(P 434) UTILIZATION OF IMAGING MASS CYTOMETRY TO EVALUATE THE IMMUNE LANDSCAPE OF PEDIATRIC AND ADULT PATIENTS WITH SARCOMA TREATED WITH AN AUTOLOGOUS DENDRITIC CELL VACCINE

Objective: Sarcomas are rare, heterogeneous, aggressive neoplasms with a propensity to affect younger individuals with poor survival once refractory to standard therapies. The role of the tumor microenvironment (TME) including the immune system is a critical area of research. Immunotherapy presents promising new modalities including autologous dendritic cell (DC) vaccines, using antigen-loaded DCs, which intensify the adaptive immune response by enhancing T-cell activity.  Currently, one of the best-described predictors of response to immune checkpoint inhibitors include tumor PD-L1 expression and PD1-expressing T cells within the tumor. Recent data suggest that other factors, including intratumoral T-regulatory cells (Treg), or checkpoint proteins such as CTLA-4, TIM-3, and LAG-3 may also impact T-cell immunotherapeutic responses. We conducted a phase I trial (2014-2019) of autologous DC vaccination in patients with advanced or metastatic sarcomas (NCT01803152). Nineteen patients were enrolled with a median age of 50 years (13-75 years) and 47% female. Disease progression before or after completion of study treatment was noted in 15 patients with a median PFS of 9.5 months (95% CI 5.6-28.7). Four of 19 patients (20%) exhibited continued progression-free survival (PFS) after DC therapy with a median follow-up of two years, including two patients with pleomorphic myxofibrosarcoma, one with leiomyosarcoma and one pediatric patient with Ewing sarcoma, far exceeding outcomes expected with metastatic sarcoma. We hypothesize that pro-inflammatory immune cells and cytokines in the circulation and in the TME associate with prolonged PFS after treatment with DC vaccine.

Methods: Archival tumor tissue from all patients treated on the DC vaccine trial was retrieved. The immune milieu of TME was assessed by imaging mass cytometry using the Standard Biotools Hyperion® platform which presents a novel approach to study sarcoma samples. A panel was designed to quantify and phenotype the following immune cells: T cells, including CD8+ cytotoxic T cells, CD4+ helper T cells, FOXP3+ T reg, B cells, natural killer (NK) and NKT cells, and multiple myeloid populations including M1 pro-inflammatory tumor-associated macrophages (TAMs), M2 suppressive TAMs, and pro-inflammatory and suppressive DC and MDSC subsets. We also analyzed key immunotherapeutically targetable or otherwise functionally relevant markers including PD-1, PD-L1, CTLA-4, TIM3. Antibodies were either acquired pre-conjugated and validated from Standard Biotools or purchased in purified format and conjugated in-house with isotopically pure metals purchased from Standard Biotools®. All the antibodies were validated on human tonsillar tissue. After ablation on the Hyperion, the resulting imaging data was analyzed using the Visiopharm® Phenoplex Guided Workflow module (v2023.09.4.15595 x64).

Results: Pilot testing was performed on one responder and one non-responder sample, a 75yo male with pleomorphic myxofibrosarcoma and a 35yo female with rhabdomyosarcoma, respectively. Samples were chosen randomly and blindly to avoid bias. Interestingly, a greater percentage of immune infiltration was identified in the TME of the responder as compared to the non-responder. In particular, tissue from the responder exhibited a greater density of M2 macrophages (CD14+ CD163+), dendritic cells, (CD11c), PDL1 expression and staining for granzyme B, suggestive of NK cells and cytotoxic T cells (Figure1-4). Further testing on samples from the rest of the patients treated on the trial is ongoing. Additionally, a concurrent spatial transcriptomic analysis of the same samples using the Visium® platform is underway.

Conclusion: Sarcomas harbor marked intra and inter-tumoral immune heterogeneity. Analysis of TME and a better understanding of immune correlates is likely to yield relevant biomarkers predicting treatment response to immunotherapy and potential identification of new immunotherapeutic approaches.