The immune microenvironment of sarcomas : a comprehensive evaluation of infiltrating immune cells and checkpoint biomarkers in musculoskeletal tumors

Sarcomas are aggressive cancers of the connective tissues, such as bone, muscle, cartilage, and fat. Despite their diverse origins, sarcomas are predominantly treated by surgery and radiation, as conventional chemotherapy has limited benefit for most subtypes. When sarcomas recur or metastasize, there are few options for systemic therapy, and prognosis is very poor. Despite advancements in our understanding of the molecular drivers of sarcomas, almost no new treatments have proven benefit for metastatic sarcomas. Immunotherapy has shown value for other cancers, such as melanoma and lung cancer; however, sarcomas lag behind the common cancers in our understanding of their immune microenvironment and potential for treatment with immunotherapeutics. Early trials using single-agent immune checkpoint inhibitors in sarcomas delivered mixed results, but these studies somewhat indiscriminately lumped together different sarcoma subtypes that might have critical immunological differences. My study employs tissue microarrays incorporating 1360 sarcoma specimens (spanning 23 subtypes) to characterize immune infiltrates and expression of targetable immune biomarkers, using immunohistochemistry. Genomically-complex sarcoma types – driven by mutations and/or copy-number alterations – are found to have much higher levels of lymphocytic and phagocytic immune infiltrates than translocation-associated sarcomas. Across nearly all subtypes, tumor-associated macrophages outnumber tumor-infiltrating lymphocytes, predominately M2 (anti-inflammatory) macrophages. Expression of the target of first-generation immune checkpoint PD-(L)1 is uniformly low, but expression of LAG-3 and TIM-3 – emerging immune checkpoints – is significantly more common. Expression of anti-phagocytic immune checkpoint CD47 is yet more predominant, displaying all-or-nothing expression with 100% positivity seen in over half of positive cases. To further characterize the lymphocytic response, T-cell receptor (TCR) sequencing was performed on specimens from 25 sarcoma patients on a clinical trial of tremelimumab (anti-CTLA-4) with durvalumab (anti-PD-L1). We found that the TCR repertoire is richer and more diverse among the genomically-complex sarcomas relative to the translocation-associated sarcomas, and following immune checkpoint blockade, we observed an overall increase in the clonality of the peripheral TCR repertoire. My study demonstrates a tangible positive relationship between genomic complexity and immunogenicity, and highlights novel immune checkpoints of relevance to sarcomas. As such, this work provides the essential translational background to direct the use of immunotherapy in sarcoma management.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat