Human soft tissue sarcomas harbor an intratumoral viral microbiome which is linked with natural killer cell infiltrate and prognosis.

BACKGROUND: Groundbreaking studies have linked the gut microbiome with immune homeostasis and antitumor immune responses. Mounting evidence has also demonstrated an intratumoral microbiome, including in soft tissue sarcomas (STS), although detailed characterization of the STS intratumoral microbiome is limited. We sought to characterize the intratumoral microbiome in patients with STS undergoing preoperative radiotherapy and surgery, hypothesizing the presence of a distinct intratumoral microbiome with potentially clinically significant microbial signatures. METHODS: We prospectively obtained tumor and stool samples from adult patients with non-metastatic STS using a strict sterile collection protocol to minimize contamination. Metagenomic classification was used to estimate abundance using genus and species taxonomic levels across all classified organisms, and data were analyzed with respect to clinicopathologic factors. RESULTS: Fifteen patients were enrolled. Most tumors were located at an extremity (67%) and were histologic grade 3 (87%). 40% were well-differentiated/dedifferentiated liposarcoma histology. With a median follow-up of 24 months, 4 (27%) patients developed metastases, and 3 (20%) died. Despite overwhelming human DNA (>99%) intratumorally, we detected a small but consistent proportion of bacterial DNA (0.02-0.03%) in all tumors, including Proteobacteria, Bacteroidetes, and Firmicutes, as well as viral species. In the tumor microenvironment, we observed a strong positive correlation between viral relative abundance and natural killer (NK) infiltration, and higher NK infiltration was associated with superior metastasis-free and overall survival by immunohistochemical, flow cytometry, and multiplex immunofluorescence analyses. CONCLUSIONS: We prospectively demonstrate the presence of a distinct and measurable intratumoral microbiome in patients with STS at multiple time points. Our data suggest that the STS tumor microbiome has prognostic significance with viral relative abundance associated with NK infiltration and oncologic outcome. Additional studies are warranted to further assess the clinical impact of these findings.

Analysis of tumor-infiltrating NK and T cells highlights IL-15 stimulation and TIGIT blockade as a combination immunotherapy strategy for soft tissue sarcomas.

PURPOSE: Given the unmet need for novel immunotherapy in soft tissue sarcoma (STS), we sought to characterize the phenotype and function of intratumoral natural killer (NK) and T cells to identify novel strategies to augment tumor-infiltrating lymphocyte (TIL) function. EXPERIMENTAL DESIGN: Using prospectively collected specimens from dogs and humans with sarcomas, archived specimens, and The Cancer Genome Atlas (TCGA) data, we evaluated blood and tumor NK and T cell phenotype and function and correlated those with outcome. We then assessed the effects of interleukin 15 (IL-15) stimulation on both NK and T cell activation and TIGIT upregulation. Finally, we evaluated cytotoxic effects of IL-15 combined with TIGIT blockade using a novel anti-TIGIT antibody. RESULTS: TILs were strongly associated with survival outcome in both archived tissue and TCGA, but higher TIL content was also associated with higher TIGIT expression. Compared with blood, intratumoral NK and T cells showed significantly higher expression of both activation and exhaustion markers, in particular TIGIT. Ex vivo stimulation of blood and tumor NK and T cells from patients with STS with IL-15 further increased both activation and exhaustion markers, including TIGIT. Dogs with metastatic osteosarcoma receiving inhaled IL-15 also exhibited upregulation of activation markers and TIGIT. Ex vivo, combined IL-15 and TIGIT blockade using STS blood and tumor specimens significantly increased cytotoxicity against STS targets. CONCLUSION: Intratumoral NK and T cells are prognostic in STS, but their activation is marked by significant upregulation of TIGIT. Our data suggest that combined IL-15 and TIGIT blockade may be a promising clinical strategy in STS.