(P 367) CHARACTERIZING HYPOXIA AND ASSOCIATED MOLECULAR FEATURES IN EXTREMITY SOFT TISSUE SARCOMA

Objective: Undifferentiated pleomorphic sarcoma (UPS) and myxofibrosarcoma (MFS) are common extremity soft tissue sarcomas (STS) in adults with an approximate 50% risk of distant failure. Thus, there is a need to better understand the mechanisms of metastasis to increase therapeutic options and eventually improve outcomes. One known factor that correlates to aggressive STS disease is tumor hypoxia. We conducted a study to determine if hypoxia was detectable in extremity STS using exogenous hypoxia markers ([18F] fluoroazomycin-arabinoside (FAZA) and pimonidazole) and to evaluate associated epigenetic and expression changes.

Methods: We conducted a feasibility clinical trial (NCT03418818) to measure hypoxia in extremity or truncal STS with FAZA-PET/MRI. Patients in Arm A underwent a FAZA-PET/MRI prior to neoadjuvant radiation (RT) and patients in Arm B underwent a FAZA-PET/MRI prior to surgery and received adjuvant RT. We preferentially enrolled patients with UPS and MFS, however, other STS types were included. Prior to surgery, patients in Arm B received pimonidazole, and after tumor resection tissue was collected for correlative studies. Viable tumor regions were stained for pimonidazole and known endogenous hypoxia markers HIF-1α and CA-IX. To evaluate molecular changes due to hypoxia, we sorted dissociated tumor cells into pimonidazole + (hypoxic) and pimonidazole - (normoxic) populations by fluroescence activated cell sorting flow cytometry. Next, we performed DNA methylation analysis on hypoxic and normoxic clinical specimens (n=4) and on primary cell lines (UPS (n= 4) and MFS (n= 5)) exposed to hypoxic (0.1% O2) or normoxic conditions for 72 hours using EPIC microarrays (Illumina). We also performed bulk RNA sequencing of the same primary cell lines (Illumina). We conducted exploratory analyses of DNA methylation and RNA sequencing data using unsupervised clustering and principal component analysis. For differential methylation analysis we employed the ChAMP package, and for differential expression analysis we used the DESeq2 package in R. To identify significantly over- or underrepresented pathways in the gene expression data, we performed Gene Set Enrichment Analysis (GSEA).

Results: Twenty-five patients were enrolled (Arm A (n=15), Arm B (n=10)), the majority of whom had UPS (n=9) or MFS (n=10). Of the patients who had a successful baseline FAZA-PET/MRI, 14/20 patients demonstrated quantitative evidence of hypoxia, which correlated inversely with disease-free survival (p < 0.05, log rank test). Pimonidazole staining was heterogenous and colocalized with HIF-1α and CA-IX. We sorted dissociated tumor cells from six patients, but only four had enough DNA to proceed with the methylation analysis. Unsupervised and supervised analyses of DNA methylation data did not show any significant changes associated with hypoxia in patient samples or cell lines. GSEA of RNA sequencing data identified significant enrichment of 102 genes involved in hypoxia and 54 genes involved in glycolysis in cell lines cultured in hypoxic conditions. Differential expression analysis identified 99 upregulated and 20 downregulated genes in cell lines cultured in hypoxic conditions compared to normoxic conditions (adjusted p value < 0.05). Ranked GSEA performed on the differentially expressed genes pointed to significant enrichment of genes activated by the HIF1α pathway in the cell lines cultured in hypoxic conditions, including CA9, FOS, NDRG1, LEP and EGLN3.

Conclusion: Overall, hypoxia was detectable in extremity STS using FAZA-PET/MRI and associated with worse disease-free survival. However, hypoxia was not associated with any DNA methylation changes in STS under the conditions evaluated. Future efforts will aim to identify hypoxia-specific expression alterations in UPS and MFS in this clinical cohort.