Sarcomas: Immune biomarker expression and checkpoint inhibitor trials

Open AccessPublished:October 19, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.102115

      Highlights

      • Karyotypically complex pleomorphic sarcomas are the most likely to be immune hot.
      • Alveolar soft part sarcoma show response to immune checkpoint inhibitors.
      • Multiplexed and spatially resolved technologies can aid immune biomarker discovery.
      • Sarcoma subtype and/or biomarker specific Phase 3 trials are required.

      Abstract

      Sarcomas are a heterogenous group of mesenchymal cancers comprising over 100 subtypes. Current chemotherapy for all but a very few subtypes has limited efficacy, resulting in 5-year relative survival rates of 16% for metastatic patients. While sarcomas have often been regarded as an “immune cold” tumor category, recent biomarker studies have confirmed a great deal of immune heterogeneity across sarcoma subtypes. Reports from the first generation of clinical trials treating sarcomas with immunotherapy demonstrate a few positive responses, supporting efforts to stratify patients to optimize response rates. This review summarizes recent advances in knowledge around immune biomarker expression in sarcomas, the potential use of new technologies to complement these study results, and clinical trials particularly of immune checkpoint inhibitor therapy in sarcomas.
      Each of the immune biomarkers assessed was reviewed for subtype-specific expression patterns and correlation with prognosis. Overall, there is extensive heterogeneity of immune biomarker presence across sarcoma subtypes, and no consensus on the prognostic effect of these biomarkers. New technologies such as multiplex immunohistochemistry and high plex in situ profiling may offer more insights into the sarcoma microenvironment. To date, clinical trials using immune checkpoint inhibitor monotherapy have not shown compelling clinical benefits. Combination therapy with dual checkpoint inhibitors or in combinations with other agents has yielded more promising results in dedifferentiated liposarcoma, undifferentiated pleomorphic sarcoma, angiosarcoma and alveolar soft-part sarcoma. Better understanding of the sarcoma immune status through biomarkers may help decipher the reasons behind differential responses to immunotherapy.

      Keywords

      Introduction

      Sarcomas are a heterogenous group of cancers stemming from mesenchymal tissues, and can be classified into soft-tissue and bone sarcomas [
      • WHO Classification of Tumours Editorial Board
      ]. These broad tumor categories are further divided based on distinct morphology and genetic changes into more than 100 subtypes as defined by the World Health Organization [
      • WHO Classification of Tumours Editorial Board
      ]. While most sarcomas are detected as local tumors and treated with excisional surgery and radiation, metastasis will occur in up to 50% within the first five years (depending on the site, grade and subtype) [
      • Brennan M.
      • Antonescu C.
      • Moraco N.
      • Singer S.
      Lessons learned from the study of 10,000 patients with soft tissue sarcoma.
      ]. Given the limited efficacy of current systemic treatment options, the 5-year relative survival rate for patients with distant metastasis is 16% [

      Howlader N, Noone AM, Krapcho M, Miller D, Brest A, Yu M, et al. SEER cancer statistics review, 1975-2016;2019 ASI 4474-35; 2019.

      ]. Only a few sarcoma subtypes have experienced increase cure rates from specific systemic therapies, most prominently tyrosine kinase inhibitors for gastrointestinal stromal tumors (GIST) [
      • Poveda A.
      • García del Muro X.
      • López-Guerrero J.A.
      • Cubedo R.
      • Martínez V.
      • Romero I.
      • et al.
      GEIS guidelines for gastrointestinal sarcomas (GIST).
      ] and combination cytotoxic chemotherapy for rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma [
      • Young R.J.
      • Natukunda A.
      • Litière S.
      • Woll P.J.
      • Wardelmann E.
      • van der Graaf W.T.A.
      First-line anthracycline-based chemotherapy for angiosarcoma and other soft tissue sarcoma subtypes: Pooled analysis of eleven european organisation for research and treatment of cancer soft tissue and bone sarcoma group trials.
      ]. The majority of metastatic sarcomas are restricted to using chemotherapy (typically doxorubicin and/or ifosphamide), which remains of limited and controversial value [
      • Graham D.S.
      • Onyshchenko M.
      • Eckardt M.A.
      • DiPardo B.J.
      • Venigalla S.
      • Nelson S.D.
      • et al.
      Low rates of chemotherapy use for primary, high-grade soft tissue sarcoma: a national cancer database analysis.
      ].
      Historically most sarcomas have been viewed as “immune cold” tumors, despite research beginning in the 1800s that documented anecdotally-successful treatment through infection-induced antitumor immune responses [
      • Coley II., W.B.
      Contribution to the knowledge of sarcoma.
      ,
      • Graeber C.
      The breakthrough: immunotherapy and the race to cure cancer.
      ]. More recently, it has become recognized that at the level of DNA structure, sarcomas can be broadly categorized into two main groups: (1) sarcomas with simple and specific chromosomal changes (often translocations) and a low mutational burden, and (2) karyotypically-complex sarcomas with numerous copy number aberrations and moderate mutational burdens [
      • Dancsok A.R.
      • Asleh-Aburaya K.
      • Nielsen T.O.
      Advances in sarcoma diagnostics and treatment.
      ,
      • Taylor B.S.
      • Barretina J.
      • Maki R.G.
      • Antonescu C.R.
      • Singer S.
      • Ladanyi M.
      Advances in sarcoma genomics and new therapeutic targets.
      ]. Common translocation-associated subtypes include synovial sarcoma, myxoid liposarcoma, Ewing sarcoma, alveolar soft-part sarcoma (ASPS), and alveolar rhabdomyosarcoma, while copy number-driven subtypes include undifferentiated pleomorphic sarcoma (UPS), dedifferentiated liposarcoma (DDLPS), malignant peripheral nerve sheath tumor (MPNST) and leiomyosarcoma [
      • Taylor B.S.
      • Barretina J.
      • Maki R.G.
      • Antonescu C.R.
      • Singer S.
      • Ladanyi M.
      Advances in sarcoma genomics and new therapeutic targets.
      ,
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. The biologic features of these two groups has suggested particular potential for immunotherapy in the copy number-driven subtypes [
      • Lim J.
      • Poulin N.M.
      • Nielsen T.O.
      New strategies in sarcoma: linking genomic and immunotherapy approaches to molecular subtype.
      ,
      • Abeshouse A.
      • Adebamowo C.
      • Adebamowo S.N.
      • Akbani R.
      • Akeredolu T.
      • Ally A.
      • et al.
      Comprehensive and integrated genomic characterization of adult soft tissue sarcomas.
      ].
      Existing cancer immunotherapy modalities include chimeric antigen receptor T-cell therapy, oncolytic viruses, cancer vaccines, and immune checkpoint inhibitors (ICI). In general, while pre-clinical data suggest there are some responses of sarcomas to immunotherapy, compared to cancers such as melanoma and non-small cell lung cancer [
      • Murciano-Goroff Y.R.
      • Warner A.B.
      • Wolchok J.D.
      The future of cancer immunotherapy: microenvironment-targeting combinations.
      ], clinical results in sarcoma have not shown dramatic improvements and trials have been slower in development. The positive, albeit few responses to immunotherapy, among patients with sarcoma suggest that more effort should be used to identify which patients are most likely to respond.
      Immune biomarkers have been applied to many cancers to inform prognosis, and more importantly to predict response to immunotherapy. PD-L1 tumor expression has been used in non-small cell lung cancer [
      • Mok T.S.K.
      • Wu Y.
      • Kudaba I.
      • Kowalski D.M.
      • Cho B.C.
      • Turna H.Z.
      • et al.
      Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial.
      ], breast cancer [
      • Schmid P.
      • Adams S.
      • Rugo H.S.
      • Schneeweiss A.
      • Barrios C.H.
      • Iwata H.
      • et al.
      Atezolizumab and nab-paclitaxel in advanced triple-negative breast cancer.
      ] and gastric cancer [
      • Fuchs C.S.
      • Doi T.
      • Jang R.W.
      • Muro K.
      • Satoh T.
      • Machado M.
      • et al.
      Safety and efficacy of pembrolizumab monotherapy in patients With previously treated advanced gastric and gastroesophageal junction cancer: Phase 2 clinical KEYNOTE-059 Trial.
      ] to select for immunotherapy treatment, and mismatch repair deficiency is an FDA-approved criterion for using ICI to treat any solid tumor [
      • Prasad V.
      • Kaestner V.
      • Mailankody S.
      Cancer drugs approved based on biomarkers and not tumor type—FDA approval of pembrolizumab for mismatch repair-deficient solid cancers.
      ]. Multiple studies have attempted to identify the immune biomarkers expressed in sarcomas that are relevant to clinical management. This review will summarize recent advances in knowledge of sarcoma immune biomarker expression, new technologies for their investigation, and ICI clinical trials focussing on sarcoma subtypes for which effective targeted therapies do not yet exist. Immune augmentation strategies like CAR-T cell therapy and cancer vaccines will not be covered in this review, as others have recently reviewed the clinical development of such modalities in sarcoma [
      • Ayodele O.
      • Abdul Razak A.R.
      Immunotherapy in soft-tissue sarcoma.
      ,
      • Thanindratarn P.
      • Dean D.C.
      • Nelson S.D.
      • Hornicek F.J.
      • Duan Z.
      Chimeric antigen receptor T (CAR-T) cell immunotherapy for sarcomas: From mechanisms to potential clinical applications.
      ,
      • Zuo W.
      • Zhao L.
      Recent advances and application of PD-1 blockade in sarcoma.
      ,
      • Pollack S.M.
      • Ingham M.
      • Spraker M.B.
      • Schwartz G.K.
      Emerging targeted and immune-based therapies in sarcoma.
      ].

      Markers of infiltration

       T cell infiltration: CD3, CD4, CD8, FOXP3

      Tumor infiltrating lymphocytes (TILs) have become widely studied given their vital role in antitumor immunity. In numerous carcinomas [
      • Jochems C.
      • Schlom J.
      Tumor-infiltrating immune cells and prognosis: the potential link between conventional cancer therapy and immunity.
      ], increased CD8+ cytotoxic T cell [
      • Guidoboni M.
      • Gafà R.
      • Viel A.
      • Doglioni C.
      • Russo A.
      • Santini A.
      • et al.
      Microsatellite instability and high content of activated cytotoxic lymphocytes identify colon cancer patients with a favorable prognosis.
      ,
      • Sato E.
      • Olson S.H.
      • Ahn J.
      • Bundy B.
      • Nishikawa H.
      • Qian F.
      • et al.
      Intraepithelial CD8 super(+) tumor-infiltrating lymphocytes and a high CD8 super(+)/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer.
      ] and CD4+ T helper cell infiltrations [
      • Gu-Trantien C.
      • Loi S.
      • Garaud S.
      • Equeter C.
      • Libin M.
      • de Wind A.
      • et al.
      CD4+ follicular helper T cell infiltration predicts breast cancer survival.
      ,
      • Badoual C.
      • Hans S.
      • Rodriguez J.
      • Peyrard S.
      • Klein C.
      • Agueznay N.E.H.
      • et al.
      Prognostic value of tumor-infiltrating CD4 super(+) T-Cell subpopulations in head and neck cancers.
      ] are associated with improved survival and response to therapy due to their roles in an immune active environment (Fig. 1). On the other hand, an increase in FOXP3+ regulatory T cells may reflect an immunosuppressed environment, or the resolution of a robust anti-tumor response [
      • Jochems C.
      • Schlom J.
      Tumor-infiltrating immune cells and prognosis: the potential link between conventional cancer therapy and immunity.
      ,
      • Martin F.
      • Ladoire S.
      • Mignot G.
      • Apetoh L.
      • Ghiringhelli F.
      Human FOXP3 and cancer.
      ].
      Figure thumbnail gr1
      Fig. 1Immune cells in the tumor environment can exhibit anti-tumor response or have immune-suppressive activity. Dendritic cells activate CD8+ cytotoxic T cells through CD8/ MHC I/ T cell receptor (TCR or CD3) complex and helper T cells through the CD4/ MHC II/ TCR complex. Helper T cells interact with and activate CD20+ B cells which induces plasma cell differentiation and antibody class switching. Macrophages can be pro-inflammatory and anti-tumor CD68+ “M1-like”, or anti-inflammatory and pro-tumor CD163+ M2 macrophages which make up most of the tumor-associated macrophages (TAM). In addition to M2 TAMs, FOXP3+ Regulatory T cells are also highly immunosuppressive. Immune checkpoints such as PD-1, CTLA-4, TIM-3, and LAG-3 can be found on the surface of a variety of immune cells. The PD-1/PD-L1 axis and CD47/SIRPα axis are two immune checkpoint pathways that interact with tumor cells, and can lead to an immunosuppressive response if activated.
      Studies characterizing T cell infiltrates in bone sarcomas have been published [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,
      • Fritzsching B.
      • Fellenberg J.
      • Moskovszky L.
      • Sápi Z.
      • Krenacs T.
      • Machado I.
      • et al.
      CD8+/FOXP3+-ratio in osteosarcoma microenvironment separates survivors from non-survivors: a multicenter validated retrospective study.
      ,
      • Brinkrolf P.
      • Landmeier S.
      • Altvater B.
      • Chen C.
      • Pscherer S.
      • Rosemann A.
      • et al.
      A high proportion of bone marrow T cells with regulatory phenotype (CD4+CD25hiFoxP3+) in Ewing sarcoma patients is associated with metastatic disease.
      ], with more recent studies focussing on soft-tissue sarcomas. A majority of studies find CD8+ T cells to be more abundant [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ,
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ,
      • Tsagozis P.
      • Augsten M.
      • Zhang Y.
      • Li T.
      • Hesla A.
      • Bergh J.
      • et al.
      An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.
      ,
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ] relative to FOXP3+  T regulatory cells [
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ,
      • Tsagozis P.
      • Augsten M.
      • Zhang Y.
      • Li T.
      • Hesla A.
      • Bergh J.
      • et al.
      An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.
      ,
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ,
      • Shanes E.
      • Friedman L.
      • Mills A.
      PD-L1 Expression and Tumor-infiltrating Lymphocytes in Uterine Smooth Muscle Tumors: Implications for Immunotherapy.
      ,
      • Kather J.N.
      • Hörner C.
      • Weis C.
      • Aung T.
      • Vokuhl C.
      • Weiss C.
      • et al.
      CD163+ immune cell infiltrates and presence of CD54+ microvessels are prognostic markers for patients with embryonal rhabdomyosarcoma.
      ]. When divided based on karyotype complexity, copy number-driven sarcomas (DDLPS [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ], leiomyosarcoma [
      • Shanes E.
      • Friedman L.
      • Mills A.
      PD-L1 Expression and Tumor-infiltrating Lymphocytes in Uterine Smooth Muscle Tumors: Implications for Immunotherapy.
      ,
      • Cohen J.E.
      • Eleyan F.
      • Zick A.
      • Peretz T.
      • Katz D.
      Intratumoral immune-biomarkers and mismatch repair status in leiyomyosarcoma -potential predictive markers for adjuvant treatment: a pilot study.
      ], UPS, and myxofibrosarcoma [
      • Wunder J.S.
      • Lee M.J.
      • Nam J.
      • Lau B.Y.
      • Dickson B.C.
      • Pinnaduwage D.
      • et al.
      Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation.
      ]) had higher T cell infiltration than translocation-associated subtypes [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. In rhabdomyosarcomas, the copy number- and mutation-driven embryonal subtype has more CD3+ and CD8+ T cell infiltration than its translocation-associated counterpart, alveolar rhabdomyosarcoma [
      • Kather J.N.
      • Hörner C.
      • Weis C.
      • Aung T.
      • Vokuhl C.
      • Weiss C.
      • et al.
      CD163+ immune cell infiltrates and presence of CD54+ microvessels are prognostic markers for patients with embryonal rhabdomyosarcoma.
      ].
      However, increased T cell infiltration among karyotypically complex subtypes is inconsistent across studies, with one publication reporting almost no T cells in leiomyosarcomas [
      • Wunder J.S.
      • Lee M.J.
      • Nam J.
      • Lau B.Y.
      • Dickson B.C.
      • Pinnaduwage D.
      • et al.
      Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation.
      ]. CD8+  infiltration was only found in 57% (8/14) of pleomorphic dermal sarcoma specimens [
      • Klein S.
      • Mauch C.
      • Wagener-Ryczek S.
      • Schoemmel M.
      • Buettner R.
      • Quaas A.
      • et al.
      Immune-phenotyping of pleomorphic dermal sarcomas suggests this entity as a potential candidate for immunotherapy.
      ], lower than what might be expected in a pleomorphic subtype. Studies on synovial sarcoma – a translocation-associated subtype – also lack consensus, with one reporting CD8+  T cell infiltration in 45% (10/22) of cases [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ], while another documented the presence of CD8+  infiltrates in 97% (35/36) of cases [
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ]. This large apparent discrepancy however is easily explained by different methodologies and definitions of what constitutes a positive case, as the former study was based on 1–2 mm tissue microarray cores with a cutoff of ≥10 CD8+  cells [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ] whereas the latter assessed hot spot areas identified on whole sections and employed a cutoff of ≥1 CD8+  cell[
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ]). Immune infiltrates can only be reliably compared among sarcoma types when they are described within the same study employing the same definitions of positive; tissue biomarker publications ideally should report results per mm2 of examined tissue to facilitate comparisons between studies[
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ].
      In retroperitoneal liposarcomas [
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ], soft-tissue sarcomas [
      • Nafia I.
      • Toulmonde M.
      • Bortolotto D.
      • Chaibi A.
      • Bodet D.
      • Rey C.
      • et al.
      IDO targeting in sarcoma: biological and clinical implications.
      ] and a small uterine leiomyosarcoma cohort [
      • Shanes E.
      • Friedman L.
      • Mills A.
      PD-L1 Expression and Tumor-infiltrating Lymphocytes in Uterine Smooth Muscle Tumors: Implications for Immunotherapy.
      ] there were no significant prognostic associations with T cell infiltrates (Table 1). However, studies on individual subtypes suggest that CD8+  and FOXP3+  infiltrates may be prognostic. Newly diagnosed soft-tissue sarcomas have higher numbers of CD8+  T cells than recurrent tumors, and subsequent decreases in CD8+  T cell count indicate poor survival among patients with multiple recurrences [
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ]. In primary UPS, high densities of CD8+  and CD3+  TILs are independent prognostic factors for favourable survival [
      • Boxberg M.
      • Steiger K.
      • Lenze U.
      • Rechl H.
      • von Eisenhart-Rothe R.
      • Wörtler K.
      • et al.
      PD-L1 and PD-1 and characterization of tumor-infiltrating lymphocytes in high grade sarcomas of soft tissue - prognostic implications and rationale for immunotherapy.
      ]. In synovial sarcoma, higher continuous counts of CD8+  T cell infiltration have been associated with favourable overall survival in one cohort [
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ], but worse metastasis-free survival in another cohort [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ]. In DDLPS and MPNST, patients with CD8+  and FOXP3+  concurrent infiltration had better overall survival compared to CD8+ only [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. In contrast, concurrent infiltration of CD8+  and FOXP3+ T cells in myxoid liposarcoma was correlated with worse overall survival than single CD8+ infiltration [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ].
      Table 1Summary of IHC-based immune biomarker studies in sarcoma. Boxes represent prognostic effect of corresponding biomarker: positive (solid green), negative (striped red), no association (white).
      *Immune biomarkers associated with prognosis as single markers and as co-infiltrates.
      **Immune biomarkers associated with prognosis only as co-infiltrates.
      Thus to date, publications on the T cell infiltration in specific histologic subtypes are difficult to compare due to the varying definitions of what constitutes a positive case [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ] and the different tissue formats and areas examined in the analysis [
      • Shanes E.
      • Friedman L.
      • Mills A.
      PD-L1 Expression and Tumor-infiltrating Lymphocytes in Uterine Smooth Muscle Tumors: Implications for Immunotherapy.
      ,
      • Wunder J.S.
      • Lee M.J.
      • Nam J.
      • Lau B.Y.
      • Dickson B.C.
      • Pinnaduwage D.
      • et al.
      Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation.
      ]. There is a trend for copy-number driven subtypes (DDLPS, MPNST, and UPS) to have positive association between T cell infiltration and prognosis, and translocation-associated subtypes (synovial sarcoma and myxoid liposarcoma) to have mixed results [
      • Taylor B.S.
      • Barretina J.
      • Maki R.G.
      • Antonescu C.R.
      • Singer S.
      • Ladanyi M.
      Advances in sarcoma genomics and new therapeutic targets.
      ]. The studies that reported no prognostic association with T cell infiltration had either merged dissimilar histologic subtypes or very few patients, suggesting subtype-dependent prognostic significance needs to be addressed with larger subtype-specific cohorts.

       B cell infiltration: CD20

      B cells play a crucial role in tumor-related immunity, and have recently garnered increased attention [
      • Sharonov G.V.
      • Serebrovskaya E.O.
      • Yuzhakova D.V.
      • Britanova O.V.
      • Chudakov D.M.
      B cells, plasma cells and antibody repertoires in the tumour microenvironment.
      ,
      • Wouters M.C.A.
      • Nelson B.H.
      Prognostic significance of tumor-infiltrating B cells and plasma cells in human cancer.
      ]. B cells contribute to tumor clearance through cancer-targeting antibodies that mediate antibody-dependent cell cytotoxicity and phagocytosis, and form immune complexes [
      • Sharonov G.V.
      • Serebrovskaya E.O.
      • Yuzhakova D.V.
      • Britanova O.V.
      • Chudakov D.M.
      B cells, plasma cells and antibody repertoires in the tumour microenvironment.
      ]. One of the earliest studies applying the B-cell biomarker CD20 in soft-tissue sarcoma specimens demonstrated B cell infiltration to associate with improved prognosis (Table 1) [
      • Sorbye S.W.
      • Kilvaer T.
      • Valkov A.
      • Donnem T.
      • Smeland E.
      • Al-Shibli K.
      • et al.
      Prognostic impact of lymphocytes in soft tissue sarcomas.
      ]. Correspondingly, a smaller soft-tissue cohort found B cells to associate with improved metastasis-free and overall survival, with results validated in The Cancer Genome Atlas cohort based on expression levels of MS4A1 (encoding for CD20) [
      • Tsagozis P.
      • Augsten M.
      • Zhang Y.
      • Li T.
      • Hesla A.
      • Bergh J.
      • et al.
      An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.
      ].
      However, it should be noted that most studies using immunohistochemistry (IHC) have reported sparse staining of CD20 in sarcomas, and no prognostic association [
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ,
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ]. Few CD20+  B cells were observed in soft-tissue sarcomas [
      • Tsagozis P.
      • Augsten M.
      • Zhang Y.
      • Li T.
      • Hesla A.
      • Bergh J.
      • et al.
      An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.
      ], leiomyosarcoma [
      • Lee A.T.J.
      • Chew W.
      • Wilding C.P.
      • Guljar N.
      • Smith M.J.
      • Strauss D.C.
      • et al.
      The adequacy of tissue microarrays in the assessment of inter- and intra-tumoural heterogeneity of infiltrating lymphocyte burden in leiomyosarcoma.
      ], or pleomorphic dermal sarcoma [
      • Klein S.
      • Mauch C.
      • Wagener-Ryczek S.
      • Schoemmel M.
      • Buettner R.
      • Quaas A.
      • et al.
      Immune-phenotyping of pleomorphic dermal sarcomas suggests this entity as a potential candidate for immunotherapy.
      ]. In retroperitoneal liposarcomas (well-differentiated, dedifferentiated, myxoid/round cell and pleomorphic liposarcoma), CD20+  B cells had the lowest frequency among all analyzed lymphocyte biomarkers (including CD8, CD4 and FOXP3) [
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ]. CD20+  B cells in embryonal and alveolar rhabdomyosarcoma were only observed in peritumoral regions, with no intratumoral infiltration [
      • Kather J.N.
      • Hörner C.
      • Weis C.
      • Aung T.
      • Vokuhl C.
      • Weiss C.
      • et al.
      CD163+ immune cell infiltrates and presence of CD54+ microvessels are prognostic markers for patients with embryonal rhabdomyosarcoma.
      ].
      More recently, a study published in Nature using randomized clinical trial materials highlights the potential significance of B cells in sarcoma [
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ]. Using the microenvironment cell populations-counter method extracted from gene expression data, the authors found that a specific immune-high group comprising 18% of tested sarcomas has high expression of B cell lineage signatures [
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ]. This B cell signature was associated with improved survival across sarcoma subtypes and with the highest objective response rate to pembrolizumab [
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ]. Protein expression of B cell biomarkers was validated using multispectral fluorescence, showing CD20+  cells existing in high densities in tumors with tertiary lymphoid structures [
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ].
      This positive correlation between CD20+  B cells and tertiary lymphoid structures in sarcomas, confirmed by another study [
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ], may explain seemingly contradicting observations in other studies. B cells are mostly found in high densities in tumors with tertiary lymphoid structures, which are only identified in 12–16% of sarcoma tumor specimens [
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ,
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ]; thus, small studies will lack the power to demonstrate significant associations with other patient data [
      • Kather J.N.
      • Hörner C.
      • Weis C.
      • Aung T.
      • Vokuhl C.
      • Weiss C.
      • et al.
      CD163+ immune cell infiltrates and presence of CD54+ microvessels are prognostic markers for patients with embryonal rhabdomyosarcoma.
      ,
      • Klein S.
      • Mauch C.
      • Wagener-Ryczek S.
      • Schoemmel M.
      • Buettner R.
      • Quaas A.
      • et al.
      Immune-phenotyping of pleomorphic dermal sarcomas suggests this entity as a potential candidate for immunotherapy.
      ,
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ]. Therefore, while B cell infiltration in sarcoma is mostly rare, the presence of B cells may be an important biomarker for an antitumor immune response amenable to reactivation by checkpoint inhibitor therapy.

       Macrophage infiltration: CD163, CD68, CD204

      A part of the innate immune system, macrophages are usually the most abundant leukocyte in tumors [
      • Poh A.R.
      • Ernst M.
      Targeting macrophages in cancer: from bench to bedside.
      ]. Traditionally, macrophages are classified as pro-inflammatory and anti-tumor M1 macrophages or anti-inflammatory and pro-tumor M2 macrophages [
      • Yang M.
      • McKay D.
      • Pollard J.W.
      • Lewis C.E.
      Diverse functions of macrophages in different tumor microenvironments.
      ]. Tumor-associated macrophages (TAMs) mostly display an M2 phenotype, and are associated with poor survival in carcinomas of the lung, breast, and pancreas [
      • Poh A.R.
      • Ernst M.
      Targeting macrophages in cancer: from bench to bedside.
      ,
      • Yang M.
      • McKay D.
      • Pollard J.W.
      • Lewis C.E.
      Diverse functions of macrophages in different tumor microenvironments.
      ]. Phenotypes can be partially distinguished by IHC, as CD163 preferentially stains M2-like macrophages, whereas CD68 detects both but is often more strongly expressed in M1 macrophages [
      • Poh A.R.
      • Ernst M.
      Targeting macrophages in cancer: from bench to bedside.
      ,
      • Yang M.
      • McKay D.
      • Pollard J.W.
      • Lewis C.E.
      Diverse functions of macrophages in different tumor microenvironments.
      ].
      CD68+  and CD163+ macrophages are consistently detected across all sarcomas [
      • Tsagozis P.
      • Augsten M.
      • Zhang Y.
      • Li T.
      • Hesla A.
      • Bergh J.
      • et al.
      An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.
      ,
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ,
      • Wunder J.S.
      • Lee M.J.
      • Nam J.
      • Lau B.Y.
      • Dickson B.C.
      • Pinnaduwage D.
      • et al.
      Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation.
      ,
      • Klein S.
      • Mauch C.
      • Wagener-Ryczek S.
      • Schoemmel M.
      • Buettner R.
      • Quaas A.
      • et al.
      Immune-phenotyping of pleomorphic dermal sarcomas suggests this entity as a potential candidate for immunotherapy.
      ,
      • Lee C.
      • Espinosa I.
      • Vrijaldenhoven S.
      • Subramanian S.
      • Montgomery K.D.
      • Zhu S.
      • et al.
      Prognostic significance of macrophage infiltration in leiomyosarcomas.
      ]. Like other cancer types, sarcomas contain more macrophages than lymphocytes [
      • Tsagozis P.
      • Augsten M.
      • Zhang Y.
      • Li T.
      • Hesla A.
      • Bergh J.
      • et al.
      An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.
      ,
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ,
      • Dancsok A.R.
      • Gao D.
      • Lee A.F.
      • Steigen S.E.
      • Blay J.
      • Thomas D.M.
      • et al.
      Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas.
      ]. Similar to observations on T cell infiltration, there are more macrophages found among copy number-driven sarcomas compared to translocation-associated sarcomas, which display macrophage density levels comparable with benign mesenchymal tumors [
      • Dancsok A.R.
      • Gao D.
      • Lee A.F.
      • Steigen S.E.
      • Blay J.
      • Thomas D.M.
      • et al.
      Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas.
      ]. More specifically, UPS, DDLPS, myxofibrosarcoma, and leiomyosarcoma have the highest macrophage counts [
      • Dancsok A.R.
      • Gao D.
      • Lee A.F.
      • Steigen S.E.
      • Blay J.
      • Thomas D.M.
      • et al.
      Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas.
      ].
      Sarcoma TAMs have a predominantly M2-polarized phenotype [
      • Tsagozis P.
      • Augsten M.
      • Zhang Y.
      • Li T.
      • Hesla A.
      • Bergh J.
      • et al.
      An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.
      ,
      • Dancsok A.R.
      • Gao D.
      • Lee A.F.
      • Steigen S.E.
      • Blay J.
      • Thomas D.M.
      • et al.
      Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas.
      ] much like most carcinomas [
      • Poh A.R.
      • Ernst M.
      Targeting macrophages in cancer: from bench to bedside.
      ]. In particular, UPS has the highest M2 to total macrophage ratio compared to GIST and leiomyosarcoma [
      • Toulmonde M.
      • Penel N.
      • Adam J.
      • Chevreau C.
      • Blay J.
      • Le Cesne A.
      • et al.
      Use of PD-1 targeting, macrophage infiltration, and IDO pathway activation in sarcomas: A phase 2 clinical trial.
      ]. In keeping with the protumor role of M2 macrophages, low levels of CD163+  macrophages are associated with favourable survival in synovial sarcoma [
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ] and solitary fibrous tumors [
      • Dancsok A.R.
      • Gao D.
      • Lee A.F.
      • Steigen S.E.
      • Blay J.
      • Thomas D.M.
      • et al.
      Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas.
      ] (Table 1). However, the opposite effect was observed in embryonal rhabdomyosarcoma [
      • Kather J.N.
      • Hörner C.
      • Weis C.
      • Aung T.
      • Vokuhl C.
      • Weiss C.
      • et al.
      CD163+ immune cell infiltrates and presence of CD54+ microvessels are prognostic markers for patients with embryonal rhabdomyosarcoma.
      ] and DDLPS [
      • Dancsok A.R.
      • Gao D.
      • Lee A.F.
      • Steigen S.E.
      • Blay J.
      • Thomas D.M.
      • et al.
      Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas.
      ], in which increased CD163+  macrophages were associated with improved survival. Despite research attempting to dichotomize macrophage phenotypes, the inconsistent correlations with survival suggest that macrophage polarization is not a definitive characterization [
      • Nahrendorf M.
      • Swirski F.K.
      Abandoning M1/M2 for a network model of macrophage function.
      ], and further investigation is needed.
      In addition to phenotype markers, macrophages also express SIRPα which can recognize CD47 on host tumor cells to activate a “don’t eat me” signal [
      • Weiskopf K.
      Cancer immunotherapy targeting the CD47/SIRPα axis.
      ]. Only one sarcoma study to date has investigated SIRPα, which found DDLPS, angiosarcoma, chordoma, and well-differentiated liposarcoma to have the highest levels of SIRPα+ macrophages [
      • Dancsok A.R.
      • Gao D.
      • Lee A.F.
      • Steigen S.E.
      • Blay J.
      • Thomas D.M.
      • et al.
      Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas.
      ]. The presence of SIRPα+ macrophages is associated with worse prognosis in synovial sarcoma and myxofibrosarcoma [
      • Dancsok A.R.
      • Gao D.
      • Lee A.F.
      • Steigen S.E.
      • Blay J.
      • Thomas D.M.
      • et al.
      Tumor-associated macrophages and macrophage-related immune checkpoint expression in sarcomas.
      ]. Given the expanding development of CD47/SIRPα axis-based immunotherapy [
      • Weiskopf K.
      Cancer immunotherapy targeting the CD47/SIRPα axis.
      ], future studies should investigate SIRPα as potential predictive biomarker.

      Immune checkpoints

       PD-1

      The programmed cell death-1 (PD-1) checkpoint pathway has been one of the most researched targets in immunotherapy. PD-1 is expressed mostly on immune cells such as activated T cells, B cells, natural killer cells, activated monocytes, and dendritic cells [
      • Keir M.E.
      • Butte M.J.
      • Freeman G.J.
      • Sharpe A.H.
      PD-1 and its ligands in tolerance and Immunity.
      ,
      • Sharpe A.H.
      • Pauken K.E.
      The diverse functions of the PD1 inhibitory pathway.
      ] (Fig. 1). Tumor cells can express the ligand PD-L1 to interact with PD-1 and thereby inhibit immune cell activity, resulting in an effective immune-evasion strategy [
      • Sharpe A.H.
      • Pauken K.E.
      The diverse functions of the PD1 inhibitory pathway.
      ].
      Across three sarcoma subtypes (DDLPS, leiomyosarcoma, UPS), tumors with high immunogenic gene signatures have high expression levels for the genes encoding both PD-1 and PD-L1 (PDCD1 and CD274, respectively) [
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ]. PD-1 expression as detected via IHC is also significantly higher in copy-number driven than translocation-associated sarcomas [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. Independent cohorts of specific subtypes have yielded supportive findings, as more than half of pleomorphic dermal sarcoma specimens have positive PD-1 expression on immune cells [
      • Klein S.
      • Mauch C.
      • Wagener-Ryczek S.
      • Schoemmel M.
      • Buettner R.
      • Quaas A.
      • et al.
      Immune-phenotyping of pleomorphic dermal sarcomas suggests this entity as a potential candidate for immunotherapy.
      ], whereas few PD-1+  TILs were observed in synovial sarcoma [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ] and none in Ewing sarcoma (both of which are translocation-associated) [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,

      Spurny C, Kailayangiri S, Jamitzky S, Altvater B, Wardelmann E, Dirksen U, et al. Programmed cell death ligand 1 (PD‐L1) expression is not a predominant feature in Ewing sarcomas. Pediatric Blood Cancer 2018;65:e26719,n/a.

      ]. PD-1 expression has also been observed on sarcoma cells in select studies, although the significance of this remains unclear [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ,

      Spurny C, Kailayangiri S, Jamitzky S, Altvater B, Wardelmann E, Dirksen U, et al. Programmed cell death ligand 1 (PD‐L1) expression is not a predominant feature in Ewing sarcomas. Pediatric Blood Cancer 2018;65:e26719,n/a.

      ]. Two recent studies [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ,
      • Zheng B.
      • Ren T.
      • Huang Y.
      • Sun K.
      • Wang S.
      • Bao X.
      • et al.
      PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.
      ] agree with earlier results [
      • Kim J.R.
      • Moon Y.J.
      • Kwon K.S.
      • Bae J.S.
      • Wagle S.
      • Kim K.M.
      • et al.
      Tumor infiltrating PD1-positive lymphocytes and the expression of PD-L1 predict poor prognosis of soft tissue sarcomas.
      ] associating PD-1 expression with worse survival, while other authors did not observe any significant prognostic value (Table 1) [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ,
      • Boxberg M.
      • Steiger K.
      • Lenze U.
      • Rechl H.
      • von Eisenhart-Rothe R.
      • Wörtler K.
      • et al.
      PD-L1 and PD-1 and characterization of tumor-infiltrating lymphocytes in high grade sarcomas of soft tissue - prognostic implications and rationale for immunotherapy.
      ]. Consequently, efforts have also been made to address other components of the PD-1/PD-L1 pathway in sarcomas.

       PD-L1

      PD-L1 is expressed on stromal, malignant, and immune cells, and has been shown in other cancers to be predictive for checkpoint inhibitor therapy response and patient survival [
      • Teng F.
      • Meng X.
      • Kong L.
      • Yu J.
      Progress and challenges of predictive biomarkers of anti PD-1/PD-L1 immunotherapy: A systematic review.
      ]. Indeed, PD-L1 expression is included as an FDA-approved companion diagnostic for PD-1/PD-L1 blockade therapy in lung cancer [
      • D'Incecco A.
      • Andreozzi M.
      • Ludovini V.
      • Rossi E.
      • Capodanno A.
      • Landi L.
      • et al.
      PD-1 and PD-L1 expression in molecularly selected non-small-cell lung cancer patients.
      ]. In sarcoma, past notable studies have shown PD-L1 copy number gain [
      • Budczies J.
      • Mechtersheimer G.
      • Denkert C.
      • Klauschen F.
      • Mughal S.S.
      • Chudasama P.
      • et al.
      PD-L1 (CD274) copy number gain, expression, and immune cell infiltration as candidate predictors for response to immune checkpoint inhibitors in soft-tissue sarcoma.
      ] and tumoral PD-L1 expression to associate with poor prognosis [
      • Kim J.R.
      • Moon Y.J.
      • Kwon K.S.
      • Bae J.S.
      • Wagle S.
      • Kim K.M.
      • et al.
      Tumor infiltrating PD1-positive lymphocytes and the expression of PD-L1 predict poor prognosis of soft tissue sarcomas.
      ,
      • Kim C.
      • Kim E.K.
      • Jung H.
      • Chon H.J.
      • Han J.W.
      • Shin K.
      • et al.
      Prognostic implications of PD-L1 expression in patients with soft tissue sarcoma.
      ].
      More recently, a study of 203 sarcoma specimens identified PD-L1 expression on malignant and immune cells in 23% of all cases [
      • Nafia I.
      • Toulmonde M.
      • Bortolotto D.
      • Chaibi A.
      • Bodet D.
      • Rey C.
      • et al.
      IDO targeting in sarcoma: biological and clinical implications.
      ], consistent with another large sarcoma cohort study [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. Reports from IHC studies suggest high heterogeneity between histologic subtypes [
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ]. Ewing sarcoma [

      Spurny C, Kailayangiri S, Jamitzky S, Altvater B, Wardelmann E, Dirksen U, et al. Programmed cell death ligand 1 (PD‐L1) expression is not a predominant feature in Ewing sarcomas. Pediatric Blood Cancer 2018;65:e26719,n/a.

      ], chondrosarcoma [
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ], pediatric [
      • Bertolini G.
      • Bergamaschi L.
      • Ferrari A.
      • Renne S.L.
      • Collini P.
      • Gardelli C.
      • et al.
      PD-L1 assessment in pediatric rhabdomyosarcoma: a pilot study.
      ] and adult [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,
      • Kather J.N.
      • Hörner C.
      • Weis C.
      • Aung T.
      • Vokuhl C.
      • Weiss C.
      • et al.
      CD163+ immune cell infiltrates and presence of CD54+ microvessels are prognostic markers for patients with embryonal rhabdomyosarcoma.
      ] alveolar and embryonal rhabdomyosarcoma, and synovial sarcoma [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,
      • Oike N.
      • Kawashima H.
      • Ogose A.
      • Hotta T.
      • Hatano H.
      • Ariizumi T.
      • et al.
      Prognostic impact of the tumor immune microenvironment in synovial sarcoma.
      ,
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ,
      • Zheng B.
      • Ren T.
      • Huang Y.
      • Sun K.
      • Wang S.
      • Bao X.
      • et al.
      PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.
      ,

      Wedekind MF, Haworth KB, Arnold M, Stanek JR, Lee D, Cripe TP. Immune profiles of desmoplastic small round cell tumor and synovial sarcoma suggest different immunotherapeutic susceptibility upfront compared to relapse specimens. Pediatric Blood Cancer 2018;65:e27313,n/a.

      ] show little to no PD-L1 expression on malignant cells. Studies on DDLPS and pleomorphic liposarcomas [
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ,
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ], myxofibrosarcoma [
      • Wunder J.S.
      • Lee M.J.
      • Nam J.
      • Lau B.Y.
      • Dickson B.C.
      • Pinnaduwage D.
      • et al.
      Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation.
      ,
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ], fibrosarcoma [
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ], desmoplastic small round cell tumor [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,

      Wedekind MF, Haworth KB, Arnold M, Stanek JR, Lee D, Cripe TP. Immune profiles of desmoplastic small round cell tumor and synovial sarcoma suggest different immunotherapeutic susceptibility upfront compared to relapse specimens. Pediatric Blood Cancer 2018;65:e27313,n/a.

      ], osteosarcoma [
      • Wunder J.S.
      • Lee M.J.
      • Nam J.
      • Lau B.Y.
      • Dickson B.C.
      • Pinnaduwage D.
      • et al.
      Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation.
      ,
      • Zheng B.
      • Ren T.
      • Huang Y.
      • Sun K.
      • Wang S.
      • Bao X.
      • et al.
      PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.
      ,
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ,
      • Le Cesne A.
      • Marec-Berard P.
      • Blay J.
      • Gaspar N.
      • Bertucci F.
      • Penel N.
      • et al.
      Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study.
      ], UPS [
      • Wunder J.S.
      • Lee M.J.
      • Nam J.
      • Lau B.Y.
      • Dickson B.C.
      • Pinnaduwage D.
      • et al.
      Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation.
      ,
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ], and pleomorphic dermal sarcoma [
      • Klein S.
      • Mauch C.
      • Wagener-Ryczek S.
      • Schoemmel M.
      • Buettner R.
      • Quaas A.
      • et al.
      Immune-phenotyping of pleomorphic dermal sarcomas suggests this entity as a potential candidate for immunotherapy.
      ] report less than half the cases show PD-L1 expression on the sarcoma cells. In comparison, PD-L1 was expressed on the neoplastic cells in more than half of leiomyosarcoma [
      • Shanes E.
      • Friedman L.
      • Mills A.
      PD-L1 Expression and Tumor-infiltrating Lymphocytes in Uterine Smooth Muscle Tumors: Implications for Immunotherapy.
      ,
      • Cohen J.E.
      • Eleyan F.
      • Zick A.
      • Peretz T.
      • Katz D.
      Intratumoral immune-biomarkers and mismatch repair status in leiyomyosarcoma -potential predictive markers for adjuvant treatment: a pilot study.
      ,
      • Zheng B.
      • Wang J.
      • Cai W.
      • Lao I.
      • Shi Y.
      • Luo X.
      • et al.
      Changes in the tumor immune microenvironment in resected recurrent soft tissue sarcomas.
      ] and giant cell tumor specimens [
      • Zheng B.
      • Ren T.
      • Huang Y.
      • Sun K.
      • Wang S.
      • Bao X.
      • et al.
      PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.
      ].
      The prognostic value of PD-L1 expression in sarcomas also varies with histologic subtype. PD-L1 expression measured by IHC was not significantly associated with survival in leiomyosarcoma [
      • Shanes E.
      • Friedman L.
      • Mills A.
      PD-L1 Expression and Tumor-infiltrating Lymphocytes in Uterine Smooth Muscle Tumors: Implications for Immunotherapy.
      ] or retroperitoneal liposarcomas that included dedifferentiated, well-differentiated, myxoid and pleomorphic liposarcoma (Table 1) [
      • Yan L.
      • Wang Z.
      • Cui C.
      • Guan X.
      • Dong B.
      • Zhao M.
      • et al.
      Comprehensive immune characterization and T-cell receptor repertoire heterogeneity of retroperitoneal liposarcoma.
      ]. PD-L1 expression correlated with lower IRS grade and better survival in alveolar rhabdomyosarcoma [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ]. In contrast, PD-L1 expression associated with worse survival in the specific entity of DDLPS [
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ]. Curiously, PD-L1 protein [
      • Zheng B.
      • Ren T.
      • Huang Y.
      • Sun K.
      • Wang S.
      • Bao X.
      • et al.
      PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.
      ,
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ] and CD274 gene expression [
      • Wunder J.S.
      • Lee M.J.
      • Nam J.
      • Lau B.Y.
      • Dickson B.C.
      • Pinnaduwage D.
      • et al.
      Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation.
      ] was associated with different effects on survival in osteosarcoma and UPS. Such conflicting results highlight the need for more studies to validate published findings and suggest a nonlinear relationship between PD-L1 gene and protein expression in sarcoma.
      Finally, it should be noted that not only is PD-L1 expressed on malignant cells, it is also expressed on immune cells present in leiomyosarcoma [
      • Shanes E.
      • Friedman L.
      • Mills A.
      PD-L1 Expression and Tumor-infiltrating Lymphocytes in Uterine Smooth Muscle Tumors: Implications for Immunotherapy.
      ], pleomorphic dermal sarcoma [
      • Klein S.
      • Mauch C.
      • Wagener-Ryczek S.
      • Schoemmel M.
      • Buettner R.
      • Quaas A.
      • et al.
      Immune-phenotyping of pleomorphic dermal sarcomas suggests this entity as a potential candidate for immunotherapy.
      ], and pediatric alveolar and embryonal rhabdomyosarcomas [
      • Bertolini G.
      • Bergamaschi L.
      • Ferrari A.
      • Renne S.L.
      • Collini P.
      • Gardelli C.
      • et al.
      PD-L1 assessment in pediatric rhabdomyosarcoma: a pilot study.
      ]. The role of PD-L1 expression on immune cells infiltrating sarcomas has not been extensively explored and may have important implications on prognosis as reported in carcinomas [
      • Kim H.R.
      • Ha S.
      • Hong M.H.
      • Heo S.J.
      • Koh Y.W.
      • Choi E.C.
      • et al.
      PD-L1 expression on immune cells, but not on tumor cells, is a favorable prognostic factor for head and neck cancer patients.
      ].

       Others: IDO, LAG3, TIM3

      In addition to PD-1/PD-L1, other targetable immune checkpoints including CTLA-4, IDO, LAG-3, and TIM-3 offer additional directions for immune-oncology research. As expected, expression of these checkpoints is significantly associated with other immune infiltrates and checkpoint biomarker expression in sarcomas [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. The immune-hot groups of soft-tissue sarcoma identified by immune cell gene signatures display high expression of CTLA4, HAVCR2, and LAG3 encoding CTLA-4, TIM-3, and LAG-3 respectively [
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ]. Likewise, LAG-3 and TIM-3 expression were more common in copy-number driven sarcomas compared to translocation-associated sarcomas [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. In pleomorphic dermal sarcoma, all cases identified as having high CD8 infiltration had LAG-3+ immune cells [
      • Klein S.
      • Mauch C.
      • Wagener-Ryczek S.
      • Schoemmel M.
      • Buettner R.
      • Quaas A.
      • et al.
      Immune-phenotyping of pleomorphic dermal sarcomas suggests this entity as a potential candidate for immunotherapy.
      ]. Similarly, high CD8+  TILs correlated positively with IDO-1 staining in soft-tissue sarcomas [
      • Nafia I.
      • Toulmonde M.
      • Bortolotto D.
      • Chaibi A.
      • Bodet D.
      • Rey C.
      • et al.
      IDO targeting in sarcoma: biological and clinical implications.
      ]. Furthermore, pretreatment specimens from a phase 2 clinical trial showed that two pleomorphic sarcoma subtypes, leiomyosarcoma and UPS, had around 70% cases with IDO-1 expression, while only 29% of other soft-tissue sarcomas met the same criteria for IDO-1 [
      • Toulmonde M.
      • Penel N.
      • Adam J.
      • Chevreau C.
      • Blay J.
      • Le Cesne A.
      • et al.
      Use of PD-1 targeting, macrophage infiltration, and IDO pathway activation in sarcomas: A phase 2 clinical trial.
      ]. Currently, limited evidence reports that novel immune checkpoints LAG-3 and TIM-3 lack independent correlation with survival in sarcomas [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. Nonetheless, given the close relationship between sarcoma immune status and these checkpoints, multiplex approaches to biomarker detection may be required to better understand the role of immune checkpoints in sarcoma.

       Tumor mutation burden and microsatellite instability

      Tumor mutation burden (TMB) and microsatellite instability are two other biomarkers that positively correlate with immunotherapy responses [
      • Goodman A.M.
      • Kato S.
      • Bazhenova L.
      • Patel S.P.
      • Frampton G.M.
      • Miller V.
      • et al.
      Tumor mutational burden as an independent predictor of response to immunotherapy in diverse cancers.
      ,
      • Zhao P.
      • Li L.
      • Jiang X.
      • Li Q.
      Mismatch repair deficiency/microsatellite instability-high as a predictor for anti-PD-1/PD-L1 immunotherapy efficacy.
      ]. Sarcoma studies consistently show TMB is low [
      • Abeshouse A.
      • Adebamowo C.
      • Adebamowo S.N.
      • Akbani R.
      • Akeredolu T.
      • Ally A.
      • et al.
      Comprehensive and integrated genomic characterization of adult soft tissue sarcomas.
      ,
      • Doyle L.A.
      • Nowak J.A.
      • Nathenson M.J.
      • Thornton K.
      • Wagner A.J.
      • Johnson J.M.
      • et al.
      Characteristics of mismatch repair deficiency in sarcomas.
      ,
      • Chalmers Z.R.
      • Connelly C.F.
      • Fabrizio D.
      • Gay L.
      • Ali S.M.
      • Ennis R.
      • et al.
      Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden.
      ] and microsatellites are stable [
      • Cote G.M.
      • He J.
      • Choy E.
      Next-generation sequencing for patients with sarcoma: A single center experience.
      ,
      • Bonneville R.
      • Krook M.A.
      • Kautto E.A.
      • Miya J.
      • Wing M.R.
      • Chen H.
      • et al.
      Landscape of microsatellite instability across 39 cancer types.
      ] across subtypes. Low TMB has been observed in subtype-specific cohorts of alveolar soft-part [
      • Groisberg R.
      • Roszik J.
      • Conley A.P.
      • Lazar A.J.
      • Portal D.E.
      • Hong D.S.
      • et al.
      Genomics, morphoproteomics, and treatment patterns of patients with alveolar soft part sarcoma and response to multiple experimental therapies.
      ], Ewing [
      • Gröbner S.N.
      • Worst B.C.
      • Weischenfeldt J.
      • Buchhalter I.
      • Kleinheinz K.
      • Rudneva V.A.
      • et al.
      The landscape of genomic alterations across childhood cancers.
      ,
      • Agelopoulos K.
      • Richter G.H.S.
      • Schmidt E.
      • Dirksen U.
      • von Heyking K.
      • Moser B.
      • et al.
      Deep sequencing in conjunction with expression and functional analyses reveals activation of FGFR1 in Ewing Sarcoma.
      ], synovial [
      • He M.
      • Abro B.
      • Kaushal M.
      • Chen L.
      • Chen T.
      • Gondim M.
      • et al.
      Tumor mutation burden and checkpoint immunotherapy markers in primary and metastatic synovial sarcoma.
      ], and osteosarcoma [
      • Gröbner S.N.
      • Worst B.C.
      • Weischenfeldt J.
      • Buchhalter I.
      • Kleinheinz K.
      • Rudneva V.A.
      • et al.
      The landscape of genomic alterations across childhood cancers.
      ]. Tumor mutational burden was not associated with immune biomarkers in pleomorphic dermal sarcoma [
      • He M.
      • Abro B.
      • Kaushal M.
      • Chen L.
      • Chen T.
      • Gondim M.
      • et al.
      Tumor mutation burden and checkpoint immunotherapy markers in primary and metastatic synovial sarcoma.
      ,
      • Klein S.
      • Quaas A.
      • Noh K.
      • Cartolano M.
      • Abedpour N.
      • Mauch C.
      • et al.
      Integrative analysis of pleomorphic dermal sarcomas reveals fibroblastic differentiation and susceptibility to immunotherapy.
      ] or survival in synovial sarcoma [
      • He M.
      • Abro B.
      • Kaushal M.
      • Chen L.
      • Chen T.
      • Gondim M.
      • et al.
      Tumor mutation burden and checkpoint immunotherapy markers in primary and metastatic synovial sarcoma.
      ], but negatively correlated with survival in pediatric Ewing sarcoma [
      • Liu K.X.
      • Lamba N.
      • Hwang W.L.
      • Niemierko A.
      • DuBois S.G.
      • Haas-Kogan D.A.
      Risk stratification by somatic mutation burden in Ewing sarcoma.
      ]. Currently there are no prognostic data on microsatellite instability.
      Of note, studies do report select cases with high TMB in angiosarcoma [
      • Painter C.A.
      • Jain E.
      • Tomson B.N.
      • Dunphy M.
      • Stoddard R.E.
      • Thomas B.S.
      • et al.
      The angiosarcoma project: enabling genomic and clinical discoveries in a rare cancer through patient-partnered research.
      ,
      • Campbell B.B.
      • Light N.
      • Fabrizio D.
      • Zatzman M.
      • Fuligni F.
      • de Borja R.
      • et al.
      Comprehensive analysis of hypermutation in human cancer.
      ], malignant peripheral nerve sheath tumor [
      • Campbell B.B.
      • Light N.
      • Fabrizio D.
      • Zatzman M.
      • Fuligni F.
      • de Borja R.
      • et al.
      Comprehensive analysis of hypermutation in human cancer.
      ], and pleomorphic dermal sarcoma [
      • Klein S.
      • Quaas A.
      • Noh K.
      • Cartolano M.
      • Abedpour N.
      • Mauch C.
      • et al.
      Integrative analysis of pleomorphic dermal sarcomas reveals fibroblastic differentiation and susceptibility to immunotherapy.
      ]. Angiosarcoma tumors with high TMB are superficially located and show UV-dominant mutations [
      • Painter C.A.
      • Jain E.
      • Tomson B.N.
      • Dunphy M.
      • Stoddard R.E.
      • Thomas B.S.
      • et al.
      The angiosarcoma project: enabling genomic and clinical discoveries in a rare cancer through patient-partnered research.
      ,
      • Campbell B.B.
      • Light N.
      • Fabrizio D.
      • Zatzman M.
      • Fuligni F.
      • de Borja R.
      • et al.
      Comprehensive analysis of hypermutation in human cancer.
      ] as are seen in melanomas, a tumor type responding to checkpoint inhibitors. Preliminary data from two patients with angiosarcoma demonstrated clinical benefit from pembrolizumab treatment [
      • Painter C.A.
      • Jain E.
      • Tomson B.N.
      • Dunphy M.
      • Stoddard R.E.
      • Thomas B.S.
      • et al.
      The angiosarcoma project: enabling genomic and clinical discoveries in a rare cancer through patient-partnered research.
      ], suggesting this may be a sarcoma worthy of assessment in clinical trials.

       New technologies

      Lymphocyte counts on hematoxylin and eosin stained slides are the simplest and most established method to assess the immune microenvironment in sarcomas. Given the heterogeneity of immune cell composition, even within the category of lymphocytes, more specific approaches are needed to identify immune responses with different implications for prognosis and therapy [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ]. Despite the specificity of conventional IHC, the fundamental limitation of interpreting a complex multicellular reaction by measuring a single biomarker per slide means that more sophisticated approaches are needed to properly characterize the sarcoma tumor immune microenvironment [
      • Taylor C.R.
      • Levenson R.M.
      Quantification of immunohistochemistry—issues concerning methods, utility and semiquantitative assessment II.
      ].
      Multiplex IHC/Immunofluorescence approaches are designed to detect multiple biomarkers on a single tissue section. Such techniques identify biomarker co-expression on individual cells, reveal the spatial distribution of immune, malignant, and stromal cells and reduce consumption of precious tissue specimens. Beyond employing conventional chromogenic detection systems or fluorescently labelled antibodies, newer techniques that assess multiple biomarkers in situ take advantage of DNA barcoded [
      • Goltsev Y.
      • Samusik N.
      • Kennedy-Darling J.
      • Bhate S.
      • Hale M.
      • Vazquez G.
      • et al.
      Deep profiling of mouse splenic architecture with CODEX multiplexed imaging.
      ,

      Manesse M, Patel KK, Bobrow M, Downing SR. The InSituPlex ® Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples. Methods Mol Biol (Clifton, N.J.) 2020;2055:585.

      ] or metal-tagged (Imaging Mass Cytometry [
      • Giesen C.
      • Wang H.A.O.
      • Schapiro D.
      • Zivanovic N.
      • Jacobs A.
      • Hattendorf B.
      • et al.
      Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.
      ] and Multiplexed Ion Beam Imaging [
      • Keren L.
      • Bosse M.
      • Marquez D.
      • Angoshtari R.
      • Jain S.
      • Varma S.
      • et al.
      A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging.
      ]) antibodies [
      • Tan W.C.C.
      • Nerurkar S.N.
      • Cai H.Y.
      • Ng H.H.M.
      • Wu D.
      • Wee Y.T.F.
      • et al.
      Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy.
      ]. New studies using multiplex approaches are identifying specific immune cells in the tumor microenvironment of sarcomas with predictive and prognostic value [
      • Tsagozis P.
      • Augsten M.
      • Zhang Y.
      • Li T.
      • Hesla A.
      • Bergh J.
      • et al.
      An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma.
      ,
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ,
      • Keung E.Z.
      • Burgess M.
      • Salazar R.
      • Parra E.R.
      • Rodrigues-Canales J.
      • Bolejack V.
      • et al.
      Correlative analyses of the SARC028 trial reveal an association between sarcoma-associated immune infiltrate and response to pembrolizumab.
      ].
      At the RNA level, whole transcriptome sequencing has been used to discover novel immune biomarkers and has already found significant clinical applications [
      • Buzdin A.
      • Sorokin M.
      • Garazha A.
      • Glusker A.
      • Aleshin A.
      • Poddubskaya E.
      • et al.
      RNA sequencing for research and diagnostics in clinical oncology.
      ]. Limitations of bulk RNA sequencing, which does not attribute expression to particular cells in the tumor microenvironment, are addressed by single-cell RNAseq [
      • Haque A.
      • Engel J.
      • Teichmann S.A.
      • Lönnberg T.
      A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.
      ], although this loses morphological context. Spatial transcriptomics [
      • Thrane K.
      • Eriksson H.
      • Maaskola J.
      • Hansson J.
      • Lundeberg J.
      Spatially resolved transcriptomics enables dissection of genetic heterogeneity in stage III cutaneous malignant melanoma.
      ], direct in situ sequencing [
      • Ke R.
      • Mignardi M.
      • Hauling T.
      • Nilsson M.
      Fourth generation of next-generation sequencing technologies: promise and consequences.
      ], laser capture microdissected RNAseq [
      • Farris S.
      • Wang Y.
      • Ward J.M.
      • Dudek S.M.
      Optimized method for robust transcriptome profiling of minute tissues using laser capture microdissection and low-input RNA-seq.
      ] and in situ RNA hybridization assays [
      • Bakheet A.M.H.
      • Zhao C.
      • Chen J.
      • Zhang J.
      • Huang J.
      • Du Y.
      • et al.
      Improving pathological early diagnosis and differential biomarker value for hepatocellular carcinoma via RNAscope technology.
      ] circumvent this constraint [
      • Wang Y.
      • Mashock M.
      • Tong Z.
      • Mu X.
      • Chen H.
      • Zhou X.
      • et al.
      Changing technologies of RNA sequencing and their applications in clinical oncology.
      ].
      Lastly, Digital Spatial Profiling enables multiplexed measurement of proteins or RNAs within microscopically-identified regions of interest, at the resolution of a single cell type, by utilizing UV-cleavable oligonucleotide conjugated antibodies or RNA probes. Analysing immune cells in this sarcoma microenvironment using this technology has already been used to characterize mechanisms of doxorubicin resistance [
      • Gundle K.R.
      • Deutsch G.B.
      • Goodman H.J.
      • Pollack S.M.
      • Thompson M.J.
      • Davis J.L.
      • et al.
      Multiplexed evaluation of microdosed antineoplastic agents in situ in the tumor microenvironment of patients with soft tissue sarcoma.
      ]. Going forward, these new technologies will increasingly be used to characterize the complex sarcoma tumor microenvironment to guide development of immuno-oncology strategies.

      Immune checkpoint inhibitor clinical trials

       Mono and dual immune checkpoint inhibitor therapy

      While monoclonal ICI have demonstrated tremendous success in cancer types such as melanoma and non-small cell lung cancer [
      • Murciano-Goroff Y.R.
      • Warner A.B.
      • Wolchok J.D.
      The future of cancer immunotherapy: microenvironment-targeting combinations.
      ], clinical trials involving patients with sarcoma have largely trailed behind. A single-centre phase II study using ipilimumab in adult patients with synovial sarcoma was one of the first ICI trials in sarcoma, but was terminated early due to slow accrual and lack of response: all six patients had progressive disease after the first cycle of therapy [
      • Maki R.G.
      • Jungbluth A.A.
      • Gnjatic S.
      • Schwartz G.K.
      • D’Adamo D.R.
      • Keohan M.L.
      • et al.
      A pilot study of anti-CTLA4 antibody ipilimumab in patients with synovial sarcoma.
      ]. A few years later, a phase I trial of ipilimumab in 33 pediatric patients (NCT01445379) including 17 with sarcomas [
      • Merchant M.S.
      • Wright M.
      • Baird K.
      • Wexler L.H.
      • Rodriguez-Galindo C.
      • Bernstein D.
      • et al.
      Phase 1 clinical trial of ipilimumab in pediatric patients with advanced solid tumors.
      ]. Of the six patients with confirmed stable disease for more than six weeks, two were patients with osteosarcoma [
      • Merchant M.S.
      • Wright M.
      • Baird K.
      • Wexler L.H.
      • Rodriguez-Galindo C.
      • Bernstein D.
      • et al.
      Phase 1 clinical trial of ipilimumab in pediatric patients with advanced solid tumors.
      ]. However, the authors concluded that the high frequency of immune-related adverse events (transaminitis, colitis) in the pediatric subjects precluded ipilimumab's effective use as a monotherapy [
      • Merchant M.S.
      • Wright M.
      • Baird K.
      • Wexler L.H.
      • Rodriguez-Galindo C.
      • Bernstein D.
      • et al.
      Phase 1 clinical trial of ipilimumab in pediatric patients with advanced solid tumors.
      ]. Similarly, a phase II study of nivolumab (NCT02428192) in advanced uterine leiomyosarcoma demonstrated lack of response in all 12 enrolled patients [
      • Ben-Ami E.
      • Barysauskas C.M.
      • Solomon S.
      • Tahlil K.
      • Malley R.
      • Hohos M.
      • et al.
      Immunotherapy with single agent nivolumab for advanced leiomyosarcoma of the uterus: Results of a phase 2 study.
      ]. However, the authors reported an increase in PD-L1 and PD-L2 expression post-treatment, suggesting a modest immune reaction [
      • Ben-Ami E.
      • Barysauskas C.M.
      • Solomon S.
      • Tahlil K.
      • Malley R.
      • Hohos M.
      • et al.
      Immunotherapy with single agent nivolumab for advanced leiomyosarcoma of the uterus: Results of a phase 2 study.
      ]. Nonetheless, since then there have been no new sarcoma clinical trials of ipilimumab or nivolumab monotherapy.
      On the other hand, pembrolizumab showed more promising results in SARC028, a phase II trial of advanced soft-tissue and bone sarcoma (NCT02301039) [
      • Tawbi H.A.
      • Burgess M.
      • Bolejack V.
      • Van Tine B.A.
      • Schuetze S.M.
      • Hu J.
      • et al.
      Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial.
      ]. Out of the 40 patients with soft-tissue sarcoma (ten each of soft tissue leiomyosarcoma, DDLPS, UPS, and synovial sarcoma), there was one complete response (UPS) and six partial responses (three UPS, two DDLPS, and one synovial sarcoma) using RECIST criteria (the primary endpoint) [
      • Tawbi H.A.
      • Burgess M.
      • Bolejack V.
      • Van Tine B.A.
      • Schuetze S.M.
      • Hu J.
      • et al.
      Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial.
      ]. In the 40 patients with bone sarcoma (13 Ewing, 22 osteosarcoma, and 10 dedifferentiated or mesenchymal chondrosarcomas), only two patients (osteosarcoma and chondrosarcoma) had a partial response [
      • Tawbi H.A.
      • Burgess M.
      • Bolejack V.
      • Van Tine B.A.
      • Schuetze S.M.
      • Hu J.
      • et al.
      Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial.
      ]. While neither cohort met the primary endpoint, the soft-tissue sarcoma arm met criteria to be considered an active therapy with a 12-week progression survival of 55% [
      • Tawbi H.A.
      • Burgess M.
      • Bolejack V.
      • Van Tine B.A.
      • Schuetze S.M.
      • Hu J.
      • et al.
      Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial.
      ]. Consequently, the SARC028 trial added two expansion cohorts in advanced UPS and DDLPS [
      • Burgess M.A.
      • Bolejack V.
      • Schuetze S.
      • Van Tine B.A.
      • Attia S.
      • Riedel R.F.
      • et al.
      Clinical activity of pembrolizumab (P) in undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated/pleomorphic liposarcoma (LPS): Final results of SARC028 expansion cohorts.
      ]. The UPS cohort successfully met its primary endpoints with an objective response rate of 23%, as reported at the 2019 American Society of Clinical Oncology (ASCO) meeting [
      • Burgess M.A.
      • Bolejack V.
      • Schuetze S.
      • Van Tine B.A.
      • Attia S.
      • Riedel R.F.
      • et al.
      Clinical activity of pembrolizumab (P) in undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated/pleomorphic liposarcoma (LPS): Final results of SARC028 expansion cohorts.
      ]. Further investigations remain underway to characterize the clinical effectiveness of PD-1/PDL-1 monotherapy in UPS (Table 2).
      Table 2Clinical trials of immune checkpoint inhibitor monotherapy for patients with sarcoma.
      Immune checkpoint inhibitorNCT trial numberPhaseStatusParticipantsSarcoma subtype(s)Includes pediatric patients
      Ipilimumab (anti-CTLA4)NCT01445379ICompleted33Sarcoma, Wilm's tumor, lymphoma, neuroblastomaYes: 3–21 years
      ZKAB001 (anti-PD-L1)NCT03676985I/IIRecruiting15High-grade osteosarcomaNo
      Atezolizumab (anti-PD-L1)NCT03141684IIRecruiting46Metastatic alveolar soft part sarcomaYes: 2 years and older
      NCT03474094IIActive, not recruiting22Liposarcoma, undifferentiated pleomorphic sarcoma, leiomyosarcoma, myxofibrosarcoma, angiosarcoma, all translocation sarcoma except Ewing, rhabdomyosarcoma and myxoid liposarcomaNo
      Avelumab (anti-PD-L1)NCT03006848IIActive, not recruiting15Recurrent or refractory osteosarcomaYes: 12–49 years
      MDX-010 (anti-CTLA4)NCT00140855IITerminated (study discontinued due to poor accrual)17Synovial sarcomaYes: 13 years and older
      NivolumabNCT02428192IICompleted12Advanced uterine leiomyosarcomaNo
      Pembrolizumab(anti-PD-1)NCT02301039 (SARC028)IIActive, not recruiting146Bone and soft-tissue sarcomaYes: 12 years and older
      NCT03013127IIActive, not recruiting12OsteosarcomaNo
      NCT03316573IIRecruiting30Histiocytic Sarcoma, follicular dendritic cell sarcoma, interdigitating dendritic cell sarcoma, lymphomaNo
      TSR-042 (anti-PD-1)NCT04274023IINot yet recruiting16Clear cell sarcomaNo
      Going beyond ICI monotherapy, the phase II trial Alliance A091401 trial (NCT02500797) demonstrated that combination therapy using two ICIs can be effective in metastatic sarcoma [
      • D'Angelo S.P.
      • Mahoney M.R.
      • Van Tine B.A.
      • Atkins J.
      • Milhem M.M.
      • Jahagirdar B.N.
      • et al.
      Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401): two open-label, non-comparative, randomised, phase 2 trials.
      ]. In this study, the nivolumab monotherapy arm did not meet its pre-defined primary endpoint, as only three (ASPS, non-uterine leiomyosarcoma, and sarcoma NOS) of 38 patients achieved a partial response [
      • D'Angelo S.P.
      • Mahoney M.R.
      • Van Tine B.A.
      • Atkins J.
      • Milhem M.M.
      • Jahagirdar B.N.
      • et al.
      Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401): two open-label, non-comparative, randomised, phase 2 trials.
      ]. On the other hand, the combination therapy arm using nivolumab and ipilimumab had six of 38 patients achieve confirmed objective responses, including two complete responses (myxofibrosarcoma and uterine leiomyosarcoma) [
      • D'Angelo S.P.
      • Mahoney M.R.
      • Van Tine B.A.
      • Atkins J.
      • Milhem M.M.
      • Jahagirdar B.N.
      • et al.
      Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401): two open-label, non-comparative, randomised, phase 2 trials.
      ]. The median overall survival on the monotherapy arm was 10.7 months, compared to 14.3 months in the combination arm [
      • D'Angelo S.P.
      • Mahoney M.R.
      • Van Tine B.A.
      • Atkins J.
      • Milhem M.M.
      • Jahagirdar B.N.
      • et al.
      Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401): two open-label, non-comparative, randomised, phase 2 trials.
      ]. However, since this trial was not set up to directly compare the efficacy of the two arms, the authors could only conclude that the combination arm is comparable to standard sarcoma chemotherapy [
      • D'Angelo S.P.
      • Mahoney M.R.
      • Van Tine B.A.
      • Atkins J.
      • Milhem M.M.
      • Jahagirdar B.N.
      • et al.
      Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401): two open-label, non-comparative, randomised, phase 2 trials.
      ]. The potential of using combination ICI therapy as first or second line treatment for sarcoma led the Alliance cooperative group to expand the trial. At the 2020 ASCO meeting, preliminary results show that both the UPS and DDLPS expansion cohort reached the primary response endpoint in the combination ICI arm, each with two confirmed responses out of twelve patients [
      • Chen J.L.
      • Mahoney M.R.
      • George S.
      • Antonescu C.R.
      • Liebner D.A.
      • Van Tine B.A.
      • et al.
      A multicenter phase II study of nivolumab +/ ipilimumab for patients with metastatic sarcoma (Alliance A091401): Results of expansion cohorts.
      ].
      Given the limited response rates observed with established ICIs, currently active monotherapy clinical trials are focusing on exploring novel agents, including anti-PD-L1 antibodies ZKAB001 (NCT03676985) and atezolizumab (NCT03141684; NCT03474094), and anti-PD-1 antibody TSR-042 (NCT04274023) (Table 2). Other trials are adopting combination strategies to involve dual ICI regimens (NCT02500797; NCT02815995; NCT03365791; NCT04095208), or an ICI with cancer vaccination therapy (NCT02609984; NCT03069378) or cytokines (NCT03063632; NCT03282344) (Table 3).
      Table 3Clinical trials of immune checkpoint inhibitor in combination therapy for patients with sarcoma.
      Immune checkpoint inhibitorOther agentsNCT trial numberPhaseStatusParticipantsSarcoma subtype(s)Includes pediatric patients
      Immune checkpoint inhibitor in combination with another immunotherapy agent
      Atezolizumab (anti-PD-L1)CMB305 (dendritic cell-targeting viral vector expressing the NY-ESO-1 gene and G305 [NY-ESO-1 recombinant protein plus GLA-SE])NCT02609984IICompleted88Metastatic/ recurrent/ locally advanced soft tissue sarcomaNo
      Avelumab (anti-PD-L-1)DCC-3014 (CSF1R inhibitor)NCT04242238IRecruiting48Advanced or metastatic sarcomaNo
      Nivolumab (anti-CTLA-4)NKTR-214 (CD122 agonist)NCT03282344IIActive, not recruiting85Metastatic and/or locally advanced sarcomaYes: 12 years and older
      Pembrolizumab(anti-PD-1)GSK3377794 (genetically engineered NY-ESO-1 Specific [c259] T Cells)NCT03697824IIWithdrawn
      Internal decision, study will be replaced with a larger monotherapy trial.
      0Synovial sarcomaYes: 10 years and older
      INFγ-1bNCT03063632IIActive, not recruiting46Advanced synovial sarcoma, previously treated mycosis fungoides and Sezary syndromeYes: 12 years and older
      Metronomic chemotherapy +/− intratumoral G100 (toll-like receptor 4 agonist)NCT02406781IIRecruiting227Leiomyosarcoma, undifferentiated pleomorphic sarcoma, other sarcoma, gastrointestinal stromal tumor, osteosarcoma, advanced or metastatic soft-tissue sarcoma with presence of tertiary lymphoid structuresNo
      Talimogene Laherparepvec (T-VEC)NCT03069378IIRecruiting60Metastatic and/or locally advanced sarcomaNo
      PD-1 antibodyMASCT-I (multi-antigen autoimmune cell injection) and Apatinib (tyrosine kinase inhibitor)NCT04074564INot yet recruiting20Sarcoma, excluding bone or brain metastasesYes: 14–70 years
      Immune checkpoint inhibitor in combination with chemotherapy or radiation therapy
      AtezolizumabStereotactic body radiation therapyNCT03548428IINot yet recruiting103Soft tissue sarcomaNo
      AvelumabTrabectedinNCT03074318I/IIActive, not recruiting35Metastatic or unresectable leiomyosarcoma and liposarcomaNo
      Durvalumab (anti-PD-L1)TrabectedinNCT03085225IActive, not recruiting50Soft tissue sarcomas and ovarian carcinomasNo
      NivolumabTrabectedinNCT03590210IIRecruiting92Advanced or metastatic soft tissue sarcomaNo
      PembrolizumabDoxorubicinNCT02888665I/IIActive, not recruiting37Advanced soft tissue sarcomaNo
      GemcitabineNCT03123276I/IIRecruiting24Undifferentiated pleomorphic sarcoma or leiomyosarcomaNo
      Radiation therapyNCT03338959I/IISuspended (Administrative)26Intermediate or high-grade soft tissue sarcomaNo
      Radiation therapyNCT03092323IIRecruiting102High-risk soft-tissue sarcomaYes: 12 years and older
      Immune checkpoint inhibitor in combination with other agents
      Camrelizumab (anti-PD-1)Famitinib (tyrosine kinase inhibitor)NCT04044378I/IIRecruiting80Advanced osteosarcomaYes: 12 years and older
      Ipilimumab (anti-CTLA-4)Dasatinib (tyosine kinase inhibitor)NCT01643278ICompleted29Gastrointestinal stromal tumor, stage III and IV soft tissue sarcomaNo
      Nivolumab or ipilimumabCryoablationNCT04118166IIRecruiting30Soft tissue sarcomaN
      PembrolizumabAxitinib (tyrosine kinase inhibitor)NCT02636725IIActive, not recruiting33Advanced alveolar soft part sarcoma and other soft tissue sarcomasYes: 16 years and older
      Eribulin (tyrosine kinase inhibitor)NCT03899805IIRecruiting57Sarcoma, liposarcoma, leiomyosarcoma, undifferentiated pleomorphic sarcomaNo
      Ziv-aflibercept (VEGF inhibitor)NCT02298959IRecruiting78Unresectable stage III or stage IV melanoma, renal cell cancer, ovarian cancer, colorectal cancer, or sarcomaNo
      Toripalimab (anti-PD-1)Anlotinib (tyrosine kinase inhibitor)NCT03946943IINot yet recruiting25Undifferentiated pleomorphic sarcomaYes: 16 years and older
      Dual immune checkpoint inhibitor therapy
      Durvalumab (anti-PD-L1)Tremelimumab (anti-CTLA-4)NCT02815995IIActive, not recruiting62Adipocytic tumors (well-diff/ de-differentiated, pleomorphic and myxoid liposarcoma), vascular tumors (leiomyosarcomas, angiosarcomas, epithelioid hemangioendotheliomas, and hemangiopericytomas), undifferentiated pleomorphic sarcoma, synovial sarcoma, osteosarcoma, other sarcomasNo
      TremelimumabNCT02879162IIActive, not recruiting140Advanced rare tumorsYes: 16 years and older
      NivolumabIpilimumabNCT02500797IIActive, not recruiting164Unresectable metastatic sarcomaNo
      NCT02982486IINot yet recruiting60Bone and soft-tissue sarcoma, chondrosarcoma, gastrointestinal stromal sarcoma, Ewing's tumor, osteosarcoma, desmoplastic small round cell tumorNo
      Relatlimab (anti-LAG-3)NCT04095208IINot yet recruiting67Advanced or metastatic soft tissue sarcomaNo
      PembrolizumabEpacadostat (IDO1 inhibitor)NCT03414229IIActive, not recruiting48Metastatic and/or locally advanced sarcomaNo
      Dual immune checkpoint inhibitor therapy in combination with chemotherapy or radiation therapy
      AGEN1884 (anti-CTLA-4)+AGEN2034 (anti-PD-1)DoxorubicinNCT04028063IIRecruiting28Soft tissue sarcomaNo
      Durvalumab + TremelimumabRadiation therapy +/− surgical resectionNCT03116529I/IIRecruiting35Predominantly low-grade soft tissue sarcomaNo
      Nivolumab+ipilimumabRadiation therapy+surgical resectionNCT03463408IRecruiting24Soft-tissue sarcomaNo
      Radiation therapyNCT03307616IIRecruiting40Undifferentiated pleomorphic sarcoma or dedifferentiated liposarcomaNo
      TrabectedinNCT03138161I/IIRecruiting45Advanced and metastatic soft tissue sarcomaNo
      * Internal decision, study will be replaced with a larger monotherapy trial.

       Immune checkpoint inhibitors in combination with other agents

      Recent sarcoma clinical trials are combining ICI with chemotherapy agents and/or radiation therapy, a logical approach as the standard therapy for most sarcoma subtypes includes chemotherapy and radiation. Furthermore, tumor irradiation is associated with increased immune cell infiltration [
      • Dancsok A.R.
      • Setsu N.
      • Gao D.
      • Blay J.
      • Thomas D.
      • Maki R.G.
      • et al.
      Expression of LYMPHOCYTE immunoregulatory biomarkers in bone and soft-tissue sarcomas.
      ], suggesting radiation to be an immune activating procedure (the abscopal effect).
      PEMBROSARC is a phase II study (NCT02406781) that used pembrolizumab and cyclophosphamide to treat four cohorts of soft tissue sarcoma (leiomyosarcoma, UPS, GIST, and others) [
      • Toulmonde M.
      • Penel N.
      • Adam J.
      • Chevreau C.
      • Blay J.
      • Le Cesne A.
      • et al.
      Use of PD-1 targeting, macrophage infiltration, and IDO pathway activation in sarcomas: A phase 2 clinical trial.
      ]. The study reported three out of 50 patients to be progression-free at six months (GIST, solitary fibrous tumor, endometrial stromal sarcoma) [
      • Toulmonde M.
      • Penel N.
      • Adam J.
      • Chevreau C.
      • Blay J.
      • Le Cesne A.
      • et al.
      Use of PD-1 targeting, macrophage infiltration, and IDO pathway activation in sarcomas: A phase 2 clinical trial.
      ]. The median progression-free survival was 1.4 months in all four cohorts [
      • Toulmonde M.
      • Penel N.
      • Adam J.
      • Chevreau C.
      • Blay J.
      • Le Cesne A.
      • et al.
      Use of PD-1 targeting, macrophage infiltration, and IDO pathway activation in sarcomas: A phase 2 clinical trial.
      ]. While these trial results are unimpressive, the authors observed an increase in kynurenine to tryptophan ratio between cycles one and three of pembrolizumab [
      • Toulmonde M.
      • Penel N.
      • Adam J.
      • Chevreau C.
      • Blay J.
      • Le Cesne A.
      • et al.
      Use of PD-1 targeting, macrophage infiltration, and IDO pathway activation in sarcomas: A phase 2 clinical trial.
      ]. Given the close association of kynurenine with IDO, the authors speculate that there may remain a role for combining IDO-1 inhibitors with pembrolizumab in sarcoma [
      • Toulmonde M.
      • Penel N.
      • Adam J.
      • Chevreau C.
      • Blay J.
      • Le Cesne A.
      • et al.
      Use of PD-1 targeting, macrophage infiltration, and IDO pathway activation in sarcomas: A phase 2 clinical trial.
      ].
      In the same trial, pembrolizumab was added to cyclophosphamide in an osteosarcoma cohort of 17 patients. Four experienced tumor shrinkage, including one confirmed partial response [
      • Le Cesne A.
      • Marec-Berard P.
      • Blay J.
      • Gaspar N.
      • Bertucci F.
      • Penel N.
      • et al.
      Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study.
      ]. The median progression-free survival was 1.4 months, and median overall survival 5.6 months [
      • Le Cesne A.
      • Marec-Berard P.
      • Blay J.
      • Gaspar N.
      • Bertucci F.
      • Penel N.
      • et al.
      Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study.
      ]. Interestingly, none of the three patients with tumor shrinkage and available tumor material had expression of PD-L1 on either sarcoma or immune cells [
      • Le Cesne A.
      • Marec-Berard P.
      • Blay J.
      • Gaspar N.
      • Bertucci F.
      • Penel N.
      • et al.
      Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study.
      ]. However, consistent with past findings of low PD-L1 expression in osteosarcoma [
      • van Erp A.E.M.
      • Versleijen-Jonkers Y.M.H.
      • Hillebrandt-Roeffen M.H.S.
      • van Houdt L.
      • Gorris M.A.J.
      • van Dam L.S.
      • et al.
      Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent.
      ,
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ,
      • Torabi A.
      • Amaya C.N.
      • Wians F.H.
      • Bryan B.A.
      PD-1 and PD-L1 expression in bone and soft tissue sarcomas.
      ], only 12% of osteosarcoma cases were PD-L1+  in this cohort [
      • Le Cesne A.
      • Marec-Berard P.
      • Blay J.
      • Gaspar N.
      • Bertucci F.
      • Penel N.
      • et al.
      Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study.
      ]. Therefore, it is unclear whether the lack of PD-L1 expression among responders has any significance. Overall, results of the PEMBROSARC trial have been underwhelming and the authors concluded that more investigation is needed to understand mechanisms of resistance to immunotherapy in sarcoma.
      One of the most successful ICI trials in sarcoma to date is a phase II study (NCT02636725), combining pembrolizumab with VEGF receptor tyrosine kinase inhibitor axitinib [
      • Wilky B.A.
      • Trucco M.M.
      • Subhawong T.K.
      • Florou V.
      • Park W.
      • Kwon D.
      • et al.
      Axitinib plus pembrolizumab in patients with advanced sarcomas including alveolar soft-part sarcoma: a single-centre, single-arm, phase 2 trial.
      ]. This study included 30 patients with advanced sarcoma, among whom twelve had ASPS; the rest included high grade pleomorphic sarcoma, soft tissue leiomyosarcoma, and DDLPS [
      • Wilky B.A.
      • Trucco M.M.
      • Subhawong T.K.
      • Florou V.
      • Park W.
      • Kwon D.
      • et al.
      Axitinib plus pembrolizumab in patients with advanced sarcomas including alveolar soft-part sarcoma: a single-centre, single-arm, phase 2 trial.
      ]. Findings were encouraging in the ASPS cohort, as six of eleven patients achieved partial response and an additional two achieved stable disease. Among patients with other histologic subtypes, there were two partial responses (epithelioid sarcoma and leiomyosarcoma) and seven with stable disease [
      • Wilky B.A.
      • Trucco M.M.
      • Subhawong T.K.
      • Florou V.
      • Park W.
      • Kwon D.
      • et al.
      Axitinib plus pembrolizumab in patients with advanced sarcomas including alveolar soft-part sarcoma: a single-centre, single-arm, phase 2 trial.
      ]. The primary endpoint of three-month progression-free survival in patients with ASPS was 72.7%, while the three-month progression-free survival for patients with other sarcomas was 61.9%. This success of ICI in ASPS was preceded by case reports [
      • Conley A.P.
      • Trinh V.A.
      • Zobniw C.M.
      • Posey K.
      • Martinez J.D.
      • Arrieta O.G.
      • et al.
      Positive tumor response to combined checkpoint inhibitors in a patient with refractory alveolar soft part sarcoma: A case report.
      ,
      • Lewin J.
      • Davidson S.
      • Anderson N.D.
      • Lau B.Y.
      • Kelly J.
      • Tabori U.
      • et al.
      Response to immune checkpoint inhibition in two patients with alveolar soft-part sarcoma.
      ] and a retrospective analysis of phase I immunotherapy trials [
      • Groisberg R.
      • Hong D.S.
      • Behrang A.
      • Hess K.
      • Janku F.
      • Piha-Paul S.
      • et al.
      Characteristics and outcomes of patients with advanced sarcoma enrolled in early phase immunotherapy trials.
      ]. However, these exciting results for ASPS have some caveats. Firstly, soft tissue sarcomas have been shown to respond to different tyrosine kinase inhibitors with median progression-free survival similar to that of this study [
      • van der Graaf W.TA.
      • Blay J.
      • Chawla S.P.
      • Kim D.
      • Bui-Nguyen B.
      • Casali P.G.
      • et al.
      Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind, placebo-controlled phase 3 trial.
      ,
      • Schoffski P.
      • Wozniak A.
      • Kasper B.
      • Aamdal S.
      • Leahy M.G.
      • Rutkowski P.
      • et al.
      Activity and safety of crizotinib in patients with alveolar soft part sarcoma with rearrangement of TFE3: European Organization for Research and Treatment of Cancer (EORTC) phase II trial 90101 'CREATE'.
      ], so the additional benefit of adding pembrolizumab is unclear. Secondly, the clinical course of ASPS is generally more indolent than other sarcomas in the first few years (although long-term prognosis is poor) [
      • Lieberman P.H.
      • Brennan M.F.
      • Kimmel M.
      • Erlandson R.A.
      • Garin-Chesa P.
      • Flehinger B.Y.
      Alveolar soft-part sarcoma. A clinico-pathologic study of half a century.
      ]. Short term progression-free survival may be an unreliable primary endpoint in this disease. Instead, tumor size or a longer period of progression-free survival could be a more relevant primary endpoint as employed by other phase II trials on patients with ASPS [
      • Schoffski P.
      • Wozniak A.
      • Kasper B.
      • Aamdal S.
      • Leahy M.G.
      • Rutkowski P.
      • et al.
      Activity and safety of crizotinib in patients with alveolar soft part sarcoma with rearrangement of TFE3: European Organization for Research and Treatment of Cancer (EORTC) phase II trial 90101 'CREATE'.
      ,
      • Judson I.
      • Morden J.P.
      • Kilburn L.
      • Leahy M.
      • Benson C.
      • Bhadri V.
      • et al.
      Cediranib in patients with alveolar soft-part sarcoma (CASPS): a double-blind, placebo-controlled, randomised, phase 2 trial.
      ].
      Nevertheless, therapies combining ICI with other agents have demonstrated promising effects in select patients, leading to many more trials to investigate combination therapies (Table 3). Current ongoing trials include single ICI with chemotherapy (NCT02888665; NCT03074318; NCT03085225; NCT03123276; NCT03590210) or radiation therapy (NCT03092323; NCT03338959; NCT03548428), single ICI with tyrosine kinase inhibitors ((NCT01643278; NCT02298959; NCT02636725; NCT03899805; NCT03946943; NCT04044378; NCT04118166), and dual ICI with chemotherapy (NCT03138161; NCT04028063) or radiation therapy (NCT03116529; NCT03307616; NCT03463408).

       Limitations of published studies

      Several important limitations exist in the current sarcoma immune biomarker literature. Due to the comparative rarity of sarcomas relative to other cancer types, most studies have small sample sizes. Of the studies cited in this review, most had fewer than 100 patient samples (Table 1). Hence it is more difficult to identify significant correlations with other biomarkers or with patient outcomes for sarcomas, and there may be more apparent inconsistencies between studies. Many studies have consequently lumped sarcoma subtypes together to increase apparent power, but in doing so lose the ability to recognize effects specific to (biologically disparate) histologic subtypes. Immune status also varies within most histologic subtypes, which could represent an even more important impediment on identifying significant differences [
      • Lee A.T.J.
      • Chew W.
      • Wilding C.P.
      • Guljar N.
      • Smith M.J.
      • Strauss D.C.
      • et al.
      The adequacy of tissue microarrays in the assessment of inter- and intra-tumoural heterogeneity of infiltrating lymphocyte burden in leiomyosarcoma.
      ,
      • Petitprez F.
      • de Reyniès A.
      • Keung E.Z.
      • Chen T.W.
      • Sun C.
      • Calderaro J.
      • et al.
      B cells are associated with survival and immunotherapy response in sarcoma.
      ]. In the same vein, the small sample sizes and response rates in existing phase II sarcoma immunotherapy trials render it difficult to investigate the predictive value of immune biomarkers. To date, there is no data showing that any specific immune biomarker can predict response to ICI in sarcomas.
      Tissue microarrays are one way to obtain higher through-put results on larger tumor datasets. However, the arrayed tissue cores are generally extracted from tumor centres whereas immune responses may be most prominent at the (unsampled) periphery. In DDLPS, no PD-L1 expression was found using tissue microarrays, whereas 22% of whole section cases were positive [
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ]. In leiomyosarcoma, a median of 11 cores was needed for an accurate estimate of the true TIL burden [
      • Lee A.T.J.
      • Chew W.
      • Wilding C.P.
      • Guljar N.
      • Smith M.J.
      • Strauss D.C.
      • et al.
      The adequacy of tissue microarrays in the assessment of inter- and intra-tumoural heterogeneity of infiltrating lymphocyte burden in leiomyosarcoma.
      ]. Apparent rates of positivity may also be hard to compare between IHC studies due to differences between antibody clones produced by different companies, staining platforms, staining conditions, interpretation and cutpoints for defining a case as positive [
      • Rimm D.L.
      • Han G.
      • Taube J.M.
      • Yi E.S.
      • Bridge J.A.
      • Flieder D.B.
      • et al.
      A prospective, multi-institutional, pathologist-based assessment of 4 immunohistochemistry assays for PD-L1 expression in non-small cell lung cancer.
      ]. In sarcoma cell lines [
      • Park H.K.
      • Kim M.
      • Sung M.
      • Lee S.E.
      • Kim Y.J.
      • Choi Y.
      Status of programmed death-ligand 1 expression in sarcomas.
      ] and leiomyosarcoma tumors [
      • Cohen J.E.
      • Eleyan F.
      • Zick A.
      • Peretz T.
      • Katz D.
      Intratumoral immune-biomarkers and mismatch repair status in leiyomyosarcoma -potential predictive markers for adjuvant treatment: a pilot study.
      ], clone 22C3 (Dako) showed less staining than clone SP142 and clone 28-8 (Abcam #ab205921), respectively. Centralized protocols for quantification of PD-L1 expression should be considered in sarcoma, similar to those established in lung cancer [
      • Rimm D.L.
      • Han G.
      • Taube J.M.
      • Yi E.S.
      • Bridge J.A.
      • Flieder D.B.
      • et al.
      A prospective, multi-institutional, pathologist-based assessment of 4 immunohistochemistry assays for PD-L1 expression in non-small cell lung cancer.
      ]. Methods for pathologists to standardize TIL counting in carcinomas have been published [
      • Salgado R.
      • Denkert C.
      • Demaria S.
      • Sirtaine N.
      • Klauschen F.
      • Pruneri G.
      • et al.
      The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014.
      ], but standardized methods for staining and scoring for even commonly-used biomarkers such as CD8 and CD20 are not so well developed.
      In terms of the sarcoma clinical trial literature, the elephant in the room is a lack of Phase III studies. The recent sarcoma literature contains multiple examples of agents that were promising in Phase II, even to the point of provisional approval, but which did not hold up in Phase III trials, the most recent and prominent example being olaratumab [
      • Tap W.D.
      • Wagner A.J.
      • Schöffski P.
      • Martin-Broto J.
      • Krarup-Hansen A.
      • Ganjoo K.N.
      • et al.
      Effect of doxorubicin plus olaratumab vs doxorubicin plus placebo on survival in patients with advanced soft tissue sarcomas: The ANNOUNCE randomized clinical trial.
      ]. ICI or other immunotherapy strategies must ultimately reach this standard to prove their real value in sarcoma therapy.

      Conclusion

      Based on the described findings from immune biomarker studies, and the observed pattern of responses in ICI trials, dedifferentiated liposarcoma, embryonal rhabdomyosarcoma, uterine and non-uterine leiomyosarcoma, osteosarcoma, and undifferentiated pleomorphic sarcoma are the most commonly immune-hot sarcoma subtypes. These karyotypically complex subtypes are the most likely to have high levels of immune infiltration and checkpoint expression, and to exhibit responses to ICI therapy. Of all sarcomas, Ewing sarcoma is the most consistently immune-cold tumor that hence would possibly need immune augmentation strategies instead. Interestingly, other translocation-associated subtypes such as synovial sarcoma and solitary fibrous tumor demonstrate conflicting results in immunogenicity, and may need more investigation to reach consensus. Patients with alveolar soft part sarcoma have shown promising responses to ICIs for which confirmatory trials are underway.
      Sarcomas have not seen the same breakthroughs in immunotherapy as seen in other malignancies. More subtype and/or biomarker specific studies are required, as published studies still inappropriately combine biologically different sarcoma subtypes. More emphasis should be given to B cell markers as they have recently demonstrated potentially stronger prognostic and predictive values than the T-cell biomarkers that have been the focus of most studies to date. New technologies can be used to discover novel immune biomarker relationships and to enable more complete understanding of the sarcoma microenvironment by employing multiplexed, spatially resolved RNA and protein techniques. Perhaps these technologies will be able to identify predictive sarcoma biomarkers for the ICI regimens with promising early results, including pembrolizumab monotherapy, nivolumab with ipilimumab, and axitinib with pembrolizumab. Subtypes such as alveolar soft part sarcoma, angiosarcoma, dedifferentiated liposarcoma and undifferentiated pleomorphic sarcoma have shown some impressive responses to checkpoint inhibitors and are particularly worthy of extra investigation in more advanced clinical trials linked to correlative science efforts employing new biomarker technologies.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgements

      The authors would like to thank Drs. Robin Jones, William Tap, David Thomas and Shane Zaidi for helpful discussions about active clinical trials. This research was supported by grants from the Canadian Cancer Society (705615) and the Terry Fox Research Institute (1082).

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