Supplementary Figure S2. Classification of cancer cells according to WD/DD liposarcoma signatures.

Heatmaps of expression of 29 key gene signatures in (A) SARC028 and (B) Stanford RNA sequencing data

Pleomorphic liposarcoma (PLPS) is an aggressive high-grade sarcoma that often shows diverse morphological features and can mimic high-grade undifferentiated pleomorphic sarcoma (UPS)/spindle cell sarcoma or myxofibrosarcoma (MFS), especially when pleomorphic lipoblasts are sparse. The molecular profile of PLPS is distinct from well differentiated/dedifferentiated liposarcoma and myxoid liposarcoma. In this study, we investigate 39 cases of PLPS by comprehensive genomic profiling, occurring in 32 patients with available molecular data. Cases were reviewed and morphologic parameters-lipoblastic component, UPS-like, and MFS-like areas were estimated. The genomic findings were collected and compared to UPS and MFS groups studied using the same platform. The cohort included 15 females and 17 males, with a median age of 56.5 (range, 34-78). The lower extremity (n = 17) was the most common site involved, followed by upper extremity (n = 5) and pelvis (n = 5). UPS-like and MFS-like patterns were the most common morphologic variants, ranging from 15% to 95% and 20% to 90%, respectively. TP53 (87%) and RB1 (51%) mutations and copy number alterations were the most common alterations seen, followed by ATRX (36%). Compared to UPS and MFS, TP53 and RB1 gene alterations were significantly more common in PLPS. Conversely, CDKN2A/B deletions were infrequent in PLPS. Survival analysis showed that MYC amplification was associated with significantly shorter overall survival in PLPS. Among histologic variants, CYSLTR2 alterations were found to be highest in cases with predominantly pleomorphic lipoblasts; additionally, strong correlations were found between gene alteration frequencies of MFS and MFS-like PLPS, and between UPS and UPS-like PLPS. RB1 allele-specific copy number analysis showed loss of heterozygosity in 82% of cases. Our cohort of PLPS showed a complex molecular landscape with distinct genetic alterations, histologic correlations, and clinical outcomes, highlighting its unique position among genomically complex sarcomas and providing insights that may inform future diagnostic and therapeutic approaches.

ABSTRACT Background Concomitant medications (CMs) influence outcomes in patients receiving immune checkpoint inhibitors (ICIs), but their impact in sarcoma remains undefined. We assessed the association between CM use and ICI outcomes in patients with advanced or metastatic sarcoma. Methods This pooled analysis included patients from seven investigator‐initiated phase II trials of ICI‐based therapy for sarcoma. CMs within 30 days of treatment were defined as baseline; on‐treatment exposure was captured longitudinally. The primary endpoint was progression‐free survival (PFS); secondary endpoints included overall survival (OS), objective response rate (ORR), and immune‐related adverse events (irAEs). Multivariable Cox models adjusted for age, ECOG performance status, histological subtype, treatment regimen, and race. Results Among 321 patients (median follow‐up: 47.4 months), in time‐dependent analyses, exposure to anti‐infective medications was associated with shorter PFS (adjusted hazard ratio [aHR] 1.54, 95% CI 1.05–2.27). Baseline use of vitamins/minerals/supplements/herbal products was associated with longer PFS (aHR 0.73, 95% CI 0.56–0.95) and OS (aHR 0.67, 95% CI 0.51–0.89). Baseline statin use was associated with longer PFS (aHR 0.64, 95% CI 0.47–0.87) but not OS. Baseline use of opioids was associated with shorter OS (aHR 1.71, 95% CI 1.12–2.61). No CM class was significantly associated with ORR or irAE occurrence. Conclusions CM use is associated with differential efficacy outcomes in sarcoma patients receiving ICIs. These findings highlight the need for prospective studies to define the impact of commonly prescribed medications on ICI outcomes and to optimize clinical trial stratification.

Histologic distribution by immunotype and response

BACKGROUND:Aggressive angiomyxoma (AA) is a rare, locally infiltrative mesenchymal tumor with frequent local recurrence. Optimal management remains poorly defined, particularly the roles of surgery, endocrine therapy, and observation. METHODS:We retrospectively analyzed patients with histologically confirmed AA treated at a single institution from 2000 to 2024. Clinical, pathologic, and treatment data were collected. Overall survival (OS), relapse-free survival (RFS), duration of treatment (DOT), and radiographic response by quasi-RECIST 1.1 were assessed descriptively. RESULTS:Forty patients were included; median age was 42 years, 87.5% were female, and median tumor size was 9.9 cm. Initial management was surgery in 30 patients, systemic therapy in 8, and observation in 2. Among patients initially managed with surgery, median OS from the time of first resection was not reached at a median follow-up of 68.6 months; estimated 10-year OS was 95% (95% CI, 68%-99%). Among patients with R0/R1 resection, median RFS was 49.8 months (95% CI, 21.0-64.7), with 3-year RFS of 56% (95% CI, 31%-75%). Tumor size and margin status were not significantly associated with recurrence. Thirteen patients received systemic therapy, predominantly endocrine-based; ORR was 25% and median DOT was 15.4 months. Two patients were initially observed, and three with residual macroscopic disease after R2 resection remained free from further intervention for prolonged periods. Radiation therapy was rarely used. CONCLUSIONS:AA was associated with excellent long-term survival despite frequent recurrence or subsequent intervention after surgery. Endocrine therapy showed objective activity in a subset of patients. Observation or delayed intervention may be feasible in selected, clinically stable patients with untreated or residual macroscopic disease, supporting individualized, function-preserving management focused on symptoms, morbidity, and patient preferences.

T cell subpopulations associated with shorter survival after accounting for histologic subtype and treatment

BACKGROUND:The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib delays disease progression in dedifferentiated liposarcoma (DDLPS) by inducing tumor cell quiescence or senescence, though most tumors ultimately progress. Preclinical data suggest CDK4/6 inhibition enhances intratumoral inflammation and may synergize with immune checkpoint inhibitors. METHODS:This non-randomized, open-label, phase 2 study was conducted at Memorial Sloan Kettering Cancer Center. Patients with metastatic or unresectable DDLPS, or those expected to benefit from systemic therapy prior to surgery, were eligible. Patients received palbociclib (125 mg orally, days 1-21 of a 28-day cycle) plus retifanlimab (500 mg intravenous flat dose every 4 weeks). The primary endpoint was to assess the best objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. RESULTS:30 patients were treated and evaluable. Median age was 61 years (range 36-81), 67% were male, and 63% were treatment-naive. The median follow-up was 14.8 months. ORR was 20% (95% CI 8% to 39%) and the clinical benefit rate was 53% (95% CI 34% to 72%). Median progression-free survival and overall survival were 4.8 (95% CI 1.71 to 15.7) and 27 months (95% CI 21.7 to not reached (NR)), respectively. Median duration of response was 18.4 months (95% CI 9.4 months to NR). Grade ≥3 treatment-related adverse events occurred in 61% of patients, including one Grade 4 neutropenia. There were no Grade 5 events. Retifanlimab and palbociclib were discontinued due to toxicity in 23% and 17% of patients, respectively. Tumor sequencing identified JUN amplification in 6 of 14 patients with progressive disease versus 2 patients with stable disease or partial response. Patients with progressive disease had a higher median copy number alteration burden compared with those with stable disease or partial response. CONCLUSIONS:Palbociclib plus retifanlimab demonstrated deep and durable responses in a subset of patients with advanced DDLPS, with an ORR exceeding that historically observed with either agent alone. Adverse events were generally manageable. Copy number alteration burden and JUN amplification merit further evaluation as potential biomarkers of resistance. TRIAL REGISTRATION NUMBER:https://clinicaltrials.gov/study/NCT04438824.

Detailed histologic diagnoses according to TME subtype

Supplementary Figure S3. T cell state contour plots.

Abstract Introduction: Identifying novel approaches that harness and augment anti-tumor immune response is an important area of ongoing research and drug development. Based on their performance in pre-clinical models, orthosteric activators of the Stimulator of Interferon Genes (STING) have been investigated in human trials. The activation of STING by its ligand cGAMP leads to both IFN dependent innate immune signaling as well as IFN independent pharmacology associated with STING's proton transport activity that leads to autophagy and pyroptosis. CRD3874 is a first in class small molecule allosteric STING agonist that binds to an allosteric site within STING's proton channel and decouples the IFN and non-IFN consequences of STING activation by blocking STING's non-IFN actions. CRD3874-SI, is a proprietary intravenous formulation of CRD3874, potent activator of all five human STING variants that has demonstrated promising pre-clinical anti-cancer activity in serval mouse tumor models when systemically administered as mono therapy or in combination with checkpoint therapy. Drug administration led to elevated plasma levels of CXCL10 and IFNβ in mice and monkeys. High IV doses up to 75mg/kg were systemically tolerated in cynomolgus monkeys. Methods: This is a single institution, phase Ia/b study of CRD3874-SI in patients with advanced solid tumors who received at least one line of prior therapy. The dose escalation phase will explore the safety and tolerability of CRD3874-SI following standard 3+3 design. CRD3874-SI is administered by intravenous infusion once per week for two cycles. From cycle 3 onwards, participants will received 3 or 4 consecutive weekly infusions, over a 28-day treatment cycle. The primary objective is to assess the safety and tolerability of CRD3874-SI by determining the maximum tolerated dose, recommended phase 2 dose and schedule of administration. Secondary objectives include further defining the safety profile, examining the pharmacokinetics and pharmacodynamics (CXCL10 analysis) of CRD3874-SI and evaluating the efficacy of CRD3874-SI as determined by the best objective response rate and clinical benefit rate per RECIST v1.1. Treatment will continue until disease progression or unacceptable toxicity. This study is currently open to enrollment. 21 patients have received treatment across 4 dose levels to date. Expansion cohorts are planned after determining the appropriate dose (RP2D). Clinical trial information: NCT 06021626. Research sponsor: Curadev Pharma, Inc. Citation Format: Ciara M. Kelly, Reinhard von Roemeling, Monali Banerjee, Viswatej Avutu, Olayade Babatunde, Lauren Banks, Ping Chi, Mark A. Dickson, Mrinal M. Gounder, Grace Gray, Camron Clark, Mary Louise Keohan, Robert G. Maki, Sujana Movva, Damon Reed, Kelly Schroeder, Li-Xuan Qin, Phillip Wong, Sandip Middya, Ritesh Shrivastava, Debjani Chakraborty, Rajib Ghosh, Sourav Basu, Arjun Surya, William D. Tap, Sandra P. D'Angelo. Phase I trial of CRD3874-SI, a systemically administered third generation allosteric STING agonist, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT295.

Supplementary Table S3. Representativeness of Study Participants

Proportion of PBMC immunotypes in sarcoma, melanoma, and urothelial carcinoma

Supplementary Table S1. Flow cytometry antibodies.

TRAEs occurring in ≥10% of patients.

Purpose: Immune checkpoint blockade (ICB) benefits only a subset of patients with sarcoma. Biomarkers of response and resistance are needed to help guide patient selection.Experimental Design: We analyzed peripheral blood and tumor samples from patients with sarcoma treated in five ICB-based clinical trials. Baseline peripheral blood mononuclear cells (PBMC) underwent 11-color flow cytometry to define T-cell immunotypes. Baseline tumor tissue underwent RNA sequencing (RNA-seq) to classify tumors into four tumor microenvironment (TME) subtypes using consensus clustering of 29 functional gene expression signatures. Associations between immune features and clinical outcomes were assessed. A deep learning model was applied to baseline hematoxylin and eosin (H&E) slides to detect and quantify lymphoid aggregates in patients with available RNA-seq.Results: Among 178 patients with PBMC available for analysis, a proliferative (PRO) circulating T-cell immunotype was associated with poorer overall survival (OS) than lymphocyte-activation gene 3 (LAG)- or LAG+ immunotypes. RNA-seq from 67 tumors identified an immune-enriched/nonfibrotic TME subtype associated with a higher response rate, longer progression-free survival, and longer OS compared with immune-enriched/fibrotic, immune-depleted, and fibrotic subtypes. Automated analysis of 48 baseline H&E slides identified lymphoid aggregates in five tumors; four were classified as immune-enriched and two of these responded to ICB.Conclusions: Patients with sarcoma and a PRO circulating T-cell immunotype had inferior outcomes to ICB, whereas those with an immune-enriched/nonfibrotic TME had superior outcomes. Automated analysis of H&E slides showed promise in identifying patients with an immune-enriched TME. These findings support the use of a multimodal approach to identify predictors of response to immunotherapy in sarcoma.

Supplementary Table S5. Transcriptome-wide differential gene expression between cluster 8 of P12 vs. all other P12 clusters.

Changes in tumor and immune cell populations and phenotypes with anti–PD-1. A,CDK4 and MDM2 expression in tumor cells according to best response. PD, P03, before and after retifanlimab initiation (IO); SD, three before IO and six after IO. B, Signature scores in tumor cells according to best response. Same samples as in A. C, Integrated clustering of cancer cells from patients with paired samples and sufficient tumor fraction. D and E, Intracluster signature score changes with initiation of retifanlimab, plotted (D) along the x-axis or (E) as dot size. F, Volcano plot of differential expression of genes related to immune response in cluster 8 vs. all other clusters from P12.