Supplementary Figure S2. Variation of HMGB1 protein levels between samples collected after treatment (week 6) and before treatment (baseline).

Background: Clear cell sarcoma (CCS) is an ultrarare sarcoma driven by a specific chromosomal translocation, most commonly the EWS RNA binding protein 1-activating transcription factor 1 fusion (EWSR1::ATF1), for which chemotherapy shows limited activity, with a median progression-free survival (PFS) of approximately 3 months in retrospective series. In the present trial, a CCS cohort was selected based on signals of activity observed in the IMMUNOSARC I phase I/II trial evaluating nivolumab in combination with sunitinib in sarcomas. Methods: Patients aged 12 to 80 years with advanced, progressive, and measurable CCSs were enrolled after central pathology review, and molecular confirmation of an EWSR1 rearrangement was required. Sunitinib was administered at 37.5 mg/d during the first 2 weeks and then at 25 mg/d along with nivolumab at 240 mg every 2 weeks. The primary end point was the 6-month PFS rate, defined under the null (H0) and alternative (H1) hypotheses as 25% and 55%, respectively. Under Simon's 2-stage minimax design (α = 0.05, power = 0.90), a minimum of 10 of 23 patients needed to be progression-free at 6 months. Results: At the time of cutoff, 23 patients were evaluable for the primary end point. With a median follow-up of 23.0 months (95% confidence interval [CI], 10.0 to 35.0 months), the 6-month PFS rate was 50.1% (95% CI, 29.1% to 71.1%), while the median PFS was 6.2 months (95% CI, 3.0 to 9.3 months). Of 21 patients who underwent at least 1 radiological assessment, 3 (14.3%) achieved partial response, 14 (66.7%) had stable disease, and 4 (19.0%) had progressive disease. The median overall survival was 17.0 months (95% CI, 95% CI, 5.6 to 28.5 months). The main all-grade drug-related toxicities were lymphocytopenia (46.2%), leukopenia (38.5%), anemia (38.5%), and neutropenia (38.5%). Two grade 4 toxicities were reported: Alanine aminotransferase increased and ischemia (each 3.8%), while 31 grade 3 toxicities occurred, with anemia and lymphocytopenia being the most common (each 23.1%). A higher programmed death-ligand 1 composite score was associated with better PFS: 21.2 months (95% CI, 6.0 to 36.4 months) versus 4.2 months (95% CI, 2.7 to 5.6 months), P = 0.045. Conclusions: While further studies are needed, initial findings suggest that nivolumab plus sunitinib could be a valuable addition to the current armamentarium for CCS management. Trial registration: ClinicalTrials.gov ID NCT03277924 (date of registration: 2017 September 6).

Supplementary Table S3. Univariate Analysis of Different Potential Prognostic Factors on PFS and OS.

PURPOSE:This single-arm, phase Ib trial aimed to evaluate the safety and preliminary efficacy of epirubicin, ifosfamide, and nivolumab as first-line treatment for advanced undifferentiated pleomorphic sarcoma (UPS). PATIENTS AND METHODS:Adult patients with a centrally confirmed diagnosis of advanced UPS were eligible. Patients received epirubicin 60 mg/m2 (days 1-2), ifosfamide 3 g/m2 (days 1-3) every 21 days for up to six cycles, and nivolumab 360 mg flat dose intravenously (day 3/each cycle), followed by maintenance nivolumab for one year. The primary endpoint was the determination of the recommended phase II dose (RP2D). RESULTS:Sixteen patients were enrolled with no dose-limiting toxicities observed; the RP2D was established at full-dose epirubicin, ifosfamide, and nivolumab 360 mg. Grade 3 to 4 treatment-related adverse events included neutropenia (62.5%) and anemia (43.8%). The overall response rate was 68.8%, with 94% of patients experiencing tumor shrinkage. The median progression-free survival was 9.9 months (95% confidence interval, 7-12.7), and the median overall survival was not reached. CONCLUSIONS:The combination of epirubicin, ifosfamide, and nivolumab is a safe, feasible, and highly active treatment for advanced UPS.

Supplementary Figure S1. Kaplan–Meier survival analyses. A) progression-free survival (PFS) and B) overall survival (OS).

Supplementary Table S1. Representativeness of Study Participants

INTRODUCTION:Osteosarcomas (OS), the most common primary malignant bone tumours, are classified as low-grade (characterised by MDM2 amplification) or high-grade (with complex karyotypes). Accurate diagnosis is essential for treatment and prognosis. This study evaluates pre-analytical variables associated with the success or failure of epigenetic analyses in osteosarcoma samples and proposes a standardised preparation protocol. METHODS:Retrospective cohort study of adult patients with OS diagnosed at our sarcoma reference centre (CSUR) over the past 20 years. Pre-analytical variables: year of diagnosis, histological subtype, tissue type, site of origin, sample type (core needle biopsy or surgical specimen with or without chemotherapy), decalcification method (none, EDTA, or nitric acid), and FISH availability. Five 5-μm sections were obtained from each paraffin block. DNA methylation profiling was performed using the Infinium MethylationEPIC v2.0 platform (Illumina). Univariate and multivariate analyses were performed to identify failure predictors. RESULTS:A total of 103 samples from 79 patients were analysed: 58 conventional OS, 14 extraskeletal, 24 parosteal, and 7 dedifferentiated OS. Of the 95 formalin-fixed, paraffin-embedded (FFPE) samples, 43 (45.2%) were suitable for epigenetic analysis, whereas all frozen samples were adequate (100%). Decalcification affected success rates, although not significantly: nitric acid was associated with the highest failure rate (68.97%), followed by EDTA (57.14%) and non-decalcified samples (46.15%). CONCLUSION:FFPE samples are suitable for epigenetic studies, although performance depends on pre-analytical factors. Frozen tissue remains the gold standard. Nitric acid should be avoided. A protocol is proposed that prioritises frozen tissue, documents decalcification methods, excludes strong acids, incorporates quality control measures, and favours samples less than five years old.

e23520 Background: Solitary fibrous tumor (SFT) is a rare sarcoma subtype characterized by limited therapeutic options in advanced disease. Tyrosine kinase inhibitors (TKIs) with antiangiogenic properties show clinical activity, but predictive biomarkers of response are lacking. ISG15, an interferon-stimulated gene involved in immune and stress responses, has been implicated in resistance mechanisms to antiangiogenic TKIs, including pazopanib, in previous studies. Methods: Plasma ISG15 levels were quantified by ELISA in paired blood samples from 13 SFT patients within GEIS-69, treated with sunitinib followed by combination with nivolumab. Samples were collected at baseline and after 3 weeks of sunitinib monotherapy. Overall survival (OS) and progression-free survival (PFS) were estimated using Kaplan–Meier methodology. Survival differences were assessed using the log-rank test and univariate Cox proportional hazards regression. Optimal cut-offs for baseline ISG15 were defined using maxstat. Results: Baseline plasma ISG15 levels ranged from 162 to 3358 pg/mL (median 731 pg/mL). Patients with high baseline ISG15 levels showed significantly shorter PFS compared with those with low levels (median 2.8 vs 5.1 months; p = 0.049), while no statistically significant differences in OS were observed. In univariate Cox regression analysis, high baseline plasma ISG15 levels were associated with an increased risk of progression (HR 3.52, 95% CI 0.93–13.32; p = 0.064). Following sunitinib treatment, ISG15 levels increased in most patients (10/12 evaluable cases), with dynamic changes ranging up to 19.2-fold. However, neither OS nor PFS differed significantly according to the magnitude of ISG15 induction during treatment. Conclusions: High baseline plasma ISG15 levels are associated with shorter PFS in SFT patients treated with sunitinib, supporting its potential role as a predictive biomarker in the antiangiogenic setting. Antiangiogenic therapy represents the current standard first-line treatment for unresectable or advanced disease. In contrast, treatment-induced ISG15 upregulation does not appear to correlate with clinical outcome. These findings warrant validation in larger cohorts.

e23526 Background: Immunogenic cell death (ICD) is a functionally unique form of apoptosis that triggers a robust tumor-specific immune response by releasing damage-associated molecular patterns (DAMPs). High Mobility Group Box 1 (HMGB1) is a canonical DAMP and a key biomarker of ICD. Anthracycline-based chemotherapy, the standard of care for soft-tissue sarcomas (STS) such as leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS), is a known inducer of ICD. We sought to determine the prognostic value of HMGB1 expression variation for predicting progression-free survival (PFS) in patients treated with an anthracycline-based chemotherapy regimen combined with an anti-PD-1 inhibitor. Methods: We analyzed HMGB1 expression levels in a cohort of 32 patients with LMS and UPS undergoing anthracycline-based treatment combined with nivolumab, within the IMMUNOSARC clinical trial. Maximally selected log-rank statistics (Maxstat) were utilized to identify the optimal cut-off for HMGB1 expression variation (percentage of variation between baseline and the first radiologic evaluation) to segregate patients into two distinct prognostic groups for PFS. The variation in HMGB1 expression was also correlated with overall survival (OS). Results: The Maxstat analysis identified an optimal prognostic cut-off for HMGB1 expression variation at 13.2%. The group with HMGB1 expression variation > 13.2% demonstrated significantly superior PFS compared to the HMGB1 expression variation ≤13.2% group (13.5 months [95% CI, 11.1-16.0] vs. 8.4 months [95% CI, 6.2-10.5], p = 0.006). This threshold did not demonstrate significant prognostic value for OS (not reached, vs. 17.7 months [95% CI, not reached], p = 0.076). Conclusions: Increased variation in HMGB1 expression is a significant predictor of improved PFS in patients with LMS and UPS treated with chemo-immunotherapy regimens, suggesting that HMGB1 may serve as a valuable circulatory biomarker to monitor treatment efficacy and guide clinical decision-making. Ongoing analyses are evaluating HMGB1 variation at the time of disease progression to determine if the dynamic expression of this protein can predict clinical progression.

Supplementary Table S2. Analysis of prognostic factors related with better clinical response.

Retroperitoneal sarcoma (RPS) resection remains challenging, with up to 50

Background Advanced soft tissue sarcoma (STS) is a group of rare and heterogeneous malignancies, requiring better therapeutic strategies. Pazopanib, a widely used second-line treatment, has been suggested to exert immunomodulatory effects, which may be relevant for enhancing response. Specifically, pazopanib reduces immunosuppressive cells like myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) while enhancing the function of dendritic cells, T cells, and NK effectors. Based on this knowledge, we hypothesized that proteins involved in immunomodulation could serve as predictive biomarkers of response to pazopanib. Objectives To explore immune-related biomarkers of prognosis and pazopanib response in advanced soft tissue sarcoma using paired pre- and post-treatment tumour samples and complementary preclinical models. Design Exploratory translational study integrating differential gene expression profiling of clinical specimens with in silico prognostic evaluation and in vitro functional assays in soft tissue sarcoma cell lines treated with pazopanib. Methods HTG transcriptome-direct profiling was performed on paired FFPE tumour samples from pazopanib-treated patients, followed by bioinformatic identification of differentially expressed genes and survival correlations, and subsequent validation of candidate biomarkers, at RNA and protein levels in sarcoma cell lines exposed to pazopanib. Results Using HTG transcriptomics, we identified 38 differentially expressed genes in post-pazopanib STS samples. Among them, SERPINE1 was the most consistent dynamic biomarker, showing a 4.19-fold increase (FDR = 0.016) and correlating with poor survival (progression-free survival (PFS), p = 0.033; disease-free survival (DFS), p = 0.004). In vitro , SERPINE1 upregulation was observed in STS cell lines CP0024 (24h, 1.47-fold; 48h, 1.71-fold), ICP059 (48h, 1.15-fold), and 93T449 (72h, 2.29-fold), with earlier expression in more sensitive cell lines. Protein validation confirmed these results after 48h of treatment. Conclusions Our results support SERPINE1 as a biomarker for poor prognosis and suggest its role as an exploratory, hypothesis-generating candidate marker for pazopanib response, warranting further validation in larger cohorts.

Soft tissue sarcomas (STSs) are a rare and highly heterogeneous group of tumours originating from mesenchymal cells, characterized by significant clinical and biological diversity. Due to this complexity, STSs present considerable challenges in both diagnosis and treatment, requiring an expert, multidisciplinary, and coordinated approach. To address these challenges, a multidisciplinary team of experts from the Spanish Group for Research in Sarcomas (GEIS) has developed a comprehensive, evidence-based consensus guideline that incorporates the latest advancements in biology, imaging techniques, and treatment options. The primary objective of this guideline is to provide clear and practical, up-to-date recommendations addressing the key questions that arise in the management of STSs. This approach aims to support therapeutic decision-making, enhance overall patient management, and ultimately improve clinical outcomes for patients affected by STSs. Key recommendations include the use of MRI as the preferred imaging modality prior to biopsy and ensuring that all patients are referred to specialised sarcoma centres with a multidisciplinary team for diagnosis and treatment.

INTRODUCTION:Gastrointestinal stromal tumors (GISTs) are the most common sarcoma subtype. Patients with unresectable or metastatic GISTs harboring the D842V mutation in the PDGFRA gene have a poor prognosis due to intrinsic resistance to imatinib and all other approved tyrosine kinase inhibitors. Avapritinib, targeting this mutation, is the first agent approved for patients with unresectable or metastatic GIST that have the PDGFRA D842V mutation. This study assesses the effectiveness and safety of avapritinib in real-world clinical scenarios involving Spanish patients with this mutation. MATERIALS AND METHODS:The AVARWE study is a descriptive, retrospective, multicenter observational study of 21 patients treated with avapritinib across 13 Spanish centers from June 9, 2023, to December 18, 2023. Data collected included patient demographics, disease characteristics, treatment history, and response rates based on RECIST criteria. The main outcomes, progression-free survival (PFS) and overall survival (OS), were measured, with safety assessed through adverse events documentation according to CTCAE criteria. RESULTS:Median PFS 35.6 was months and median OS was 42.2 months, with survival rates at 1, 5, and 3 years demonstrating avapritinib effectiveness. The objective response rate was 76.2% for partial response and 4.8% for complete response. Avapritinib enabled surgical intervention in previously unresectable cases and was generally well-tolerated, with manageable adverse events. CONCLUSION:Avapritinib extends PFS and OS among patients with PDGFRA D842V-mutant GIST in real-world practice, mirroring pivotal trial outcomes. Its substantial activity supports its use as a first-line therapy for this subgroup. The manageable safety profile reinforces avapritinib viability for routine use. Given the rarity of these cases, it is advised to consult sarcoma-expert units.

PurposeCombined BRAF/MEK inhibition with encorafenib (E) plus binimetinib (B) has demonstrated efficacy and tolerability in phase III clinical trials, and is the standard of care for advanced/metastatic BRAFV600-mutant melanoma. However, real-life evidence is limited, particularly in patients pre-treated with immune checkpoint inhibitors (ICI).Patients and methodsBECARE GEM 2002 was a retrospective, non-interventional study aimed at investigating the real-world effectiveness and tolerability of EB in patients with unresectable or metastatic BRAFV600-mutant melanoma conducted at 21 sites in Spain. The primary objective of this study was to characterise the population of patients receiving EB and assess the efficacy and tolerability of EB in real life. The study included patients treated according to standard clinical practice with EB as the 1st line or 2nd line after progression to ICI for an unresectable or metastatic stage. Patients who previously received treatment with BRAF and/or MEK inhibitor, other than as adjuvants, that ended ≥ 6 m before EB were not eligibleResultsFrom September 2021 to March 2023, 117 patients were included; 89 (76.1%) and 28 (23.9%) patients received EB as 1st line and 2nd line, respectively. The median follow-up was 13.8 months (95% CI: 12.0-17.4). In patients with EB as 1st line treatment, ORR and median PFS were 75% and 12 months (95% CI: 9.4-18.6), respectively. In patients with EB as 2nd line treatment after ICI, ORR and median PFS were 77.8% and 12.5 months (95% CI: 6.6-NA), respectively. In patients with brain metastasis ORR and median PFS were 70.8% and 6.3 months (95% CI: 6.1-10.3). Treatment-related adverse events of grade ≥3 were reported in 17 (14.5%) patients; transaminitis (9.4%) and diarrhoea (2.6%) were the most frequent adverse events.ConclusionIn this real-world study, EB treatment demonstrated effectiveness and a consistent safety profile in patients with BRAFV600-mutant melanoma treated according to standard clinical practice, including in those with prior ICI treatment and of brain metastasis; therefore, EB is a feasible treatment option for unresectable and metastatic melanoma.Clinical trial identification: REec: 0004-2021-OBSClinical trial identificationREec: 0004-2021-OBS.

Sarcomas are rare malignant tumors of mesenchymal origin with a high misdiagnosis rate due to their heterogeneity and low incidence. Conventional diagnostic techniques, such as Fluorescence In Situ Hybridization (FISH) and Next-Generation Sequencing (NGS), have limitations in detecting structural variations (SVs), copy number variations (CNVs), and predicting clinical behavior. Optical genome mapping (OGM) provides high-resolution genome-wide analysis, improving sarcoma diagnosis and prognosis assessment. This study analyzed 53 sarcoma samples using OGM. Ultra-high molecular weight (UHMW) DNA was extracted from core and resection biopsies, and data acquisition was performed with the Bionano Saphyr platform. Bioinformatic pipelines identified structural variations, comparing them with known alterations for each sarcoma subtype. OGM successfully analyzed 62.3% of samples. Diagnostic-defining alterations were found in 95.2% of cases, refining diagnoses and revealing novel oncogenic and tumor suppressor gene alterations. The challenges included DNA extraction and quality issues from some tissue samples. Despite these limitations, OGM proved to be a powerful diagnostic and predictive tool for bone and soft tissue sarcomas, surpassing conventional methods in resolution and scope, enhancing the understanding of sarcoma genetics, and enabling better patient stratification and personalized therapies.

11514 Background: It was hypothesized that anthracycline-based chemotherapy plus anti-PD1 (nivolumab) could enhance the activity of upfront chemotherapy in advanced UPS, based on a double-hit in the immunogenic cell death circuit. This peculiar tumor cell death eventually activates an adaptive immune response through particular molecular changes in dying tumor cells and microenvironment, triggered by specific drugs such as anthracyclines. We previously reported a phase Ib trial in leiomyosarcoma patients with the combination of doxorubicin, dacarbazine, and nivolumab, obtaining 56.5% of ORR. We present here the phase Ib, cohort 7a, of the ImmunoSarc2 trial. Methods: Adult patients (pts), with ECOG 0-1, naïve of previous anthracycline-containing treatments, and with a centrally confirmed diagnosis of advanced/metastatic UPS were eligible. Initial dose level 0 (L0) was defined as epirubicin 60 mg/m 2 /d 20 min on D1 and D2 followed by ifosfamide 3 g/m 2 /d 3-h on D1-3, plus nivolumab (NIV) 360 mg on D3 after chemotherapy. Cycles were given Q3W with GCSF and MESNA support. This combo would be given up to 6 courses of 21-day cycles, followed by 1-year NIV maintenance. A -1 dose level (L-1) was defined with the same regimen but with NIV 240 mg. A classic 3+3 phase 1 design was used to determine the MTD based on DLTs (main endpoint) observed during the first 21-day cycle. The cohort was foreseen to be extended with the RP2D to include up to a maximum of 20 evaluable patients. Secondary endpoints included ORR and safety profile among others. Results: Between January 2022 and June 2024, 16 patients M/F (9/7), ECOG 0/1 (15/1), with median age 56 years (29-77) were enrolled. All patients were treated with the initial L0 scheme and no DLTs were observed, being L0 the RP2D. Grade 3-4 toxicities were neutropenia 62.5%, febrile neutropenia 18.8%, anemia 31.3%, and thrombocytopenia 25%. A patient died following a subarachnoid hemorrhage in the context of grade 4 thrombocytopenia and an accidental fall. Of 16 patients, RECIST ORR according to local clinical site assessment was 68.8% distributed as 1 CR (6%), 10 PR (63 %), 4 SD (25%), and 1 PD (6%). With a median follow-up of 16.3 months (95% CI, 7.2-25.4), the median of PFS was 9.9 months (95% CI 7-12.7), while the median OS was not reached, and the 1-year OS rate was 81% (95% CI 62-100). Conclusions: Epirubicin 60 mg/m 2 /d d1-2 plus Ifosfamide 3 g/m 2 /d d1-3 plus NIV 360 mg on d3 Q3W, followed by 1 year of NIV is a feasible and manageable scheme that exhibits relevant activity as an upfront line in advanced UPS patients. A phase II/III trial is designed aiming to confirm the advantage of chemo-immunotherapy over chemotherapy alone in this context. Clinical trial information: NCT03277924 .

PURPOSE:Doxorubicin, alongside a select group of cytotoxic agents, is capable of inducing an adaptive immune response via a well-established peculiar type of tumor cell death called immunogenic cell death (ICD). We hypothesize that combining doxorubicin and dacarbazine with nivolumab may enhance therapeutic efficacy by exerting synergy in the ICD circuit. We hereby present a phase Ib trial with this combination. PATIENTS AND METHODS:Patients with advanced leiomyosarcoma and anthracycline-naïve were eligible. The initial dose level consisted of doxorubicin 75 mg/m2 once on day 1, once every three weeks, followed by dacarbazine 400 mg/m2 once on days 1 and 2, once every three weeks, plus nivolumab 360 mg once on day 2, once every 3 weeks, for six courses and then 1 year of nivolumab. A (-1) dose level was the same regimen but with nivolumab 240 mg. A classic 3 + 3 phase-I design was used to determine the recommended phase-II dose (RP2D). Secondary end points included overall response rate, safety profile, survival, and translational research. RESULTS:From January 2002 to July 2023, 24 patients were enrolled and 23 were evaluable for efficacy, excluding one patient because of noncompliant dose. All patients were treated with the initial dose level, then the RP2D. Toxicity was mild, with the most frequent being grade 4 toxicity neutropenia (16.7%) and thrombocytopenia (8.3%), while no grade 5 toxicity occurred. The centrally reviewed objective response rate was as follows: partial response 56.5%, stable disease 39.1%, and progression 4.4%. The 6-month progression-free survival (PFS) rate was 80% (95% CI, 63 to 98). Dynamic increases of HMGB1 in blood significantly correlated with longer PFS. CONCLUSION:This scheme of doxorubicin, dacarbazine, and nivolumab is feasible and well tolerated. Clinical activity is encouraging and the prognostic impact of HMGB1 supports the relevance of ICD activation. Further clinical research is already underway with this concept in leiomyosarcoma.