(P 222) ANALYSIS OF THE CITN-13 SARCOMA ARM: PEMBROLIZUMAB AND INTERFERON GAMMA FOR SYNOVIAL SARCOMA AND MYXOID/ ROUND CELL LIPOSARCOMA

Objective: Synovial sarcomas (SS) and myxoid/ round cell liposarcomas (MRCL) have a high expression of endogenous immunogenic antigens such as NY-ESO-1, MAGE – A4 and PRAME. However they also have an inhibitory tumor microenvironment with low MHC expression and few infiltrating immune cells. We previously reported that systemic weekly Interferon Gamma (IFN) can increase class I MHC expression on tumor cells as well as T cell infiltration in these tumors with demonstrated induction of antigen specific responses. However, we also noted up-regulation of PD-L1, particularly in the myeloid cells that infiltrated these cancers. To counteract PD-L1 upregulation, we launched a sub-arm of the Cancer Immunotherapy Trials Network Study-13 (CITN-13; NCT03063632) combining IFN with Pembrolizumab in patients with SS and MRCL.

Methods: The CITN-13 study was a two-stage phase II trial evaluating the response of advanced (unresectable or metastatic) SS and MRCL patients to combined IFN and PD-L1 inhibitor therapy. Included patients were treated weekly with 100 mcg/m2 and 200 mg pembrolizumab administered every three weeks. Response to treatment was monitored by CT scans every twelve weeks and assessed using RECIST v1.1criteria.
Peripheral blood and tumor biopsy samples were collected prior to and at 3 weeks intervals after therapy initiation. Evaluable FFPE tumor samples were assessed with multiplex IHC for CD3, CD4, CD8, PD-1 and MHC class I and gene expression was assessed using the Nanostring platform. Peripheral blood samples were used to run TCR sequencing and cytokine release assays. Lastly digital spatial profiling using GeoMx platform was conducted on selected cases wherein the gene expression profile of CD3, CD68 and tumor cells (TLE1 as a marker of SS tumor cells) were analyzed.

Results: A total of twelve patients were enrolled in the study; five of them had SS, and seven with MRCL. Patients had a median of 93 days as duration of treatment and an average of 133 days; though one patient was able to stay on trial with disease stability for 721 days. No clinical responses were seen. Given that no responses were seen, the trial was closed after its first stage.
Assessing gene expression using Nanostring (n=7), we saw a relative decrease of CD3e in all but one patient post-treatment. We also saw that the post treatment levels of genes associated with facilitating T cell infiltration were reduced; a significant decrease in genes associated with immune cell infiltration (CX3CL1 and IFIT3), and a relative decreased in other genes associated with T cell infiltration such as (CXCL10, CX3CR1, ITGA4, and KDR) were found. To confirm these findings at the protein level, we assessed T cell infiltration by looking at cell surface markers using multiplex IHC (n=6). Interestingly, multiplex IHC also showed decreased CD3+ cells in five of six patients and a decrease in CD4+ in all patients. Moreover, we saw an increase of PD-1in all but one case. Furthermore, 4 of the 6 cases had decreased expression of MHC I while two cases showed an increase in expression. To examine these finding within specific cell compartments of the tumor, we performed digital spatial profiling using the GeoMx platform on two SS cases. This assessment found that post-treatment CD3+ cells had decreased expression of CX3CL1 and KDR genes. Tumor cells compartment had notable decreases in CX3CR1 and IFIT3 and CD68+ cell compartment demonstrated a decrease in CX3CR1, CX3CL1, CXCL10, IFIT3 and KDR.
To assess the systemic changes in immunity occurring outside of the tumor, we analyzed serum changes in cytokines (n=9). Interestingly, no significant change in IFN was noticed; however a relative increase of CXCL10 molecule was seen. Lastly to assess the diversity of T cell Receptor (TCR) repertoire post treatment (n=9), TCR sequencing of PBMCs showed an overall decrease in diversity of TCR repertoire in SS patients while the opposite was noticed amongst MRCL patients. Furthermore, in two third of the cases there appears to be a decrease in V-beta family mean frequency usage after the treatment.

Conclusion: IFN and Pembrolizumab are not an effective combination for SS and MRCL in this regimen. Despite our prior results showing clearly increased tumor MHC class I expression, tumor antigen presentation and T cell infiltration with weekly systemic IFN, we did not see this in the current study possibly because of differences in biopsy time points and a more prolonged course with systemic IFN. Given the clear findings in our prior Phase 0 trial that was the basis of this study, this study highlights the complexity of the "simple" SS and MRCL tumor microenvironments and the importance of small pilot studies to test big ideas in a "go" or "no-go" fashion.