Autologous tumor-infiltrating lymphocyte (TIL) cell therapy is showing promising efficacy against immunologically "hot" tumors such as melanoma, cervical cancer, and renal cancer. However, generation of tumoricidal TILs from "cold" tumors with a low tumor mutational burden, such as many sarcoma types, poses a challenge due to limited infiltration of the tumor microenvironment (TME) with lymphocytes, low frequencies of tumor antigen-specific, high-affinity T cells, and incompletely understood mechanisms of immune-resistance prevailing in the TME. Here, we report the successful generation and expansion of TILs engineered with regulatable, membrane-bound IL15 (cytoTIL15 cells) from immune-excluded, paucicellular chondrosarcoma biopsies largely consisting of collagenous matrix and demonstrate that these cells have potent tumor-killing capacity in cell culture and in tumor spheroid models in the absence of exogenous IL2. Comprehensive spatial profiling of the TME revealed ubiquitous collagen and myeloid infiltration as major resistance mechanisms, whereas lymphocytic infiltration was largely restricted to peripheral regions of the tumors, a relevant consideration when sampling these tumors for TIL harvest. Moreover, we demonstrate that IL15 reduced the signaling threshold of T-cell receptors isolated from TIL clonotypes, increasing their infiltration and cytotoxicity in autologous 3D tumor models. These results suggest the possibility of developing an effective IL2-free TIL therapy for patients with immune-excluded tumors.

Supplementary Figure S4: Gating strategy for phosphor-ERK analysis.

Supplementary figure S9: Additional flow analysis on sarcoma TIL.

Supplementary Figure S5: Nanostring analysis of different sarcoma types.

Supplementary Figure S1: Gating strategy for pre-REP phenotyping.

Video S2: Real-time tracking of TIL infiltration into tumor spheroids. Autologous tumor spheroids are labeled by Deep Red Cell Tracker dye (Red) and co-culture with TIL pre-labeled with CFSE tracker dye (Green). TIL infiltration into the autologous spheroids were imaged for 24 hrs with 1 hour time interval.

Supplementary Table S2: Flow cytometry antibodies clones and dulution factors

Video S1: ACZ dependent killing of autologous tumor cells by TIL. Autologous tumor cells were labeled by mCherry and co-cultured with non-engineered or cytoTIL15 cells +/- 25 µM ACZ at an effector to target ratio of 20:1. Confluency of the tumor cells as readout by mCherry was analyzed.

Video S3: Tissue clearing and whole spheroid imaging showing increased IL15 TIL infiltration and function in autolgous 3D tumor spheroid models. Immunofluorescence staining of tissue-cleared tumor spheroids 24 hours post co-culture with autologous non-engineered and IL15 engineered TIL. Spheroids are stained with DAPI (blue), CD3 (T cells, red), granzyme B (green) and SOX9 (tumor cells, yellow).

With the first series of PRRX1-rearranged tumors published in 2019, the spectrum of these so-called fibroblastic tumors has been expanded. Since then, several smaller case series have been published; however, our understanding of them continues to be quite limited given their rarity. We herein studied 18 additional cases, the largest series to date. Eighteen tumors present in 9 male, 8 female, and 1 nonbinary patient with a median age of 35 years (range: 11 to 70 y) and involved the neck (5), the chest region (4), thigh (3), back (1), shoulder (1), forehead (1), lower leg (1), axilla (1), and the parapharyngeal region (1). Clinical follow-up (9/18 tumors; 50%; median: 10 mo; range: 4 to 40 mo) showed consistent indolent behavior without local recurrences or distant metastases. On morphology, these tumors were characterized by well-circumscription and distinctive peripheral crescent-shaped vessels. They were composed of uniform spindle and round cells growing in short fascicles within often densely hyalinized collagen lacking significant mitotic activity, necrosis, or cytologic atypia. Immunohistochemically, about half of the tested tumors expressed focal to rarely diffuse S100 with occasional co-expression of SOX10. Interestingly, almost half of the tested cases also showed complete loss of RB expression. All but 1 tumor harbored a PRRX1::NCOA1 fusion, while 1 case harbored a novel PRRX1::EP300 fusion. We herein provide additional data on these exceptionally uncommon tumors, expand their molecular spectrum, and compare them to their close morphologic mimics to aid in accurate diagnosis and avoid confusion with potentially more aggressive neoplasms.

Supplementary Figure S7: Representative IHC images of Chondrosarcoma tumors.

Supplementary Figure S8: Correlation of immune infiltration (IHC) and gene expression profiles

Supplementary Table S1: IHC antibody clones and dilution factors

Supplementary Figure S6: Immune checkpoint ligand and receptor expression analysis in tumor vs peritumor regions.

Supplementary Figure S10: ACZ dependent killing of autologous tumor cells by TIL.

Supplementary Figure S3: Gating strategy for memory phenotyping.

11573 Background: Leiomyosarcomas (LMS) are aggressive mesenchymal tumors sometimes classified by primary location as uterine LMS (uLMS) versus extra-uterine LMS (eLMS). In pursuit of novel therapies, study of the tumor microenvironment and the repertoire of T-Cell Receptor (TCR) sequences from infiltrating T-lymphocytes could help guide the application of immunotherapy in LMS. Herein, we explore the first ever database of TCR-sequenced LMS tumors in one of the larger LMS cohorts analyzed to date. Methods: A single-institution retrospective study was performed including 117 adult patients with pathology-confirmed LMS diagnosed between 1998 and 2022 at Northwestern Medicine. Overall survival (OS), progression free survival (PFS), and relapse free survival (RFS) were analyzed using Kaplan-Meier Curves (GraphPad Prism). DNA was extracted from 61 eLMS and 39 uLMS formalin-fixed paraffin-embedded (FFPE) blocks using Qiagen’s AllPrep DNA/RNA FFPE kit and used for TCR sequencing. Results: 117 patients who met the inclusion criteria were classified as uLMS (n=54) and eLMS (n=63). Baseline patient demographics and disease characteristics are described in the table. Median OS significantly differed between uLMS and eLMS patients (559 versus 1548 days; p=0.001). Median PFS was not statistically different between metastatic uLMS versus eLMS (142 versus 159 days; p=0.63). Median RFS was not significantly different between localized uLMS versus eLMS (283 versus 429 days; p=0.30). The number of different TCR clones amongst TCRs was significantly different between the two tumor types: uLMS demonstrated a mean of 821 clones versus 2415 clones in eLMS (p=0.005). Mean CDR3 length in uLMS tumors was 47.9 (95% CI 46.53-49.25) versus 48.45 (95% CI 46.66-50.25) in eLMS tumors (p=0.10). TRBV20 was the most common gene in uLMS cases and the second most common gene in eLMS cases. Conclusions: This study provides the first analysis of the TCR repertoire in LMS patients and the largest analysis of TCR repertoire ever reported in any sarcoma subtype. eLMS tumors contained significantly more TCR clones than uLMS tumors. CDR3 did not significantly differ between cohorts, and the TRBV20 gene was frequently identified in both LMS subtypes. Further gene expression analysis and immunohistochemistry is currently underway. As might be expected, clinical outcomes were worse for uLMS including significantly lower OS. Updated data will be presented including additional information about the TCR repertoire as well as gene expression data to contextualize these findings. Baseline patient demographics and disease characteristics. uLMS (n=54) eLMS (n=63) Median Age (range) 55 years (25-91) 58 years (23-87) Female Sex: n (%) 54 (100%) 34 (54%) Median Tumor Size (range) 11 cm (1-34) 5 cm (0.7-25) Necrosis Present: n (%) 47 (87%) 33 (52%) Positive Margins: n (%) 5 (9%) 9 (14%) Stage IV Disease: n (%) 16 (30%) 5 (8%) Adjuvant Chemotherapy: n (%) 27 (50%) 4 (6%)

Uterine leiomyosarcoma (uLMS) is a rare and deadly gynecologic malignancy. uLMS is histologically heterogeneous and presents with a wide spectrum of tumor differentiation, with a broad range of genomic DNA instability, which can make the diagnosis and prognosis of uLMS challenging. Methylation has emerged as a useful molecular tool in tumor classification and diagnosis in certain neoplasms. We initiated this study to investigate the role of global methylation in the differential diagnosis of uLMS from its mimics in correlation with pathologic characteristics and clinical outcomes. In this study, we performed array-based global methylation profiling analysis in a total of 71 uLMS and compared the methylation signatures of uLMS with several other uterine mesenchymal tumors and soft tissue leiomyosarcoma. We found that uLMS demonstrated distinct methylation patterns differing from all other tumor types. Notably, methylation profiling defines 2 distinct subgroups of uLMS with differing copy number alterations, resulting in unique histologic and clinical behaviors, further emphasized by differences in methylation pathway analysis. This study is the first to report methylation profiling as a useful diagnostic tool in differentiating uLMS from mimics and defines 2 subtypes of uLMS based on methylation signatures.

BACKGROUND:The use of neoadjuvant therapy (NAT) is not well characterized across the entire spectrum of localized gastrointestinal stromal tumors (GISTs). This study aimed to identify treatment trends for the use of NAT and survival outcomes in patients who underwent surgery for gastric GISTs. METHODS:The National Cancer Database was queried to identify patients with stage I to III gastric GISTs between 2006 and 2020 who underwent curative-intent surgery after NAT or upfront surgery (UFS). The cohort demographics and cancer characteristics were compared using analysis of variance, generalized linear models, and chi-square analysis. Treatment trends were assessed using linear regression. Cox proportional hazards models and Kaplan-Meier curves were used for propensity score matching (3:1). RESULTS:Between 2006 and 2020, 1504 patients (8.1%) received NAT, followed by surgery, and 17,150 patients (91.9%) received UFS with curative intent. The cohort analysis revealed that, compared with patients who underwent UFS, those who underwent NAT had higher grade tumors (P =.031), larger tumors (12.0 vs 6.4 cm, respectively; P <.001), and higher clinical staging (P <.01). Linear regression indicated that the use of NAT became more common each year (0.88%; P <.05) and that NAT was administered for longer durations before surgery (increase in 5.5 d/y; SE, 0.9; P <.05). Despite NAT being used for larger and higher-grade tumors, patients who underwent NAT had similar outcomes as those who underwent UFS (UFS: hazard ratio, 0.86 [95% CI, 0.76-1.01]; P =.07). CONCLUSION:Our results demonstrate that the use of NAT has been increasing over time and that NAT is used for more advanced gastric GISTs. Although NAT is used for higher-risk tumors, its use for gastric GISTs leads to similar outcomes as UFS.