2594 Background: Enhancing the efficacy of neoadjuvant therapy (NAT) while minimizing treatment-related toxicity remains a critical unmet need for patients (pts) with HER2-positive (HER2+) breast cancer (BC). We previously reported intratumoral (IT) delivery of increasing doses (50 million and 100 million cells) of conventional type I dendritic cells (DC1) combined with anti-HER2 antibodies is safe and effective in altering the tumor microenvironment (TME) and inducing tumor regression in early-stage HER2+ BC. We conducted a phase II neoadjuvant clinical trial of IT DC1 (NCT05325632). Methods: Pts with early-stage HER2+ BC with tumor ≥ 1cm were eligible. Treatment included initial immunotherapy with IT DC1 weekly x6 (100 million) followed by paclitaxel 80 mg/m 2 IV weekly x12. Starting from day 1, pts also received trastuzumab (H) IV (8 mg/kg loading dose, then 6 mg/m 2 ) and pertuzumab (P) IV (840 mg loading dose, then 420 mg) every 3 weeks x 6 cycles. Core needle biopsies were obtained at baseline and at week 6 following the last DC1 injection and analyzed by multiplex immunofluorescence (mIF) to assess immune cell infiltration. At these timepoints and post-chemotherapy, radiologic response was assessed by breast MRI and blood was collected for biomarker and ctDNA analysis using a personalized, tumor-informed test (Signatera, Natera, Inc.). The primary end point of this study is pathologic complete response rate (pCR). Results: A total of 47 pts (24 HR+/HER2+, 23 HR-/HER2+) were enrolled between 5/2022 and 10/2025. Median age was 54 years (range 27-82). 21 pts had biopsy-proven axillary node positive disease with clinical stage I/II/III (7/30/10). All pts completed NAT, and 42 pts underwent surgery as of 1/22/2026. The pCR rates for HR+/HER2+ and HR-/HER2+ were 50% (12/24) and 89% (16/18), respectively. The most frequent toxicities related to DC1 were grade 1/2 chills, flu-like symptoms, headache, nausea, fever, and injection site reaction. IT DC1 + HP therapy was associated with a significant increase in intratumoral CD3⁺ T cell infiltration and a decrease in tumor cells assessed by mIF. ctDNA levels were evaluable for 25 patients (13 HR+ and 12 HR-). Sixteen pts (64%) had positive ctDNA (6 HR+ and 10 HR-) at baseline and 14/16 cleared ctDNA with NAT (13/16 cleared ctDNA post- IT DC1+HP, prior to chemotherapy). At present (median post-surgery follow-up: 15.1 mos.; median follow-up from last ctDNA test: 8.7 mos.), no pts have experienced disease recurrence. Conclusions: IT DC1 + HP prior to neoadjuvant paclitaxel + HP in HER2+ BC pts was well tolerated with manageable toxicities. IT DC1 led to immune cell infiltration and ctDNA clearance with improved pathologic tumor response rates, particularly in HR-/HER2+ BC. Updated surgical outcomes and biomarker results (mIF, MRI and ctDNA) will be presented at the meeting. Clinical trial information: NCT05325632 .

Hormone receptor-positive (HR+), HER2-negative breast cancer represents the most common subtype of breast cancer and is characterized by a risk of late recurrence. Neoadjuvant endocrine therapy with aromatase inhibitors (AIs) is a well-tolerated option in postmenopausal women; however, strategies to enhance its efficacy are needed. Combination of AI with immunotherapy is a promising approach. We evaluated the efficacy and safety of combining an AI with the anti-program death ligand 1 antibody durvalumab in the neoadjuvant setting. This single-arm, phase II study used a Simon two-stage design. Postmenopausal patients with early-stage HR+/HER2-negative breast cancer received durvalumab every 4 weeks plus daily AI for 6 months prior to surgery. The primary endpoint was the achievement of a modified Preoperative Endocrine Prognostic Index (mPEPI) score of 0. Seventeen patients were enrolled and received durvalumab plus daily AI for six months before surgery. Treatment was well tolerated, with most adverse events being grade 1–2. A clinical complete response was seen in 58.8

BACKGROUND:Immunotherapy is a growing treatment option for challenging breast cancer (BC) subtypes. Systemic administration can have significant adverse events (AEs), prompting interest in intratumoral injection. We evaluated the safety and feasibility of intratumoral injections at our institution. METHODS:This is an IRB-approved retrospective review of neoadjuvant patients who received intratumoral talimogene laherparepvec (TVEC) (NCT02779855) for triple negative breast cancer (TNBC), dendritic cells (DC1) for HER2+ (NCT05325632), HER2 + /ER- (NCT03387553), or TNBC (NCT05504707), or Voyager V1 virus (VV1) (NCT01042379) for high-risk lesions on ISPY2. This study outlines the safety and feasibility of intratumoral injections assessed by AEs, adherence to therapy, and ultrasound guidance. RESULTS:The study included 111 female patients (mean age 51; range 26-80). Forty-seven (42.3%) received TVEC, 62 (55.9%) received DC1, and 2 (1.8%) received VV1. Three patients missed one injection; 110 patients had ultrasound-guided injections. Mean neoadjuvant therapy length was 172 days (range 127-244). Local AEs included pain (20.7%), injection site reaction (27.9%), and hematoma (5.4%). Systemic AEs were reported more frequently, most commonly chills (52.3%), headache (39.6%), and fever (36.8%). DISCUSSION:Intratumoral IT injection is a viable treatment option that may minimize systemic exposure while maintaining therapeutic efficacy. Breast surgeons can play a role in intratumoral IT in BC.

Prolonged or indefinite systemic therapy remains standard for advanced clear cell renal cell carcinoma (ccRCC), often resulting in cumulative toxicities and treatment burden. We conducted a single-arm phase 2 trial (ClinicalTrials.gov identifier: NCT02964078) of a fixed-duration regimen of anti-PD1 pembrolizumab plus high-dose interleukin-2 in treatment-naive advanced ccRCC. Primary objectives of safety and response were previously reported. The study met its primary endpoint with an overall response rate exceeding the pre-specified threshold of 45%. Here we report long-term follow-up (median follow-up of 76.4 months) including overall response, progression-free survival, treatment-free interval, and correlative analysis. Among 26 patients treated, the objective response rate was 73%, with complete responses in 42% of patients. Median overall survival was >84 months with a 5-year restricted mean survival time of 48.6 months. Median progression-free survival was 19.3 months, and median treatment-free interval was 23.8 months. 42% of patients remained treatment-free at the 5-year timepoint. No grade 5 adverse events occurred, and no patients with durable disease control experienced persistent grade ≥2 toxicities. Correlative analyses identified exploratory immune patterns associated with durable benefit, including enrichment of CD16⁺ natural killer cells, suppression of PD-1⁺ T-cell frequencies, and coordinated chemokine, complement, and PKC/TGF-β pathway activation.

CD19-directed chimeric antigen receptor-T cell (CAR-T) therapies have transformed treatment for relapsed/refractory large B-cell lymphoma (LBCL), yet heterogeneity in toxicity and efficacy persists. To address this, we developed a novel composite endpoint: severe toxicity-free and progression-free survival at 6 months (TPFS6), defined as absence of severe CRS, ICANS, progressive/stable disease (PD/SD), and non-relapse mortality (NRM) within 6 months of CAR-T therapy. In our cohort, 37% achieved TPFS6. Events contributing to TPFS6 failure included severe CRS (6.8%), ICANS (30.3%), NRM (1.7%), and PD/SD (61.2%). Multivariable analysis demonstrated female sex (p = 0.01), ECOG < 2 (p = 0.05), and lower LDH and CRP (both p = 0.004) predicted TPFS6. With a median follow-up of 36.2 months, the estimated 2-year OS and PFS were 54.5% and 39.6%, respectively. Landmark analysis showed TPFS6 was associated with improved OS (HR = 0.19; 95% CI: 0.11-0.35; p < 0.001) and PFS (HR = 0.33; 95% CI: 0.16-0.67; p = 0.002). CAR-T product type was not significantly associated with OS but was with PFS (HR = 3.09; 95% CI: 1.27-7.50; p = 0.013). TPFS6 remained prognostic for OS and PFS even after accounting for PD/SD, underscoring the impact of severe toxicity on subsequent survival. TPFS6 is a clinically meaningful endpoint integrating efficacy and safety and may predict long-term outcomes following CAR-T therapy.

BACKGROUND/OBJECTIVES:Penile squamous cell carcinoma (PSCC) is a rare malignancy with poor prognosis in advanced and recurrent disease, and therapeutic options remain limited. Increasing evidence suggests that the tumor immune microenvironment (TIME), including immune cell composition and spatial organization, plays a critical role in tumor progression and survival outcomes. This study aimed to characterize immune cell density and geospatial clustering patterns within the TIME of PSCC and to evaluate their associations with clinical outcomes. METHODS:Multiplex immunofluorescence (mIF) was performed on tumor samples from 57 patients with PSCC using a panel of immune markers to identify lymphoid and myeloid cell populations. Immune cell densities were quantified within tumoral and stromal compartments. Spatial relationships among immune cells and between immune cells and tumor cells were analyzed using point pattern analysis. Survival outcomes, including overall survival (OS), recurrence-free survival (RFS), and cancer-specific survival (CSS), were assessed using Kaplan-Meier methods and Cox proportional hazards models, with analyses stratified by nodal and human papillomavirus (HPV) status. RESULTS:Higher intratumoral and stromal densities of pro-immunogenic M1 macrophages were associated with improved OS. Increased densities of CD3+CD4+ helper T cells in both compartments were also associated with favorable survival outcomes. In contrast, close clustering of pro-tumorigenic M2 macrophages with tumor cells and with one another was associated with worse OS, RFS, and CSS. Bivariate clustering of helper T cells with tumor cells was associated with improved OS, including among patients with node-positive disease. Survival outcomes did not differ significantly by HPV status in patients with high helper T cell clustering. CONCLUSIONS:Immune cell density and spatial organization within the TIME are associated with survival outcomes in PSCC. Favorable patterns involving helper T cells and M1 macrophages correlate with improved survival, whereas clustering of M2 macrophages is associated with poorer outcomes, supporting the relevance of spatial immune profiling in this disease.

e17035 Background: Penile squamous cell carcinoma (PSCC) is a rare malignancy with substantial heterogeneity in tumor-intrinsic biology & the immune microenvironment. While genomic studies have identified recurrent alterations, how cancer & immune programs jointly shape PSCC ecosystems & outcomes remain poorly defined. Methods: We profiled 67 PSCC tumors using the nCounter PanCancer Immune Profiling Panel, interrogating cancer-relevant genes & curated immune-response pathways. The cohort included 29 HPV-positive tumors (43.3%) & was balanced across stage (33 stage I–II; 34 stage III–IV). Highly variable genes (HVGs) were identified, followed by unsupervised clustering, pathway-based analyses using 61 curated canonical gene sets, & survival analyses for recurrence-free survival (RFS) & overall survival (OS). To model joint cancer–immune ecosystems, non-negative matrix factorization (NMF) was applied to derive cancer–immune meta-programs with program-level signals interpreted using independent single-cell RNA sequencing data. Results: HVG analysis revealed transcriptional variability spanning inflammatory & myeloid-associated genes, interferon & antigen-presentation genes, tumor-intrinsic epithelial & cancer-testis antigens. Unsupervised expression-based clustering identified four major transcriptional clusters that were not strongly associated with any clinical covariates. Pathway-level analyses demonstrated distinct transcriptional programs associated with stage, HPV status and smoking history. Survival analyses identified Th17 signaling & canonical PI3K signaling associated with RFS in univariable analyses, canonical PI3K signaling & cytotoxic cell signatures remaining associated with RFS in multivariable models. NMF resolved this heterogeneous landscape into three orthogonal cancer–immune meta-programs that captured coordinated transcriptional structure across tumors. Program 1 was enriched for interferon-α & complement pathways, Program 2 for interferon-γ signaling, & Program 3 for E2F targets & epithelial–mesenchymal transition. These meta-programs showed clear prognostic stratification with Program 2 associated with favorable RFS and OS, whereas Program 3 associated with adverse outcomes. Integration with single-cell PSCC data demonstrated preferential enrichment of Program 1 in myeloid compartments, Program 2 across B cells, myeloid cells, and subsets of T cells, & Program 3 predominantly within epithelial, fibroblast, and endothelial compartments. Conclusions: Transcriptomic analysis in PSCC identifies coordinated cancer–immune meta-programs that define biologically distinct tumor ecosystems & stratify clinical outcomes beyond conventional clinical features supporting a promising framework for understanding penile cancer molecular heterogeneity, motivating ecosystem-informed biomarker & therapeutic strategies.

e20538 Background: Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) is commonly used to guide immunotherapy selection in advanced non–small cell lung cancer (NSCLC); however, its ability to consistently reflect tumor immune biology and treatment benefit remains limited. RNA-based immune gene expression profiling provides a complementary approach to characterize the tumor immune microenvironment using formalin-fixed paraffin-embedded specimens. We evaluated associations between RNA-based immune gene expression and duration of immunotherapy (IO) in advanced NSCLC. Methods: This retrospective biomarker study included patients with stage IV, non– EGFR , non– ALK NSCLC treated with immune checkpoint inhibitors. Tumor RNA expression profiling was performed using a multiplex, amplification-free gene expression assay quantifying 204 genes. Gene expression was analyzed as categorical variables (high expression defined as log 2 ≥1) and as continuous measures. The primary endpoint was duration of IO, defined as time from initiation of IO until progression of disease as documented by treating physician, initiation of alternative therapy, or death. Overall survival (OS) was evaluated as an exploratory secondary endpoint. Associations were assessed using univariate duration analyses, Kaplan–Meier methods, and Cox proportional hazards models. All tests were two-sided, and p<0.05 was considered significant. Analyses were exploratory and hypothesis-generating. Results: Sixty-one patients were included. Median duration of IO was 108 days (range, 7–2006). PD-L1 IHC categories (<1%, 1–49%, ≥50%) were not associated with IO duration (log-rank p=0.538). In contrast, multiple RNA-based tumor immune gene expression markers were significantly associated with duration of IO in Cox models (Table). Higher dichotomized (categorical) expressions of CDK6 , ERBB3 , MDM2 , PCSK9 , and STK11 were associated with shorter IO duration, whereas STK11 mutation status was not associated with IO duration. Of these genes, high CDK6 and PCSK9 expressions were also associated with worse OS. Conclusions: RNA expression profiling identified multiple biomarkers associated with IO duration and, for select genes, OS that were not captured by PD-L1 IHC or DNA-based mutation status. These findings suggest RNA-based assays may provide complementary biologic correlates of IO benefit in advanced NSCLC and warrant prospective validation. RNA Biomarker IO duration Hazard Ratio HR (95% CI) p Value OS HR p Value CDK6 3.25 (1.24-8.50) 0.017 28.86 (7.31-114.02) <0.001 ERBB3 2.19 (1.12-4.27) 0.022 0.70 (0.27-1.83) 0.767 MDM2 2.10 (1.08-4.08) 0.030 1.78 (0.79-4.01) 0.166 PCSK9 2.67 (1.16-6.13 0.021 3.64 (1.44-9.16) 0.006 STK11 2.57 (1.08 -6.12) 0.033 1.16 (0.40-3.33) 0.787

Abstract Purpose: This study aims to assess whether early-onset, grade 1 (G1) and low-grade (LG) treatment-related adverse events (TrAEs) can serve as predictive markers for favorable survival outcomes in advanced non-small cell lung cancer (NSCLC) across different treatment modalities. Methods: We analyzed data from 577 NSCLC patients across 11 cohorts treated at Moffitt Cancer Center: 5 immunotherapies (n=383), 3 targeted therapies (n=88), and 3 chemotherapies (n=106). Data for analysis used AE data derived from Common Terminology Criteria for Adverse Events (CTCAE v4-5), treatment response including comparison of responder (complete response (CR) and partial response (PR)) versus non-responder (stable disease (SD) and progressive disease (PD)) and comparison of disease control (DC: CR/PR/SD) versus PD using Wilcoxon two-sample test, progression-free survival (PFS) and overall survival (OS) using Kaplan-Meier survival curve with log-rank test. Our analytic approach leveraged multiple AE parameters to develop a set of innovative AE biomarkers. Early-onset G1/LG TrAEs were defined as those occurring within 30 days of treatment initiation. Results: Early-onset AE analysis across all treatment types revealed that (a) Immunotherapy had lower frequency of G1 and LG TrAEs compared to chemotherapy and targeted therapy; (b) higher frequency of G1 and LG TrAEs were associated with better treatment response in immunotherapy (responder vs non-responder with p=0.047 (G1) and 0.069 (LG); DC vs PD with p=0.005 (G1) and 0.018 (LG)), but no significant results in chemotherapy and targeted therapy; (c) For patients who did not encounter HG non-treatment related AEs (non-TrAEs), if they frequently experienced G1 and LG TrAEs, their survival outcomes tended to be better compared to the ones with less or no AE experiences in immunotherapy (median PFS: 5.5 vs 3.5 months with p=0.03 for G1 and 5.5 vs 3.3 months with p=0.015 for LG; median OS: 18.2 vs 12.2 months with p=0.008 for G1 and 16.1 vs 12.2 months with p=0.04 for LG). Please note that non-TrAEs represent a strong surrogate for compromised baseline health status. Without proper adjustment, this factor may confound the observed association between early-onset G1 and LG TrAEs and survival outcomes. For in chemotherapy and targeted therapy, both G1 and LG TrAEs did not show significant survival association. Conclusion: G1 and LG TrAEs within 30 days of therapy initiation were associated with better treatment response and improved survival in advanced NSCLC, especially in immunotherapy-treated patients. These findings support the use of early-onset G1/LG TrAE profiles as potential predictive biomarkers. Citation Format: Dung-Tsa Chen, Andreas N. Saltos, Zachary Thompson, Junmin Whiting, Sebastian Viracacha, Timothy I. Shaw, Ignacio I. Wistuba, Jhanelle E. Gray. Early-onset low-grade adverse events as predictive biomarkers in advanced NSCL: A multi-treatment cohort analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3734.

In the United States (US), only 47% of young adults (18-26 y old) had initiated the human papillomavirus (HPV) vaccine in 2022. Provider recommendation is the strongest predictor of HPV vaccination; however, limited studies have explored methods for training providers on making HPV vaccine recommendations to young adults. The purpose of this study was to obtain provider feedback on education materials for effective HPV vaccination recommendations to patients ages 18-26. Guided by the Theory of Planned Behavior and Health Belief Model, an HPV vaccine training was developed for primary care providers. Fifteen US primary care providers were recruited in 2022 for semi-structured interviews. Participants reviewed and provided feedback on the draft training. Data were analyzed using thematic analysis. Overall, feedback on the education materials was favorable. Participants noted learning new and useful information and appreciated the diversity of people throughout the training. General feedback included flexibility in the training format and timing due to busy schedules and providing incentives to encourage participation. There were mixed feelings toward participating in a role-play activity. Recommendations included using clear language and opportunities to make the training more persuasive. Future research is needed to refine the intervention based on provider suggestions and test the effectiveness of the intervention for improving provider recommendations for young adults.

842 Background: Few studies have evaluated the role of early-onset adverse events (AEs) on treatment outcomes for patients with GI cancer. This study aims to develop a pan-GI cancer AE profile to facilitate modernization of treatment strategies. Methods: We evaluated 10 study cohorts from Moffitt Cancer Center: 3 in biliary (n=115), 2 in colorectal (n=89), 2 in pancreatic r (n=44), 1 in gastric (n=23), 1 in hepatocellular (n=14), and 1 in pan GI cancers (n=16), for a total of 301 patients. Treatment included 1 cohort with immunotherapy (IO), 2 with targeted therapy (TT), 2 with combination of TT and IO, and 5 with chemotherapy (Chemo)+TT. Data for analysis used CTCAE version 4 AE data, treatment response, progression-free survival (PFS), and overall survival (OS). Our analytic approach leveraged multiple AE parameters to develop a set of innovative AE biomarkers. Early-onset AEs were defined as events that occurred between the first day and day 30 after the first treatment. Results: In the 10 aggregated GI cohorts (n=301), patients experiencing with higher frequency of grade 1 (G1) early-onset treatment related (Tr) AEs tended to improve PFS and OS (p<0.05). Other TrAEs also showed a non-significant trend toward hazard ratio (HR) <1, suggesting a potential but inconclusive benefit for PFS and OS. In contrast, all non-TrAEs had a HR>1, indicating poor survival with significant associations for grade 2 (G2) and grade > 2 (G>2) in OS. Similar findings were observed across the 9 pooled TT cohorts (n=247). For the 3 merged IO cohorts (n=143), patients presenting G1 early-onset TrAEs exhibited improved OS compared to those without AEs (p=0.02). Most grade 1-2 (G1/2) TrAEs showed HR<1 in PFS and OS. Conversely, all non-TrAEs had HR>1 with significance achieved in G2 and higher grade for OS. The 5 combined chemo cohorts (n=107) showed a nonsignificant HR <1 in PFS and OS for most G1/2 TrAEs; conversely, all non-TrAEs were associated with HR>1 with significant effects in G2 or higher grade for OS. Conclusions: This study presents compelling evidence supporting the clinical relevance of early-onset AEs in predicting pan-GI cancer patient outcomes. Specifically, G1 and G1/2 early-onset TrAEs showed robust results as potential indicators of improved OS or PFS in the entire combined cohorts and in the subset analysis for TT and IO. Even the cohorts with Chemo had a nonsignificant HR<1. Timely recognition of treatment response or disease progression is essential for optimizing clinical decision-making. The pan-GI cancer early-onset AEs may serve this function, supporting more precise treatment strategies and improved patient outcomes.

Young adults (18–26 years) are at high risk of human papillomavirus (HPV) exposure. Yet, HPV vaccine uptake is suboptimal among young adults. Provider recommendation is frequently the most influential factor in HPV vaccine acceptance. Evidence-based strategies are needed to facilitate provider recommendations for young adults. To inform provider-level strategies for recommending the HPV vaccine to young adult patients, the current study aims to understand current practices and barriers and facilitators to both recommending the HPV vaccine for young adults and for young adults to receive the HPV vaccine. Primary care providers (e.g., physicians, nurses; n = 15) in the United States completed a semi-structured interview guided by the Consolidated Framework for Implementation Research (CFIR). Data were analyzed thematically using Nvivo. Most participants identified as female (80

INTRODUCTION:The FDA approved trastuzumab deruxtecan (T-DXd) for treating human epidermal growth factor receptor 2-positive (HER2+) metastatic breast cancer (mBC) based on the phase II trial DESTINY-Breast01. The phase III trial DESTINY-Breast03 confirmed the efficacy of T-DXd with improvements in progression-free survival (PFS) (HR = 0.33) and overall survival (OS) (HR = 0.64) compared with trastuzumab emtansine (T-DM1). PATIENTS AND METHODS:This is a retrospective, single-institution, effectiveness study of patients with HER2+ mBC treated with T-DXd. Data were summarized on de-identified patients, tumors, and outcomes per treating physician. The Kaplan-Meier method was used for survival analyses. A 2-sided P value < .05 was considered statistically significant. Cox proportional hazards models were utilized. The initial model included all variables, and backward elimination was used to remove variables with P > .1 to obtain the final model. RESULTS:Eighty-four patients with progressive HER2+ mBC with a median age of 57 years (27-78 years) were treated with T-DXd. Of the total patients, 16.7% had an ECOG Performance Status ≥2, and 69.0% had 1 or 2 prior lines of therapy for mBC. Almost half (45.2%) had received prior T-DM1, and only 69.0% started T-DXd at full dose. Median real-world PFS (rwPFS) was 13.3 months (95% CI, 11.4-16.9). No significant differences in rwPFS were observed in stratified analyses. Median real-world OS (rwOS) was 38.7 months (95% CI, 24.5-61.8). CONCLUSION:T-DXd showed effectiveness for treating HER2+ mBC in a real-world cohort with pretreated patients.

Background: Patients (pts) with breast cancer (BC) harboring low expression of hormone receptors (HR) and human epidermal growth factor receptor-2 (HER2) have poorer outcomes compored to other subsets of BC. Results from the KEYNOTE-522 trial showed that activation of the immune system using a PD1/PD-L1 approach leads clinically meaningful improvement in the outcomes of patients with these high-risk tumors. Dendritic cells (DCs) are antigen presenting cells which are pivotal for robust cytotoxic responses via broader activation of the adaptive immune system. Methods: DecipHER is a dose-escalation, dose-expansion phase 1 trial designed to assess the safety and preliminary effiacy of autologous HER2- and HER3-primed DCs in combination with KEYNOTE-522 regimen in a maximum of 30 pts. Pts with clinical stage cT1-cN1/2 or cT2-4cN0/2, HR < 20, HER2-negative BCs are eligible. Pts with inflammatory BC and uncontrolled immune-mediated diseases are excluded. After collection through apheresis, autologous DCs are primed against 6 HER2 and 8 HER3 immunogenic peptides. Pts receive alternating US-guided intratumoral HER2 and HER3 DCs injections administered twice a week for 8 doses starting 2 weeks prior to neoadjuvant KEYNOTE-522 regimen. The dose-escalation phase of the study had a classic 3+3 design ( ie DL1-3 [10-20, 30-50, 80-100 million], maximum n=18). Additionally, 12 pts will be treated at the maximum tolerated dose (MTD) in the dose-expansion cohort. Dose-limiting toxicities (DLT) were defined as grade 3 or higher non-hematologic or hematologic adserve events (AEs) thought to be at least possibly related to DCs; any grade 4 nausea, vomiting or diarrhea [or grade 3 if duration > 3 days]) during the 5 weeks following treatment initiation. Secondary endpoints include absolute risk of AEs, pathological complete response (PCR) and recurrence-free survival. Tumor tissue, blood and stool samples are being collected for correlative analyses. Results: A total of 12 pts (6 on DL3) were enrolled between 08/2022 and 01/2024. The median age was 51.5 (35-71) and 25% of pts were black; 91% had grade 3, 41.7% had T3 and 75% had N1 BCs. One patient received only 2 HER2- and 4 HER3-primed DCs due to low cell yeild. Grade 1 and 2 AEs that were at least possibiliy related to DCs (>10%) were headache (66.7%) chills (50%), fatigue (33.3%), flue-like symptoms (33.5%), fever (25%), nausea (16.7%) and pain (16.7%). No DC-associated grade 3 or higher AEs were observed. None of the toxicities met the definition of DLT. For DL1, 2 out of 3 pts had an SAE ( i. cholelithiasis complicated by sepsis and ii. pneumonitis and syncope; 5 and 14 weeks after last DC injection; respectively). No SAEs were observed on dose levels 2 or 3. Immune-related AEs were pneumonitis (1pt), hypothyrodism and adrenal insuficiency (1 pt), hyperthyroidism (1pt). pCR was observed in 5 out of 10 evaluation pts. Base on thees findings, DL3 (80-100 million) was selected for the dose-expansion cohort. Conclusion: Intratumoral DCs in combination with standard neoadjuvant chemotherapy and pembrolizumab were well tolerated in pts with high-risk HR <20, HER2-negative BCs. The dose-expansion cohort portion of this trial is ongoing; correlative analyses will follow. The study is open at H. Lee Moffitt Cancer Center. Clinical trial information NCT05504707. Funding: Shulas' foundation. Citation Format: Ricardo Costa, Aixa E. Soyano, Avan Armaghani, Jennifer Childress, Loretta Loftus, Edith Abraham, Junmin Whiting, Qianxing Mo, Zena Jameel, Tracey O'Connor, Kimberley Lee, Susan Hoover, John Kiluk, Catherine Lee, Christine Laronga, Nazanin Khakpour, Hyo S. Han, Hatem H Soliman, Brian J. Czerniecki. Results of the Dose-Escalation Cohort of a Phase 1 Trial of Intratumoral HER2- and HER3-Primed Dendritic Cells Injections for the Treatment of Early-Stage TNBC and HR Low Positive Breast Cancer. DecipHER trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-08-28.

12 Background: Previous studies utilizing multiplex Immunofluorescence (mIF) have described cell density patterns of immune exhaustion implicating both innate and adaptive immune system components across varying stages of penile squamous cell carcinoma (PSCC). Here we studied geospatial clustering patterns of various immune effector cells within the tumor immune microenvironment (TIME). Methods: Tissue microarray (TMA) was constructed for 57 cases of invasive PSCC and immunostained for CD20, CD3, CD4, CD8, CD45RO, CD68, CD206, CD163, NKp46, FOXP3 using OPAL TM 7 kit (AKOYA Biosciences). Areas of tumor and stroma were identified using an image analysis system (InForm 2.2.4). Spatial analysis and co-clustering of cell phenotypes was performed using Ripley’s K. Survival curves were determined using Kaplan Meier method and tested using logrank and cox regression. Univariate and bivariate analysis describes co-clustering and proximity to tumor cells, respectively. Results: 57 PSCC patients (median age 60, [IQR (interquartile range) 31-92]); 60% HPV negative, and 31/57 with pN+ had mIF analysis. A high clustering pattern of CD68+, a general marker for macrophages, was associated with a significant OS benefit on univariate analysis (162 vs 36 mos, p=0.006) and an OS, RFS, and CSS benefit on bivariate analysis (84 vs 27 mos; NA vs 14 mos; NA vs 27 mos p=0.007, 0.014, 0.001). High clustering of CD3+CD4+, a marker for T-helper-lymphocytes, within the tumor and the tumor-stroma interface was associated with an OS benefit on bivariate analysis (84 v 20 mos, p=0.009). Low intra-tumoral clustering of CD68+CD163+, marker for M2 protumor macrophages, was associated with improved RFS on bivariate analysis (NA v 15 mos, p=<0.01). Low clustering of CD68+CD163+ in HPV+ patients was associated with a CSS benefit compared to high clustering in HPV- patients (p=0.024). High versus low clustering of CD68+CD163+ was inconsequential in HPV- patients (p=0.66).Overall, low intratumoral clustering pattern of FOXP3+, a marker for regulatory immune-suppression, was associated with an improved CSS on bivariate analysis (p=0.017). Low clustering of FOXP3+ in HPV+ patients was associated with an improved CSS versus low clustering of FOXP3+ in HPV- patients (p=0.041). Conclusions: Spatial analysis shows that proximity of CD68+ and CD3+CD4+, a general marker for macrophages and T-helper cells, to tumor cells has a positive impact survival in PSCC. Interestingly, proximity of protumor M2 macrophages to cancer cells confers a survival benefit specifically in HPV+ tumors and has no impact on survival in HPV- tumors. Decreased intratumoral FOXP3+ activity, a marker for regulatory immune-suppressive T cells, shows a preferential survival benefit in in HPV+ tumors. These findings point to diverging tumorigenic pathways related to HPV status that may be attributable to their poorer prognosis.

INTRODUCTION:Breast conserving therapy (BCT) for nonpalpable lesions can be performed using various localization devices. For larger or multifocal lesions, "bracketing" with multiple localizers is required for complete excision. SAVI Scout utilizes radar localization (RL) to target clip location(s), while SmartClip employs electromagnetic chips (EMC) to provide 3D navigation and distinguish up to three devices. This study aimed to compare the excision of breast lesions using non wire localization devices such as EMC and RL versus traditional wires. METHODS:A single institution, retrospective study was conducted from August 25, 2020 to August 6, 2024, comparing EMC, RL, and wire localization in bracketed BCT. Case length, positive margins requiring re-excision, and complete retrieval of localizers in a single specimen were analyzed. Statistical analyses were performed using Kruskal-Wallis, and χ2 or Fisher's exact tests. RESULTS:A total of 118 cases were analyzed: 43 wire, 44 RL, and 31 EMC cases. The groups were similar in lesion size and number of localizers used (P = .736 and P = 1.000, respectively). There were fewer positive margins when EMC or RL were utilized (EMC 29%, RL 22.7%, wires 50%, P = .022). EMC was also associated with significantly shorter operative times (33.9 minutes vs. RL 45.6 minutes, wire 40 minutes, P = .025). There was no significant difference in complete retrieval of localizers among the groups (EMC 93.5%, RL 97.7%, wire 100%, P = .264). CONCLUSION:Non-wire localization method is effective for bracketed BCT with lower rates of margin positivity and faster operative times with EMC. This supports their use in BCS, especially when multiple localizers are needed.

13 Background: Advanced penile cancer is a rare but aggressive malignancy. Over 70% of patients with bulky metastases relapse or have primary refractory disease with current treatments. Immunotherapy represents a promising modality, but response rates remain low. While prior studies explored single immune factors within the tumor microenvironment (TME), understanding complex interactions between T cells and macrophages and in relation to the tumor cells is crucial to best characterize the TME. Methods: We performed multiplex immunofluorescence (mIF) on 59 PSCC tissues obtained from MD Anderson to analyze expression of 12 key immune cell markers (CD3, CD8, CD68, CD86, CD206, CD163, ARG, CSF1R, MHC-II, PD-1, PD-L1). Spatial analysis and co-clustering of cell phenotypes was performed using Ripley’s K. Overall (OS), recurrence free (RFS), and cancer specific survival (CSS) curves were determined using Kaplan Meier method and tested using logrank and cox regression models. Results: The median age was 60 (IQR 24-86) and majority were HPV negative (64%). High densities and clustering of cytotoxic CD8+ T cells and CD68+ tumor associated macrophages (TAMs) were each associated with improved OS (126 vs 61 months, p= 0.03; 140 vs 61 months, p= 0.04, respectively]. Conversely, high densities of antigen-experienced CD8+PD-1+ cytotoxic T cells and antigen-experienced CD68+PD-1+ macrophages were associated with worse OS [HR 1.44 (1-2.05), p= 0.04 and HR 1.48 (1.04-2.12), p=0.03 respectively], PFS [HR 1.45 (1.03-2.05), p= 0.03 and HR 1.53 (1.06-2.19), p= 0.02 respectively ] and CSS [HR 1.52 (1.03-2.24), p= 0.03 and HR 1.63 (1.12-2.36), p= 0.01 respectively]. Clustering of CD86+ M1 macrophages was associated with improved OS (104.5 vs 29.1 months, p=0.02), while clustering of CD163+ and CD206+ M2 macrophages correlated with decreased OS (67.8 vs 199.7 months, p=0.03; 100.6 vs NA months, p=0.01 respectively). Bivariate analysis revealed improved OS associated with co-clustering of CD8+ T cells with TAMs (104 vs 42 months, p=0.03). Similarly, co-clustering of CD8+T cells with CK+ tumor cells was associated with improved OS (140 vs 61 months; p=0.02). While co-clustering of CD86+ M1 macrophages to cytokeratin (CK+) tumor cells correlated with improved OS (126 vs 42 months, p=0.03), co-clustering of CD 206+ M2 macrophages to CK+ tumor cells associated with poor RFS (48 vs NA months, p=0.03). Co-clustering of antigen experienced T cells with TAMs was also associated with decreased RFS (29 vs NA months, p =0.03). Conclusions: Using spatial analysis, we showed that interaction between T cells and macrophages impact clinical outcomes in PSCC. High densities and proximity of CD8+ T cells and M1 macrophages, and low levels of antigen experienced T cells and macrophages were associated with improved survival in PSCC.