(P 180) INVESTIGATING CLINICAL FACTORS ASSOCIATED WITH THE ESTABLISHMENT OF SUCCESSFUL PRIMARY HUMAN SOFT TISSUE SARCOMA CELL LINES

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

Methods: Tumor tissue specimens from consented sarcoma patients resected at the surgical program at Mount Sinai Hospital, Toronto, were dissociated. Most specimens were immediately used to initiate cell lines, and some were cryopreserved for future use. Paired blood or normal tissue samples from each patient were also collected along with the tumor tissue and stored in the biospecimen repository. In addition, clinical information was collected for each patient. To initiate cell cultures, fresh or viably cryopreserved tissues were physically dissociated into 1mm3 fragments using a scalpel. Depending on the histotype and composition of the specimens, they were plated in either GlutaMAX-supplemented HBSS, DMEM/F12, or Opti-MEM + 1% Penicillin / Streptomycin and 10-15% heat-inactivated fetal calf serum (FBS) media with 2-10mg/ml collagenase. After 12-24 hours, digested fragments were dissociated by pipetting before the suspension and any fragments were washed and plated in serum-containing media in a tissue culture-treated T75 flask. Cultures were allowed to rest undisturbed for 7-10 days before media was changed weekly. Once the flask was 80% confluent, cells were passaged. To further expand these cells and freeze aliquots, the flasks were continually passaged where half of the cells were replated, while the other half was cryopreserved. If cells did not grow within three months of plating, the flask was discarded. We classified cell lines into three categories based on cell growth in vitro: no growth (no cells adherent to the plate or never reached 80% confluency), initial growth (cells initially proliferated, but stopped before passage(p) 6), and persistent growth (cells proliferated beyond p6). Select cases that were used to create cell lines and others underwent bulk tumor, paired blood, and cell line Whole Exome Sequencing (WES). A Mutect2 analysis identified molecular variations that were shared between the bulk tumor and cell line but not in the normal control, typically blood.

Results: In total, our group attempted to culture 30 MFS, 58 UPS, and 79 LMS specimens (Table 1). Success rates varied between the three types, where MFS had the highest rate of overall growth, followed by UPS and LMS, respectively [n=23/30 (76.6%) MFS, n=23/58 (39.7%) UPS, and n=24/79 (30.4%) LMS, (Table 2)]. Within the cases that grew in vitro, some yielded cultures that were categorized as initial growth only, and we observed this growth profile at histotype-specific rates [n=7/30 MFS (23.3%), n=9/58 UPS (15.5%), n=7/79 LMS (8.9%)]. The remaining cases yielded cell lines that grew persistently, with the MFS type showing the highest number of persistent cell lines [n=16/30 MFS (53.3%), n=14/58 UPS (24.2%), n=17/79 LMS (21.5%)].
Our group has sequenced bulk tumors from 73 soft tissue sarcoma cases (n=34 MFS, n=36 UPS, and n=14 LMS). As expected, the most common alterations within the cases sequenced included ATRX, TP53, and RB1, consistent with the findings of other studies.
From the 167 cases that were used to initiate cell lines, 46 have sequencing information available. Some of this information was used to identify if cell lines retained tumor-specific variation (TSV) after an extended time in culture. In a pilot study where 10 UPS cell lines were evaluated for TSV retention, 6 retained TSVs beyond passage 6 (Table 3).

Conclusion: We have successfully adapted cell lines from MFS, UPS, and LMS specimens in our laboratories, albeit at different rates. Our sequencing data from bulk tumors indicated that alterations in 3 genes, ATRX, TP53, and RB1 are common in these three sarcoma types, and this is consistent with the literature. In the future, we seek to determine if these common mutations are correlated to successful cell line growth in vitro. Cell lines were also evaluated for molecular similarity to the original bulk tumor, and the retention of TSVs suggested that subpopulations of the bulk tumor have persisted in vitro. 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, is vital to improving the outcome for sarcoma patients.