(P 249) INVESTIGATING CLINICAL FACTORS ASSOCIATED WITH THE ESTABLISHMENT OF SUCCESSFUL PRIMARY HUMAN SOFT TISSUE SARCOMA CELL LINES

Objective: Soft tissue sarcomas (STS) are a heterogeneous group of neoplasms with over 70 distinct types, in aggregate accounting for 1% of adult cancers. Several of the most common adult STS include myxofibrosarcoma (MFS), undifferentiated pleomorphic sarcoma (UPS), and leiomyosarcoma (LMS). All three represent pleomorphic malignancies with complex genomic copy number alterations, and poor survival rates, which, yet lack targetable molecular aberrations for therapeutic intervention. The study of STS and search for improved therapies is further complicated by the limited availability of well characterized, representative pre-clinical human STS models, therefore our program has endeavored to develop and characterize primary STS cell lines from resected patient specimens.

Methods: Tumor specimens (n=168) were collected from consented STS patients (n=166) undergoing biopsy or resection at Mount Sinai Hospital, Toronto. Specimens were immediately used to initiate cell lines or were cryopreserved for future use. Paired blood and/or normal tissue from each patient were stored in the biospecimen repository. To initiate cell cultures, tissues were physically dissociated into 1mm3 fragments. Fragments were plated in DMEM/F12 (UPS and MFS) or Opti-MEM (LMS) + 1% Penicillin / Streptomycin and 10-15% heat inactivated fetal calf serum (FBS) media with 2-10mg/ml collagenase. After 16-24 hours, digested fragments were dissociated by pipetting, washed and plated in media in a tissue culture-treated T75 flask. Media was changed after 7-10 days. Once the flask was 80% confluent, cells were passaged. We classified cell lines in to two categories based on growth in vitro: no or little growth (no adherent cells or cells that initially proliferated, but stopped before passage 6), and persistent growth (cells proliferated beyond passage 6). Select cases with bulk tumor, paired normal and cell line samples underwent Whole Exome Sequencing (WES). A Mutect2 analysis identified tumor-specific variations (TSVs) that were shared between the patient tumor and cell line but not in the blood normal control. Clinical information was collected for each patient. To determine if clinical characteristics were correlated with growth, Pearson chi squared tests and t tests were performed, while Kaplan Meier survival analysis and log rank tests were used to evaluate disease specific survival and overall survival. Significance was considered at p < 0.05.

Results: 168 specimens were collected from predominantly untreated, high-grade, primary tumors of the extremity (41.6%) (Table 1). In total, we successfully adapted 46 cell lines from 168 specimens (27%). 10 UPS cell lines were evaluated for TSV retention, and 6 cases retained TSVs beyond passage 6 (Table 2). 145 specimens used to initiate cell cultures were from primary tumors, and the remaining were derived from metastatic (n=18) or recurrent tumors (n=5). Success rates varied between the three types, where MFS had the highest rate of persistent growth from both primary and recurrent specimens (n=17/33, p< 0.05), followed by UPS (n=14/56) and LMS (n=15/79 LMS). When considering primary specimens only, successful cell line growth was more likely to occur from extremity tumors in patients who presented with metastatic disease, where the specimen was not treated before collection (Table 3, p< 0.05).

Conclusion: We have successfully established cell lines from MFS, UPS, and LMS specimens, albeit at different rates. Clinical associations may aid in prioritizing which tumors should be used to initiate cell cultures while TSV retention confirmed the cell lines that reflected the primary tumor. The cell lines and protocols established here have facilitated functional studies to better understand these entities on a molecular level and can assist in investigating potential therapies. Overall, the development of preclinical models of rare entities, such as MFS, UPS, and LMS, remains vital to improving the outcome for STS patients.