector T cells, leading to better tumor control. In contrast, P2RX7-KO Tregs accumulated in mediastinal lymph nodes, suggesting that blocking P2RX7-KO signaling impairs the Tregs migratory ability. P2RX7+ Tregs had increased expression of T-bet and KLRG1, which are associated with greater suppressive function. Finally, Treg-specific P2RX7-KO mice (Foxp3-Cre-ERT2 P2rx7fl/fl) had reduced tumor growth and Treg tumor accumulation, indicating that P2RX7 controls lung tumor Tregs in a cell-intrinsic way. These results suggest that P2RX7 promotes lung tumor Treg infiltration and subsequent loss of cancer control. They also suggest that P2RX7 blockade can be used as a therapy to improve lung cancer control, which we are currently exploring in proof-of-concept experiments. Funding Sources Mayo Clinic and National Institutes of Health (NIH - H.B.d.S.: R01 AI170649) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Cholera toxin (CT), an exotoxin produced by Vibrio cholerae, causes severe diarrheal disease and contributes to the third leading cause of death in children under 5. Beyond its pathogenicity, CT possesses potent mucosal adjuvant properties, inducing type 2 immunity in the gastrointestinal tract. However, the immune response to acute oral CT exposure remains poorly characterized. To address this, we intragastrically administered a single nonlethal dose of CT to mice and assessed physiological and immune responses over 24 hours. In C57BL/6 mice, CT induced severe hypothermia, while BALB/c mice exhibited milder effects, with males of both strains showing heightened sensitivity. All treated animals developed diarrhea, and C57BL/6 mice experienced intestinal edema lasting up to 24 hours. Transcriptomic analysis of the small intestine revealed acute upregulation of inflammatory mediators such as IL-1β and IL-33. Immune profiling by full-spectrum flow cytometry showed CT-induced changes in the lamina propria compartment, including increased ILC3, reduced Treg RORγT+ and CD11b+CD103+ dendritic cells. In the epithelial layer, CT slightly elevated neutrophils, eosinophils, monocytes, and ILC3 while reducing CD3+ T lymphocytes and ILC1/ NK cells. These findings emphasize the broader action of CT in pathogenesis and immunity, shedding light on how immune sensing of bacterial toxins can drive protective defenses and adjuvant activity. Funding Sources Supported by Hypothesis Fund Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Developing a universal influenza vaccine requires understanding how lung-resident memory CD4+ T cells are formed. These cells remain in the lung for a long time and strengthen the immune response to secondary infections. Tissue-resident helper cells (TRH), a subset of memory T cells, share characteristics with follicular helper cells (TFH) and boost the IgA and IgG production by lung-resident B cells. TRH cells express elevated levels of the extracellular ATP (eATP) sensor P2RX7. Here we demonstrate that P2RX7 ablation on T cells impairs the establishment of flu-specific TRH cells. P2RX7-induced decrease of TRH cells negatively impacted the protection against homologous challenge, since T cell-specific P2RX7-KO mice exhibit worse control of viral load than WT mice. P2RX7-KO mice also showed a reduction in flu-specific lung-resident germinal center-like B cells and lower levels of IgA and IgG in the lungs. P2RX7-KO did not induce major changes in the numbers of TFH-induced responses in the draining lymph nodes. Our single-cell RNAseq showed reduced expression of mitochondrial genes in P2RX7-KO TRH cells, which reflects decreased mitochondrial content presence in these cells. We observed an aberrant metabolic pathway usage by P2RX7-KO TRH cells, which beyond having less mitochondria also cease to utilize glycolytic metabolism to support protein synthesis. Our findings suggest the eATP-P2RX7 axis is required to establish TRH cells that protect against influenza. Funding Sources Mayo Clinic and National Institutes of Health (NIH - H.B.d.S.: R01 AI170649) Topic Categories Mucosal and Regional Immunology (MUC)