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Addressing the Adult Soft Tissue Sarcoma Microenvironment with Intratumoral Immunotherapy

Addressing the Adult Soft Tissue Sarcoma Microenvironment with Intratumoral Immunotherapy Hindawi Sarcoma Volume 2018, Article ID 9305294, 10 pages https://doi.org/10.1155/2018/9305294 Review Article AddressingtheAdultSoftTissueSarcomaMicroenvironment with Intratumoral Immunotherapy Shailaja Raj , Lance D. Miller, and Pierre L. Triozzi Wake Forest School of Medicine, Winston-Salem, NC, USA Correspondence should be addressed to Shailaja Raj; skrimuno@gmail.com Received 28 March 2018; Accepted 16 July 2018; Published 12 August 2018 Academic Editor: Eugenie S. Kleinerman Copyright © 2018 Shailaja Raj et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sarcoma is comprised of a heterogeneous group of tumors originating from the mesenchyme. Sarcoma is also the first tumor that responded to immunotherapeutic agents often termed as “Coley’s toxins.” However, immunotherapy is yet to establish its presence in sarcomas. Complex interactions between tumor and immune cells in the tumor microenvironment play a crucial role in response to immunotherapy. (ere is a dynamic equilibrium created by the immune cells infiltrating the tumor, and this forms the basis of tumor evasion. Manipulating the intratumoral microenvironment will help overcome tumor evasion. outcome of patients with metastatic soft tissue sarcoma. (e 1. Introduction prognosis of these patients is very poor. Median overall survival is 8 to 12 months [1]. In this review, we first explore the specific oncogenic alter- ation characteristics of different subtypes of sarcoma. (is is Immunotherapy has been an attractive approach to treat followed by a description of the mechanisms by which tumor refractory cancers. Sarcoma is considered to be the first infiltrating lymphocytes affect prognosis and the specific cancer for which immunotherapy was effectively applied. immune cell populations that can be targeted and manipu- Based on observations of tumor regressions in patients with lated in different subtypes of sarcoma. We also review the concomitant streptococcal infections, William B. Coley various immune suppressive mechanisms including immune injected streptococcal organisms into tumors, especially check points, receptors, and tumor-associated macrophages sarcomas, in the last decade of the 19th century [2]. Over and their relevance in sarcoma. We then focus on intra- 50% of the inoperable sarcoma patients who are Coley tumoral immunotherapy, mechanisms of immune interac- treated were reported to respond completely. Furthermore, tions, limitations, and the types of intratumoral therapies approximately 20% survived over 20 years [2]. With their poorly characterized preparation and unpredictable toxic- including oncolytic viruses, immune cells, and cytokines. We foresee intratumoral immunotherapies being able to ities, “Coley’s toxins” never became clinically useful. Im- target and influence management of sarcomas in the future. munotherapeutic approaches have been tested in patients Soft tissue sarcomas arise from cells of mesenchymal with soft tissue sarcoma. (e results have not been as lineage, including muscle, fat, blood vessel, and nerve. Over spectacular as some of the other solid tumors. Immune 50 histological types have been identified. Localized tumors checkpoint blockade with antibodies that target cytotoxic are usually well controlled with surgery. Localized tumors T lymphocyte-associated antigen 4 (CTLA-4) and the that have high-grade histologies and those are over 5cm, programmed cell death protein 1 pathway (PD-1/PD-L1) is however, have over a 50% risk of recurrence. Patients with leading to durable clinical responses in an increasing high-risk localized tumors are usually treated with combi- number of cancers. However, responses in patients with soft nations of surgery, radiation, and/or chemotherapy. (ese tissue sarcoma have been infrequent [3–6]. Immunotherapy response is dependent on complex approaches have failed to substantially improve overall survival. Chemotherapy has not significantly impacted the interactions between tumor and immune cells within the 2 Sarcoma tumor microenvironment. Several factors determine Mutational frequencies vary by subtype. Some, including whether or not immunotherapy response will be promoted angiosarcoma, leiomyosarcoma, and undifferentiated pleo- morphic sarcoma, manifest higher frequencies, in the 2-3 or inhibited. (ese include the inherent antigenicity of the tumor. Mutations in proteins and/or aberrant proteins per Mb range. Most, including myxofibrosarcoma, lip- expressed by tumor cells and the “neoantigens” they gen- osarcoma, and synovial sarcoma, manifest very low fre- erate are the primary targets for T-cell-mediated destruction. quencies, less than 2 per Mb. Nonetheless, patients with high Tumor mutation burden has emerged as a quantitative tumor mutational burdens can be identified in nearly every marker that can help predict responses to immune check- type of soft tissue sarcoma. For example, approximately 13% point inhibition across different cancers. Immunotherapy of angiosarcomas manifest mutation frequencies of more response is also dependent on the infiltration into tumor than 20 per Mb [10]. It should be noted that tumor of immune effector cells. Specific patterns of tumor microsatellite instability, which is an indication for treat- infiltrating lymphocytes (TILs) within the tumor microen- ment with PD-1 inhibitors because of the associated increase vironment are associated with improved outcome in patients in mutations, is not considered to play a major role in soft tissue sarcoma tumorigenesis [9]. with many types of cancers, regardless of the type of therapy administered. Most importantly, a variety of processes (e fusion proteins that result from chromosomal within the microenvironment can suppress interactions translocations represent potential targets. Although there between tumors and immune effector cells and promote the may be tolerance toward epitopes within these proteins, the escape of tumors from immune surveillance. Immune area of fusion in effect represents a neoantigen. Worley et al. checkpoint ligands and receptors have emerged as the major examined the immunogenicity of these areas for sarcoma targetable mechanism of tumor immune escape. Several subtypes characterized by specific translocations, including other molecular, soluble, and cellular factors are involved, synovial, clear cell, and desmoplastic round cell [11]. Pep- and whether or not these factors are being addressed will also tides corresponding to the fusion proteins were designed determine immunotherapy sensitivity. and assessed for the ability to bind to various HLA class I Understanding the soft tissue sarcoma microenviron- molecule. (ese peptides were effectively used in vitro to generate antigen-specific T cells with cytotoxic function ment is not only critical relative to improving the efficiency of current immunotherapies but also for the development of against tumor cells expressing the fusion protein. more effective approaches. In this review, we focus on the Several soft tissue sarcoma subtypes also naturally have recent advances made in understanding the immune mi- high expression of cancer testis antigens, which are con- croenvironment in soft tissue sarcoma. We also discuss the sidered targets for immunotherapy, since in adults these rationale for building upon Coley’s work and directly proteins are expressed only by germ cells, for example, in modifying the soft tissue sarcoma microenvironment using testis tissue, and not by somatic tissue cells [11, 12]. NY- the intratumoral administration of immunologically active ESO-1 is the most notable of the cancer testis antigens agents. described in sarcoma [13, 14]. In synovial sarcoma, NY- ESO-1, as assessed by immunohistochemistry, was expressed in 20 out of 25 cases (80%) [15]. 2. Tumor Immune Microenvironment Expression has also been observed in over 90% of 2.1. Tumor Antigenicity. Soft tissue sarcomas often are di- myxoid and round-cell liposarcoma. Of note, synovial sarcoma, and myxoid and round-cell liposarcoma are vided genetically into two categories: “simple” and “com- plex.” (e simple tumors tend to have specific oncogenic translocation-driven malignancies with very low muta- tional burdens. alterations and a limited number of mutations. (ese in- clude synovial cell sarcoma, which is characterized by a specific translocation [t(X;18)(p11;q11)], and liposarcoma, which is characterized by 12q13∼15 amplification. (ey also 2.2. Tumor Infiltrating Lymphocytes (TILs). Sarcomas have include gastrointestinal stromal tumor (GIST), which is fewer TILs per gram of tissue and lower ratios of TIL in- filtration, when compared to cancers such as melanoma and characterized by activating mutations of the KIT receptor tyrosine kinases. (e “complex” tumors, such as un- renal cell carcinoma [16]. In some studies, including studies of patients with GIST, angiosarcoma, leiomyosarcoma, sy- differentiated pleomorphic and leiomyosarcoma, have nu- merous genetic mutations but no clear oncogenic driver. (e novial sarcoma, and undifferentiated pleomorphic sarcoma, the presence of TILs has been associated with improved Cancer Genome Atlas Research Network has recently re- ported on the genomic characteristics of 206 soft tissue prognosis [17–19]. Other studies of multiple soft tissue sarcomas [7]. Over 3000 soft tissue sarcomas were included sarcoma histologies, including GIST, leiomyosarcoma, and among the 100,000 cancers analyzed by a targeted genomic undifferentiated pleomorphic sarcoma, have shown either profiling reported by Chalmers et al. [8]. (ese studies, along worse survival or no effect on survival [20–22]. In several + + with a study reported by Barretina et al. of 207 tumors [9], cancer types, CD3 and CD8 TILs have been most strongly confirm that the mutational burden in soft tissue sarcoma is associated with improved survival [23]. Issels et al. reported not nearly as high as that in traditionally “immunogenic” on an analysis 341 high-risk localized soft tissue sarcoma tumors, such as melanoma. Median somatic rates of ap- patients treated with neoadjuvant chemotherapy and hy- proximately 2 per megabase (Mb) of DNA have been ob- perthermia. High TIL counts were associated with enhanced served. In melanoma, this rate is approximately 14 per Mb. progression-free and disease-free survivals [24]. Sarcoma 3 T-cell receptor sequencing, was found to have higher levels Sorbye et al. analyzed 249 soft tissue sarcomas, pre- dominantly undifferentiated pleomorphic and liposarcomas, of PD-L1 and PD-1 on immunohistochemistry, significantly + + + + + more than synovial sarcoma, which had the lowest. PD-L1 for CD3 , CD4 , CD8 , CD20 , and CD45 lymphocyte infiltration using immunohistochemistry. Only CD20 B cells and PD-1 expression again was not associated with were independently associated with improved disease-free progression-free of overall survivals. Differences in tumor + + survival [20]. Unexpectedly, low CD3 and CD4 T-cell in- PD-L1 was also not associated with survivals in the analysis filtrations were associated with better overall survival. of high-risk localized soft tissue sarcomas performed by Gene expression profiling has been used to assess Issels et al. [24]. In the Research Network analysis, the intratumoral immune response, and specific signatures that highest PD-L1 score, which also was not correlated with reflect T-cell activation have been shown to have prognostic survivals, was observed in leiomyosarcoma [8]. Character- and/or predictive value [25]. Two recent studies applying ization of the immune microenvironment of malignant peripheral nerve sheath tumor resulted in absence of PD1, molecular techniques have provided more information re- garding the immune cell infiltration into soft tissue sarco- low PDL1 expression, and minimal CD8 infiltration along with no influence on survival [31]. mas. Pollack et al. performed gene expression and T-cell receptor Vβ gene sequencing on 81 soft tissue sarcomas [26]. It has been noted in mouse fibrosarcoma models that the Undifferentiated pleomorphic and leiomyosarcomas had modest antitumor activity of anti-PD-1 therapy was in- high expression levels of genes related to antigen presen- dependent of PD-L1 staining [32]. Activity was significantly tation and T-cell infiltration. Undifferentiated pleomorphic enhanced when combining anti-PD-1 antibody with anti- sarcoma was found to have the highest T-cell infiltration body against the coinhibitory receptor, LAG-3 [33]. based on T-cell receptor sequencing, significantly more than Information regarding the expression in soft tissue sar- synovial sarcoma, which had the lowest. T-cell infiltrates in coma of LAG-3 and other immune checkpoint ligands and undifferentiated pleomorphic sarcoma also were more oli- receptors is limited. Differential gene expression was found in the Cancer Genome Atlas Research Network analysis of the goclonal compared with synovial sarcoma and liposarcoma. In the Cancer Genome Atlas Research Network study, coinhibitory ligand B7-H3 and the coinhibitor receptor TIM3, with expression highest in dedifferentiated liposarcoma, un- unsupervised clustering identified variable expression of 203 genes involved in immune response [8]. An immune in- differentiated pleomorphic sarcoma, and myxofibrosarcoma filtration score for various immune cells based on their gene [8]. Tumors evade macrophage phagocytosis through the expression signatures was developed. CD8 cell score corre- expression of antiphagocytic signals within the tumor mi- lated with improved survival in gynecologic leiomyosarco- croenvironment. (ese include the CD47-SIRPα pathway [34]. mas. Dendritic cell (DC) scores correlated with improved Antibody therapy targeting the CD47 protein was effective in survival in myxofibrosarcoma/undifferentiated pleomorphic a metastatic leiomyosarcoma model [35]. TI-621, a recombi- nant fusion protein that blocks the CD47-SIRPα axis, is being sarcoma, suggesting a role for antigen presentation in the immunologic response to these tumors. Scores related to tested in clinical trials [34]. (e role of tumor suppressor cells is also not established. Tumor-associated macrophages and natural killer (NK) cells correlated with disease-specific survival in leiomyosarcomas and myxofibrosarcoma/ regulatory T (Treg) cells suppress antitumor immune re- sponses by several mechanisms and have been shown to be undifferentiated pleomorphic sarcoma. (e infiltration of NK cells, an antigen-nonspecific immune effector, is rarely negative prognostic factors in several cancers. Tumor- noted in human solid tumors, including soft tissue sarcomas associated macrophages have been associated with negative [18, 27]. In dedifferentiated liposarcoma, a T-helper 2 sig- outcomes in uterine and nonuterine leiomyosarcoma and nature, which is associated with inhibition of T-cell cyto- myxoid liposarcoma [36–38]. (e most abundant infiltrating toxicity, was correlated with shorter disease-specific survival. immune cells in GIST include M2 macrophage, an immune- suppressive phenotype [39]. In the Cancer Genome Atlas Research Network analysis, undifferentiated pleomorphic, 2.3. Immune Suppressive Mechanisms. (e role of specific myxofibrosarcoma, and dedifferentiated liposarcoma had the highest median macrophage scores [40]. Increasedrecruitment immune checkpoints, including PD-L1/PD-1, in soft tissue sarcomas is not established. Tumor expression of PD-L1 has of macrophages in malignant peripheral nerve sheath tumors (MPNSTs) indicate that these tumors may be candidates for been associated with a worse prognosis in most cancers [28]. Kim et al. reported that the expression of PD-L1 by soft response with certain immunotherapy agents [41]. tissue sarcomas predicts a poor prognosis [24, 29]. Addi- (e majority of tumor-infiltrating Treg cells, which tionally, the degree of TIL PD-1 positivity showed similar express FoxP3, have been detected in GISTrather than non- results. D’Angelo et al. noted tumor, lymphocyte, and GIST sarcoma [8]. High FoxP3 infiltrates have been re- macrophage PD-L1 expression to be 12%, 30%, and 58%, ported to correlate with high-risk GIST [42]. Differences in respectively, with the highest prevalence, 29%, in GIST [30]. tumor infiltration of FoxP3 cells were not associated with In contrast to the work of Kim et al., there was no association survivals in the analysis of high-risk localized non-GISTsoft between clinical features, overall survival, and PD-L1 ex- tissue sarcoma performed by Issels et al. pression in tumor or immune infiltrates. Differences have Several soluble factors have been implicated. (e ex- been noted among soft tissue sarcoma subtypes. In the study pression of transforming growth factor (TGF) β, a cytokine reported by Pollack et al., undifferentiated pleomorphic that inhibits antitumor immunity by several mechanisms, sarcoma, which had the highest T-cell infiltration based on has been associated with a poorer survival in soft tissue 4 Sarcoma sarcoma [43]. Higher expression of the immune-suppressive released by tumor dying in situ are processed and presented cytokine gene TGFB1 in undifferentiated pleomorphic, to expand adaptive, antitumor Tcells systemically as well as myxofibrosarcoma, and dedifferentiated liposarcoma was to generate immunologic memory (Figure 1). Local in- noted in the Cancer Genome Atlas Research Network jections allow much higher concentrations of the immu- analysis [31]. (e proangiogenic cytokine, vascular endo- nostimulatory products in the tumor microenvironment thelial growth factor (VEGF), is a significant mediator of than do systemic infusions, which may be important in immune suppression within the tumor microenvironment, overwhelming immune suppressor mechanisms. Moreover, primarily as an inhibitor of DC function. VEGF is frequently local delivery of immunostimulating drugs should prevent overexpressed in soft tissue sarcomas [44]. their circulation at high concentrations in the blood. (us, Indoleamine-2,3-dioxygenase, which catalyzes the oxi- intratumoral immunotherapy should provide improved dative breakdown of the essential amino acid tryptophan, via efficacy and lower toxicity. Intratumoral immunotherapy the kynurenine pathway, is an inhibitor of T-cell pro- has been applied to manage accessible lesions and to induce liferation that has been implicated in immune resistance in systemic immunity in several cancers. Intratumoral Bacillus Calmette–Guerin ´ (BCG), which has been used for 40 years, several cancers, including soft tissue sarcoma [45]. Many of these processes can be operational. Peng et al. is in common use to treat nonmuscle invasive bladder recently reported an analysis of the primary tumor and the cancer. Recently, an intratumoral therapy with talimogene sole treatment-resistant metastasis of a patient with meta- laherparepvec (T-VEC), an attenuated herpes simplex virus, static uterine leiomyosarcoma who responded to the anti- type 1 (HSV-1) engineered to express human granulocyte- PD-1 antibody, pembrolizumab. (ey identified PTEN macrophage colony-stimulating factor (GM-CSF), has been mutations and reduced expression of genes encoding neo- approved to treat patients with melanoma. antigens as potential mediators of resistance to immune (e use of intratumoral immunotherapy to treat sar- checkpoint therapy [46]. It was noted that both tumors coma is supported not only by the therapeutic observations stained diffusely for PD-L2 and showed sparse PD-L1 of Coley and others but also by other clinical observations. staining. PD-1 cell infiltration significantly decreased in Lewis et al. compared 685 sarcoma patients who underwent initial definitive resection to 407 similar patients treated with the resistant tumor. (e tumor suppressor PTEN in mela- noma models leads to immunoresistance by inducing VEGF a definitive re-excision following a previous nontherapeutic excision [54]. Unexpectedly, the 5-year disease-free survival and other immunosuppressive cytokines [47]. An increase in VEGFA gene expression was observed in the treatment- for re-resected patients was significantly higher than the resistant tumor [48]. definitive patients, 88% versus 70%. (is survival difference Finally, biophysical properties of the tumor microen- could not be explained by a referral bias. When analyzed vironment can also promote immunotherapy resistance and according to stage, all re-resected patients trended toward an immune suppression. (e abnormal structure and function improved outcome in comparison with the definitively of the microvasculature that characterize solid tumors and treated group. (ese observations extended beyond the local the increases in tumor interstitial fluid pressure that result in recurrence-free survival, as there was also an improvement the tumor microenvironment act as a physiological barrier in metastasis-free survival for the re-resected group, sug- gesting a possible systemic effect. It has been postulated that to the delivery of therapeutic agents. Abnormal blood flow can also act as a barrier for immune factors and immune cell the local inflammatory response induced with incomplete migration into the tumor parenchyma. (ere is evidence initial excision may prime an immune response against that this barrier may contribute to the limited efficacy of remaining tumor cells. immunotherapy [49]. Hypoxia in the tumor microenvi- Several phenomena may limit the systemic effects of ronment, in part due to the abnormal vascularity, is con- intratumoral immunotherapy (Figure 1). Negative feedback sidered to play a central role in the suppression of immune loops designed to alleviate a local inflammatory state may effector cells and enhancement of tumor escape from im- paradoxically cause systemic immunosuppression [55]. (e mune surveillance [50]. Soft tissue sarcomas are charac- clearance of tumor cells undergoing apoptosis in situ, terized by high microvascular densities [51], very high tumor a process referred to as efferocytosis, is programmed to lead to interstitial fluid pressures [52], and significant hypoxia [53]. compartmentalization and anti-inflammatory processing of intracellular self-antigens. Efferocytosis prevents leakage of cytotoxic or antigenic intracellular contents by dying cells and 3. Intratumoral Immunotherapy results in the release of anti-inflammatory cytokines, such as (e intratumoral administration of immunologically active TGF-β, that suppress the production of proinflammatory mediators locally and systemically, resulting in decreased DC agents is one approach of directly addressing the limitations presented by the lack of tumor antigenicity and maturation and antigen-specific T cells [56]. Any tissue effector lymphocyte infiltration and the multiple immune trauma stimulates inflammatory responses that are highly suppressive mechanisms within the soft tissue sarcoma regulated and result in limiting damage both locally and microenvironment. Intratumoral immunotherapy is not systemically. Although proportional to the degree of the initial only a method of killing the treated tumor but is also an insult, even minor trauma is associated with systemic im- expedient method of generating systemic antitumor im- mune suppression, including decreases in T and NK cell responses. Just as the upregulation of PD-1 has been impli- munity. Immune affector mechanisms as well as immune effector mechanisms can be activated, so that tumor antigens cated in inflammation caused by tissue trauma, several soluble Sarcoma 5 Local tumor killing Release of tumor antigens Activation of DCs Generation of systemic antitumor immunity Inhibition of systemic antitumor immunity Trafficking of DC to draining lymph nodes Release of anti-inflammatory cytokines Activation of antitumor T cells Upregulation of immune checkpoints Generation of immunologic memory Induction of Treg cells and MDSC Figure 1: Mechanisms regulating the activity of intratumoral immunotherapy. mediators, such as TGF-β and complement products, and capsid for enhanced cancer cell transduction and a deletion cellular mediators, such as Treg cells and myeloid-derived in the E1A gene, which also promotes proliferation, that supressor cells (MDSCs), have been implicated [57, 58]. binds the tumor suppressor, the retinoblastoma protein (Rb), rendering the viral replication to cells that lack Rb, also 4. Clinical Studies commonly observed in soft tissue sarcoma [68, 69]. Because deletion of the RL1 gene and the c 34.5 gene, respectively, Whether intratumoral immunotherapy can impact the both which encode virulence factors and the herpes virus course of sarcoma has not been established. Mainly pilot and vectors HSV1716 and HSV-1 M002, is unable to replicate in phase I studies involving small numbers of patients have nondividing cells, and preferentially infect, replicate in, and been performed. €ere are no randomized studies. None- lyse rapidly dividing cells such as tumor cells [70, 71]. theless, antitumor activity has been observed. Multiple €e incorporation of immune stimulatory cytokines has approaches are being investigated in preclinical studies in also been used. GM-CSF, a potent immunostimulatory sarcoma models but have yet been advanced to clinical trials. cytokine that recruits and activates antigen-presenting cells, €e intratumoral immunotherapy approaches that have has been included in several constructs, including ONCOS- been tested clinically or are in the process of being tested 102 [68], Ad5/3-D24-GMCSF [72], and JX-594 (pex- clinically are summarized below. astimogene devacirepvec), an oncolytic vaccinia virus [73]. TNFerade is a replication-deŠcient adenovirus that ex- 4.1. Viral Vectors. Viruses engineered to exploit their in- presses TNFα, which not only inhibits tumors directly but herent antigenicity/immunogenicity as well as to express also has multiple immune e‰ects, including activation of Tcells and DC [74, 75]. €e TNF-α in TNFerade is expressed immunostimulatory molecules are emerging as clinically relevant cancer therapeutics. While direct killing of infected downstream of the radiation-inducible Egr-1 promoter tumor cells is central to their antitumor e‰ect, their ability to gene, which provides spatial and temporal control of the enhance immune responses generated to the tumor antigens cytotoxicity provided by TNF-α when administered intra- released through that process is also considered key. tumorally with radiation [76]. HSV-1 M002 is also engi- Intratumoral therapy with viruses has been shown to induce neered to expresses interleukin- (IL-) 12, a major activator of immune inŠltrates not only in the injected tumor but also in T and NK cells [71]. distant tumor [59]. Adenoviral vectors, which can accom- Several factors can be limiting in viral therapies for modate relatively large segments of DNA, have a broad host cancer. Neutralizing antibodies are highly prevalent and range and lack pathogenicity, and herpes viruses, which can can reduce the e¤cacy of repeat injections although how also accommodate relatively large segments of DNA and are antivector immunity in¥uences the clinical or biologic strongly cytolytic in human cancer cells, have been the best response of intratumoral virotherapy is not known. Ex- studied (Table 1). tracellular matrix and areas of tissue necrosis, which can be Several strategies have been used to enhance the tumor seen in high-grade tumors, may impair the spread of vi- speciŠcity. ONYX-015 is an E1B-55K gene-deleted adeno- ruses [77]. Hypoxia, a key regulator of the tumor micro- virus. ONYX-015 was originally designed to selectively environment, has been shown to decrease infectivity and replicate in and lyse p53-deŠcient cancer cells [64, 65]. cytotoxicity of HSV [78]. Macrophages can either support Sarcomas manifest a high frequency of p53 mutations and oncolytic virus therapy through proin¥ammatory stimu- functional p53 inactivation [66]. However, ONYX-015 was lation of the antitumor response at the cost of hindering later found to be e‰ective regardless of p53 status, indicating direct oncolysis or through immunosuppressive protection that other mechanisms are responsible for its tumor spec- of virus replication at the cost of hindering the antitumor iŠcity [67]. ONCOS-102 is an adenovirus with an engineered immune response [79]. 6 Sarcoma Table 1: Clinical trials of intratumoral viral vector therapy in soft parvum, an approach also tested in the 1970s [61]. (e tissue sarcoma. antitumor activity observed was not considered to be suf- ficient to warrant regulatory approval. Species of Clostridium Agent N Results Ref. bacteria are notable for their ability to lyse tumor cells Of 13 evaluable patients, 11 (85%) growing in hypoxic environments. More recently, spores TNFerade 14 objective or pathological tumor [60] from an attenuated strain of Clostridium novyi (C. novyi- responses (2 CR and 9 PR), 1 SD NT) have been developed as a therapeutic. A patient with ONYX-015 ONYX-015 viral DNA detected in 2 advanced leiomyosarcoma has been reported to respond to (+mitomycin-C, 6 patient biopsies and 5 patient plasma [61] an intratumoral injection of C. novyi-NT spores [62]. doxorubicin, after treatment cisplatin) Pathogen-associated molecular patterns (PAMPs), including 2 minor responses, 6 SD, and 4 PD in synthetic constructs that mimic compounds expressed by Ad5/3-D24- 12 evaluable patients; median survival several types of microbes, are potent immunomodulators. 15 [62] GMCSF time after treatment was 170 days. (e sequential intratumoral and intramuscular injections of One patient was alive at 1459 days the synthetic PAMP, polyinosinic-polycytidylic acid- Four of 5 patients evaluated at day polylysine-carboxymethylcellulose (Poly-ICLC), has been +14 had stable disease by cross- reported to be active in sarcoma [63]. sectional imaging. (ree of 7 patients HSV1716 9 evaluated at day +28 had stable [63] disease, and one of these patients had 5. Future Prospects a decrease in PETstandardized uptake values (e rationale as well as the feasibility and safety for the intratumoral injection of immunotherapeutics into sarcoma tumors has been established in several studies. Several 4.2. Immune Cells. Administration of DCs intratumorally clinical trials are in progress (Table 2). has been tested clinically in several cancers [80]. A Phase I Further clinical investigation is needed to better define study was conducted in 18 patients with high-risk localized how the expression and presentation of intratumoral anti- soft tissue sarcoma of the intratumoral injection of DCs gens are regulated in soft tissue sarcomas. (ere remain combined with radiation [81]. An encouraging 11 of 18 many unanswered questions about which immune cells may (61%) patients were alive with no systemic recurrence over dictate prognosis, and no available data to date correlating a period of 2 to 8 years. Ten out of 18 (56%) demonstrated immune infiltration with response to modern immuno- evidence of a systemic immune response to either tumor cell therapy in sarcoma. (e immune checkpoints operational in lysates or to survivin, a sarcoma-associated antigen. A soft tissue sarcoma progression are not established. A better freeze-stored allogeneic DC preparation, known as INTO- assessment of particular T-cell phenotypes, activation status, VAX, in which DC derived from blood of healthy donors and the presence of other suppressive immune cells and with GM-CSF and IL-4 are 17 activated with toll-like re- factors is needed to optimize intratumoral immunotherapy. ceptor (TLR) 7/8 agonist R848and TLR3 agonist Poly I:C, Although generating systemic antitumor immunity is and human recombinant interferon gamma is under in- a major goal of intratumoral immunotherapy, very few vestigation [82]. studies have examined systemic immune responses. Neo- adjuvant approaches in patients with high-risk localized soft tissue sarcomas considered candidates for surgery would be 4.3. Cytokines. Cytokines are in common use to treat many an ideal system to examine these. malignancies. As noted, viral vectors have been developed to (ere is little to suggest that intratumoral immuno- express several cytokines intratumorally, including GM-CSF, therapy that focuses only on one aspect of immune effector TNF, and IL-12. (e intratumoral administration of IL-2, activation will be highly effective either locally or systemi- a major activator of lymphocyte cytotoxicity, has also been cally. Single agent therapy is usually not completely effective, recommended as treatment options for patients with in- even in preclinical models. Studies of treatments in which transit melanoma metastases. Although tumor regressions mechanistically distinct immunotherapeutics are combined were not observed, 6 of fifteen sarcoma patients that were need to continue. Systemic chemotherapy has been shown to included in a study of the intratumoral administration of increase the antitumor activity of intratumoral immuno- direct gene transfer of an IL-2 DNA/DMRIE/DOPE lipid therapy [85, 86]. Radiation has also been effectively applied complex had stable disease lasting from 3 to 18 months and to enhance intratumoral immunotherapy [87]. (ere are continuing [83]. One of the three patients with soft tissue many questions that need to be addressed regarding the sarcoma-treated IL-2-transfected xenogeneic cells (Vero-IL-2) immune effects of chemotherapy and radiation in patients showed durable reduction of two distant, noninjected me- with soft tissue sarcoma. Other combination approaches the tastases [84]. merit study. Efferocytosis may be modifiable [56]. Ap- proaches to disrupt the sarcoma stroma by targeting, for 4.4. Microbial Products. Intratumoral injections of BCG example, matrix metalloproteinases, angiogenic factors, and were tested in patients with sarcoma in several trials con- hyaluronic acid, and to improve tumor oxygenation are ducted in the 1970s [60]. Tumor regressions were also re- under investigation [88]. (ese approaches may have ap- ported with the intratumoral injections of Corynebacterium plication to immunotherapeutics. Sarcoma 7 Table 2: Currently recruiting clinical trials of intratumoral im- have failed to demonstrate significant clinical activity due to munotherapy in soft tissue sarcoma. a tumor microenvironment characterized by low tumor antigenicity, limited infiltration of effector cells, and several ClinicalTrials.gov Treatment Phase processes that suppress immune cell function. Intratumoral identifier immunotherapy can enhance tumor antigenicity, promote HSV-1 M002 (IT or IV) I NCT00931931 TILs, and generate a systemic antitumor immune response. HSV-1 M002+concurrent radiation I/II NCT02453191 Because tumor is often easily accessible and immune HSV-1 M002+pembrolizumab II NCT03069378 mechanisms are implicated in regulating its progression, the TVEC+preoperative radiation I/II NCT02453191 JX-594+cyclophosphamide I/II NCT02630368 intratumoral application of immune modulators has been an TTI-621 I NCT02890368 attractive treatment for sarcoma. Although intratumoral INTUVAX I NCT02686944 immunotherapy has not yet been established as a standard Poly-ICLC II NCT01984892 option in the treatment in sarcoma, its feasibility, safety, and Clostridium novyi-NT spores I NCT01924689 biologic activity have been proven in clinical trials. (e ra- tionale design of immunotherapy strategies will require im- proved understanding of the regulation of immune responses Since conventional computed tomography (CT) or in soft tissue microenvironment. Given Coley’s early foray, it magnetic resonance imaging (MRI) alone may not be ade- has been disappointing that research into the immune biology quate to determine immunotherapy response, the utility of of soft tissue sarcoma has been slower than in other cancers. more functional imaging, such as positron emission to- Progress has recently been made. Novel, more effective im- mography, diffusion-weighted imaging, dynamic contrast- munotherapeutic strategies should be forthcoming. enhanced magnetic resonance imaging, and perfusion com- puted tomography, should be investigated. Furthermore, fine- Conflicts of Interest needle injection expertise is not uniformly available, even among cancer centers. Development of specific devices, in- (e authors declare that there are no conflicts of interest cluding needles suited for the administration of immunologic regarding the publication of this paper. agents, is another issue to be addressed. Newer systems, such as three-dimensional ultrasound-CTdual imaging, should be Acknowledgments tested to plan and to monitor immune drug delivery. Finally, novel animal models are needed. Most studies (e authors thank Dr. Douglas Lyles for helpful suggestions have been performed using chemically induced tumors in on the manuscript. mice. (e immune system of rodents, however, has well recognized differences from that of humans. It should be noted that most studies applied intratumoral treatments to References subcutaneous and not orthotopic tumors. It is not clear that [1] A. J. Jacobs, R. Michels, J. Stein, and A. S. Levin, “Im- the microenvironment of even orthotopic rodent sarcoma provement in overall survival from extremity soft tissue mimics the human situation. Immunotherapy studies require sarcoma over twenty years,” Sarcoma, vol. 2015, Article ID mouse models with an intact immune system. 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Addressing the Adult Soft Tissue Sarcoma Microenvironment with Intratumoral Immunotherapy

Sarcoma , Volume 2018: 10 – Aug 12, 2018

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Hindawi Publishing Corporation
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Copyright © 2018 Shailaja Raj et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2018/9305294
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Abstract

Hindawi Sarcoma Volume 2018, Article ID 9305294, 10 pages https://doi.org/10.1155/2018/9305294 Review Article AddressingtheAdultSoftTissueSarcomaMicroenvironment with Intratumoral Immunotherapy Shailaja Raj , Lance D. Miller, and Pierre L. Triozzi Wake Forest School of Medicine, Winston-Salem, NC, USA Correspondence should be addressed to Shailaja Raj; skrimuno@gmail.com Received 28 March 2018; Accepted 16 July 2018; Published 12 August 2018 Academic Editor: Eugenie S. Kleinerman Copyright © 2018 Shailaja Raj et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sarcoma is comprised of a heterogeneous group of tumors originating from the mesenchyme. Sarcoma is also the first tumor that responded to immunotherapeutic agents often termed as “Coley’s toxins.” However, immunotherapy is yet to establish its presence in sarcomas. Complex interactions between tumor and immune cells in the tumor microenvironment play a crucial role in response to immunotherapy. (ere is a dynamic equilibrium created by the immune cells infiltrating the tumor, and this forms the basis of tumor evasion. Manipulating the intratumoral microenvironment will help overcome tumor evasion. outcome of patients with metastatic soft tissue sarcoma. (e 1. Introduction prognosis of these patients is very poor. Median overall survival is 8 to 12 months [1]. In this review, we first explore the specific oncogenic alter- ation characteristics of different subtypes of sarcoma. (is is Immunotherapy has been an attractive approach to treat followed by a description of the mechanisms by which tumor refractory cancers. Sarcoma is considered to be the first infiltrating lymphocytes affect prognosis and the specific cancer for which immunotherapy was effectively applied. immune cell populations that can be targeted and manipu- Based on observations of tumor regressions in patients with lated in different subtypes of sarcoma. We also review the concomitant streptococcal infections, William B. Coley various immune suppressive mechanisms including immune injected streptococcal organisms into tumors, especially check points, receptors, and tumor-associated macrophages sarcomas, in the last decade of the 19th century [2]. Over and their relevance in sarcoma. We then focus on intra- 50% of the inoperable sarcoma patients who are Coley tumoral immunotherapy, mechanisms of immune interac- treated were reported to respond completely. Furthermore, tions, limitations, and the types of intratumoral therapies approximately 20% survived over 20 years [2]. With their poorly characterized preparation and unpredictable toxic- including oncolytic viruses, immune cells, and cytokines. We foresee intratumoral immunotherapies being able to ities, “Coley’s toxins” never became clinically useful. Im- target and influence management of sarcomas in the future. munotherapeutic approaches have been tested in patients Soft tissue sarcomas arise from cells of mesenchymal with soft tissue sarcoma. (e results have not been as lineage, including muscle, fat, blood vessel, and nerve. Over spectacular as some of the other solid tumors. Immune 50 histological types have been identified. Localized tumors checkpoint blockade with antibodies that target cytotoxic are usually well controlled with surgery. Localized tumors T lymphocyte-associated antigen 4 (CTLA-4) and the that have high-grade histologies and those are over 5cm, programmed cell death protein 1 pathway (PD-1/PD-L1) is however, have over a 50% risk of recurrence. Patients with leading to durable clinical responses in an increasing high-risk localized tumors are usually treated with combi- number of cancers. However, responses in patients with soft nations of surgery, radiation, and/or chemotherapy. (ese tissue sarcoma have been infrequent [3–6]. Immunotherapy response is dependent on complex approaches have failed to substantially improve overall survival. Chemotherapy has not significantly impacted the interactions between tumor and immune cells within the 2 Sarcoma tumor microenvironment. Several factors determine Mutational frequencies vary by subtype. Some, including whether or not immunotherapy response will be promoted angiosarcoma, leiomyosarcoma, and undifferentiated pleo- morphic sarcoma, manifest higher frequencies, in the 2-3 or inhibited. (ese include the inherent antigenicity of the tumor. Mutations in proteins and/or aberrant proteins per Mb range. Most, including myxofibrosarcoma, lip- expressed by tumor cells and the “neoantigens” they gen- osarcoma, and synovial sarcoma, manifest very low fre- erate are the primary targets for T-cell-mediated destruction. quencies, less than 2 per Mb. Nonetheless, patients with high Tumor mutation burden has emerged as a quantitative tumor mutational burdens can be identified in nearly every marker that can help predict responses to immune check- type of soft tissue sarcoma. For example, approximately 13% point inhibition across different cancers. Immunotherapy of angiosarcomas manifest mutation frequencies of more response is also dependent on the infiltration into tumor than 20 per Mb [10]. It should be noted that tumor of immune effector cells. Specific patterns of tumor microsatellite instability, which is an indication for treat- infiltrating lymphocytes (TILs) within the tumor microen- ment with PD-1 inhibitors because of the associated increase vironment are associated with improved outcome in patients in mutations, is not considered to play a major role in soft tissue sarcoma tumorigenesis [9]. with many types of cancers, regardless of the type of therapy administered. Most importantly, a variety of processes (e fusion proteins that result from chromosomal within the microenvironment can suppress interactions translocations represent potential targets. Although there between tumors and immune effector cells and promote the may be tolerance toward epitopes within these proteins, the escape of tumors from immune surveillance. Immune area of fusion in effect represents a neoantigen. Worley et al. checkpoint ligands and receptors have emerged as the major examined the immunogenicity of these areas for sarcoma targetable mechanism of tumor immune escape. Several subtypes characterized by specific translocations, including other molecular, soluble, and cellular factors are involved, synovial, clear cell, and desmoplastic round cell [11]. Pep- and whether or not these factors are being addressed will also tides corresponding to the fusion proteins were designed determine immunotherapy sensitivity. and assessed for the ability to bind to various HLA class I Understanding the soft tissue sarcoma microenviron- molecule. (ese peptides were effectively used in vitro to generate antigen-specific T cells with cytotoxic function ment is not only critical relative to improving the efficiency of current immunotherapies but also for the development of against tumor cells expressing the fusion protein. more effective approaches. In this review, we focus on the Several soft tissue sarcoma subtypes also naturally have recent advances made in understanding the immune mi- high expression of cancer testis antigens, which are con- croenvironment in soft tissue sarcoma. We also discuss the sidered targets for immunotherapy, since in adults these rationale for building upon Coley’s work and directly proteins are expressed only by germ cells, for example, in modifying the soft tissue sarcoma microenvironment using testis tissue, and not by somatic tissue cells [11, 12]. NY- the intratumoral administration of immunologically active ESO-1 is the most notable of the cancer testis antigens agents. described in sarcoma [13, 14]. In synovial sarcoma, NY- ESO-1, as assessed by immunohistochemistry, was expressed in 20 out of 25 cases (80%) [15]. 2. Tumor Immune Microenvironment Expression has also been observed in over 90% of 2.1. Tumor Antigenicity. Soft tissue sarcomas often are di- myxoid and round-cell liposarcoma. Of note, synovial sarcoma, and myxoid and round-cell liposarcoma are vided genetically into two categories: “simple” and “com- plex.” (e simple tumors tend to have specific oncogenic translocation-driven malignancies with very low muta- tional burdens. alterations and a limited number of mutations. (ese in- clude synovial cell sarcoma, which is characterized by a specific translocation [t(X;18)(p11;q11)], and liposarcoma, which is characterized by 12q13∼15 amplification. (ey also 2.2. Tumor Infiltrating Lymphocytes (TILs). Sarcomas have include gastrointestinal stromal tumor (GIST), which is fewer TILs per gram of tissue and lower ratios of TIL in- filtration, when compared to cancers such as melanoma and characterized by activating mutations of the KIT receptor tyrosine kinases. (e “complex” tumors, such as un- renal cell carcinoma [16]. In some studies, including studies of patients with GIST, angiosarcoma, leiomyosarcoma, sy- differentiated pleomorphic and leiomyosarcoma, have nu- merous genetic mutations but no clear oncogenic driver. (e novial sarcoma, and undifferentiated pleomorphic sarcoma, the presence of TILs has been associated with improved Cancer Genome Atlas Research Network has recently re- ported on the genomic characteristics of 206 soft tissue prognosis [17–19]. Other studies of multiple soft tissue sarcomas [7]. Over 3000 soft tissue sarcomas were included sarcoma histologies, including GIST, leiomyosarcoma, and among the 100,000 cancers analyzed by a targeted genomic undifferentiated pleomorphic sarcoma, have shown either profiling reported by Chalmers et al. [8]. (ese studies, along worse survival or no effect on survival [20–22]. In several + + with a study reported by Barretina et al. of 207 tumors [9], cancer types, CD3 and CD8 TILs have been most strongly confirm that the mutational burden in soft tissue sarcoma is associated with improved survival [23]. Issels et al. reported not nearly as high as that in traditionally “immunogenic” on an analysis 341 high-risk localized soft tissue sarcoma tumors, such as melanoma. Median somatic rates of ap- patients treated with neoadjuvant chemotherapy and hy- proximately 2 per megabase (Mb) of DNA have been ob- perthermia. High TIL counts were associated with enhanced served. In melanoma, this rate is approximately 14 per Mb. progression-free and disease-free survivals [24]. Sarcoma 3 T-cell receptor sequencing, was found to have higher levels Sorbye et al. analyzed 249 soft tissue sarcomas, pre- dominantly undifferentiated pleomorphic and liposarcomas, of PD-L1 and PD-1 on immunohistochemistry, significantly + + + + + more than synovial sarcoma, which had the lowest. PD-L1 for CD3 , CD4 , CD8 , CD20 , and CD45 lymphocyte infiltration using immunohistochemistry. Only CD20 B cells and PD-1 expression again was not associated with were independently associated with improved disease-free progression-free of overall survivals. Differences in tumor + + survival [20]. Unexpectedly, low CD3 and CD4 T-cell in- PD-L1 was also not associated with survivals in the analysis filtrations were associated with better overall survival. of high-risk localized soft tissue sarcomas performed by Gene expression profiling has been used to assess Issels et al. [24]. In the Research Network analysis, the intratumoral immune response, and specific signatures that highest PD-L1 score, which also was not correlated with reflect T-cell activation have been shown to have prognostic survivals, was observed in leiomyosarcoma [8]. Character- and/or predictive value [25]. Two recent studies applying ization of the immune microenvironment of malignant peripheral nerve sheath tumor resulted in absence of PD1, molecular techniques have provided more information re- garding the immune cell infiltration into soft tissue sarco- low PDL1 expression, and minimal CD8 infiltration along with no influence on survival [31]. mas. Pollack et al. performed gene expression and T-cell receptor Vβ gene sequencing on 81 soft tissue sarcomas [26]. It has been noted in mouse fibrosarcoma models that the Undifferentiated pleomorphic and leiomyosarcomas had modest antitumor activity of anti-PD-1 therapy was in- high expression levels of genes related to antigen presen- dependent of PD-L1 staining [32]. Activity was significantly tation and T-cell infiltration. Undifferentiated pleomorphic enhanced when combining anti-PD-1 antibody with anti- sarcoma was found to have the highest T-cell infiltration body against the coinhibitory receptor, LAG-3 [33]. based on T-cell receptor sequencing, significantly more than Information regarding the expression in soft tissue sar- synovial sarcoma, which had the lowest. T-cell infiltrates in coma of LAG-3 and other immune checkpoint ligands and undifferentiated pleomorphic sarcoma also were more oli- receptors is limited. Differential gene expression was found in the Cancer Genome Atlas Research Network analysis of the goclonal compared with synovial sarcoma and liposarcoma. In the Cancer Genome Atlas Research Network study, coinhibitory ligand B7-H3 and the coinhibitor receptor TIM3, with expression highest in dedifferentiated liposarcoma, un- unsupervised clustering identified variable expression of 203 genes involved in immune response [8]. An immune in- differentiated pleomorphic sarcoma, and myxofibrosarcoma filtration score for various immune cells based on their gene [8]. Tumors evade macrophage phagocytosis through the expression signatures was developed. CD8 cell score corre- expression of antiphagocytic signals within the tumor mi- lated with improved survival in gynecologic leiomyosarco- croenvironment. (ese include the CD47-SIRPα pathway [34]. mas. Dendritic cell (DC) scores correlated with improved Antibody therapy targeting the CD47 protein was effective in survival in myxofibrosarcoma/undifferentiated pleomorphic a metastatic leiomyosarcoma model [35]. TI-621, a recombi- nant fusion protein that blocks the CD47-SIRPα axis, is being sarcoma, suggesting a role for antigen presentation in the immunologic response to these tumors. Scores related to tested in clinical trials [34]. (e role of tumor suppressor cells is also not established. Tumor-associated macrophages and natural killer (NK) cells correlated with disease-specific survival in leiomyosarcomas and myxofibrosarcoma/ regulatory T (Treg) cells suppress antitumor immune re- sponses by several mechanisms and have been shown to be undifferentiated pleomorphic sarcoma. (e infiltration of NK cells, an antigen-nonspecific immune effector, is rarely negative prognostic factors in several cancers. Tumor- noted in human solid tumors, including soft tissue sarcomas associated macrophages have been associated with negative [18, 27]. In dedifferentiated liposarcoma, a T-helper 2 sig- outcomes in uterine and nonuterine leiomyosarcoma and nature, which is associated with inhibition of T-cell cyto- myxoid liposarcoma [36–38]. (e most abundant infiltrating toxicity, was correlated with shorter disease-specific survival. immune cells in GIST include M2 macrophage, an immune- suppressive phenotype [39]. In the Cancer Genome Atlas Research Network analysis, undifferentiated pleomorphic, 2.3. Immune Suppressive Mechanisms. (e role of specific myxofibrosarcoma, and dedifferentiated liposarcoma had the highest median macrophage scores [40]. Increasedrecruitment immune checkpoints, including PD-L1/PD-1, in soft tissue sarcomas is not established. Tumor expression of PD-L1 has of macrophages in malignant peripheral nerve sheath tumors (MPNSTs) indicate that these tumors may be candidates for been associated with a worse prognosis in most cancers [28]. Kim et al. reported that the expression of PD-L1 by soft response with certain immunotherapy agents [41]. tissue sarcomas predicts a poor prognosis [24, 29]. Addi- (e majority of tumor-infiltrating Treg cells, which tionally, the degree of TIL PD-1 positivity showed similar express FoxP3, have been detected in GISTrather than non- results. D’Angelo et al. noted tumor, lymphocyte, and GIST sarcoma [8]. High FoxP3 infiltrates have been re- macrophage PD-L1 expression to be 12%, 30%, and 58%, ported to correlate with high-risk GIST [42]. Differences in respectively, with the highest prevalence, 29%, in GIST [30]. tumor infiltration of FoxP3 cells were not associated with In contrast to the work of Kim et al., there was no association survivals in the analysis of high-risk localized non-GISTsoft between clinical features, overall survival, and PD-L1 ex- tissue sarcoma performed by Issels et al. pression in tumor or immune infiltrates. Differences have Several soluble factors have been implicated. (e ex- been noted among soft tissue sarcoma subtypes. In the study pression of transforming growth factor (TGF) β, a cytokine reported by Pollack et al., undifferentiated pleomorphic that inhibits antitumor immunity by several mechanisms, sarcoma, which had the highest T-cell infiltration based on has been associated with a poorer survival in soft tissue 4 Sarcoma sarcoma [43]. Higher expression of the immune-suppressive released by tumor dying in situ are processed and presented cytokine gene TGFB1 in undifferentiated pleomorphic, to expand adaptive, antitumor Tcells systemically as well as myxofibrosarcoma, and dedifferentiated liposarcoma was to generate immunologic memory (Figure 1). Local in- noted in the Cancer Genome Atlas Research Network jections allow much higher concentrations of the immu- analysis [31]. (e proangiogenic cytokine, vascular endo- nostimulatory products in the tumor microenvironment thelial growth factor (VEGF), is a significant mediator of than do systemic infusions, which may be important in immune suppression within the tumor microenvironment, overwhelming immune suppressor mechanisms. Moreover, primarily as an inhibitor of DC function. VEGF is frequently local delivery of immunostimulating drugs should prevent overexpressed in soft tissue sarcomas [44]. their circulation at high concentrations in the blood. (us, Indoleamine-2,3-dioxygenase, which catalyzes the oxi- intratumoral immunotherapy should provide improved dative breakdown of the essential amino acid tryptophan, via efficacy and lower toxicity. Intratumoral immunotherapy the kynurenine pathway, is an inhibitor of T-cell pro- has been applied to manage accessible lesions and to induce liferation that has been implicated in immune resistance in systemic immunity in several cancers. Intratumoral Bacillus Calmette–Guerin ´ (BCG), which has been used for 40 years, several cancers, including soft tissue sarcoma [45]. Many of these processes can be operational. Peng et al. is in common use to treat nonmuscle invasive bladder recently reported an analysis of the primary tumor and the cancer. Recently, an intratumoral therapy with talimogene sole treatment-resistant metastasis of a patient with meta- laherparepvec (T-VEC), an attenuated herpes simplex virus, static uterine leiomyosarcoma who responded to the anti- type 1 (HSV-1) engineered to express human granulocyte- PD-1 antibody, pembrolizumab. (ey identified PTEN macrophage colony-stimulating factor (GM-CSF), has been mutations and reduced expression of genes encoding neo- approved to treat patients with melanoma. antigens as potential mediators of resistance to immune (e use of intratumoral immunotherapy to treat sar- checkpoint therapy [46]. It was noted that both tumors coma is supported not only by the therapeutic observations stained diffusely for PD-L2 and showed sparse PD-L1 of Coley and others but also by other clinical observations. staining. PD-1 cell infiltration significantly decreased in Lewis et al. compared 685 sarcoma patients who underwent initial definitive resection to 407 similar patients treated with the resistant tumor. (e tumor suppressor PTEN in mela- noma models leads to immunoresistance by inducing VEGF a definitive re-excision following a previous nontherapeutic excision [54]. Unexpectedly, the 5-year disease-free survival and other immunosuppressive cytokines [47]. An increase in VEGFA gene expression was observed in the treatment- for re-resected patients was significantly higher than the resistant tumor [48]. definitive patients, 88% versus 70%. (is survival difference Finally, biophysical properties of the tumor microen- could not be explained by a referral bias. When analyzed vironment can also promote immunotherapy resistance and according to stage, all re-resected patients trended toward an immune suppression. (e abnormal structure and function improved outcome in comparison with the definitively of the microvasculature that characterize solid tumors and treated group. (ese observations extended beyond the local the increases in tumor interstitial fluid pressure that result in recurrence-free survival, as there was also an improvement the tumor microenvironment act as a physiological barrier in metastasis-free survival for the re-resected group, sug- gesting a possible systemic effect. It has been postulated that to the delivery of therapeutic agents. Abnormal blood flow can also act as a barrier for immune factors and immune cell the local inflammatory response induced with incomplete migration into the tumor parenchyma. (ere is evidence initial excision may prime an immune response against that this barrier may contribute to the limited efficacy of remaining tumor cells. immunotherapy [49]. Hypoxia in the tumor microenvi- Several phenomena may limit the systemic effects of ronment, in part due to the abnormal vascularity, is con- intratumoral immunotherapy (Figure 1). Negative feedback sidered to play a central role in the suppression of immune loops designed to alleviate a local inflammatory state may effector cells and enhancement of tumor escape from im- paradoxically cause systemic immunosuppression [55]. (e mune surveillance [50]. Soft tissue sarcomas are charac- clearance of tumor cells undergoing apoptosis in situ, terized by high microvascular densities [51], very high tumor a process referred to as efferocytosis, is programmed to lead to interstitial fluid pressures [52], and significant hypoxia [53]. compartmentalization and anti-inflammatory processing of intracellular self-antigens. Efferocytosis prevents leakage of cytotoxic or antigenic intracellular contents by dying cells and 3. Intratumoral Immunotherapy results in the release of anti-inflammatory cytokines, such as (e intratumoral administration of immunologically active TGF-β, that suppress the production of proinflammatory mediators locally and systemically, resulting in decreased DC agents is one approach of directly addressing the limitations presented by the lack of tumor antigenicity and maturation and antigen-specific T cells [56]. Any tissue effector lymphocyte infiltration and the multiple immune trauma stimulates inflammatory responses that are highly suppressive mechanisms within the soft tissue sarcoma regulated and result in limiting damage both locally and microenvironment. Intratumoral immunotherapy is not systemically. Although proportional to the degree of the initial only a method of killing the treated tumor but is also an insult, even minor trauma is associated with systemic im- expedient method of generating systemic antitumor im- mune suppression, including decreases in T and NK cell responses. Just as the upregulation of PD-1 has been impli- munity. Immune affector mechanisms as well as immune effector mechanisms can be activated, so that tumor antigens cated in inflammation caused by tissue trauma, several soluble Sarcoma 5 Local tumor killing Release of tumor antigens Activation of DCs Generation of systemic antitumor immunity Inhibition of systemic antitumor immunity Trafficking of DC to draining lymph nodes Release of anti-inflammatory cytokines Activation of antitumor T cells Upregulation of immune checkpoints Generation of immunologic memory Induction of Treg cells and MDSC Figure 1: Mechanisms regulating the activity of intratumoral immunotherapy. mediators, such as TGF-β and complement products, and capsid for enhanced cancer cell transduction and a deletion cellular mediators, such as Treg cells and myeloid-derived in the E1A gene, which also promotes proliferation, that supressor cells (MDSCs), have been implicated [57, 58]. binds the tumor suppressor, the retinoblastoma protein (Rb), rendering the viral replication to cells that lack Rb, also 4. Clinical Studies commonly observed in soft tissue sarcoma [68, 69]. Because deletion of the RL1 gene and the c 34.5 gene, respectively, Whether intratumoral immunotherapy can impact the both which encode virulence factors and the herpes virus course of sarcoma has not been established. Mainly pilot and vectors HSV1716 and HSV-1 M002, is unable to replicate in phase I studies involving small numbers of patients have nondividing cells, and preferentially infect, replicate in, and been performed. €ere are no randomized studies. None- lyse rapidly dividing cells such as tumor cells [70, 71]. theless, antitumor activity has been observed. Multiple €e incorporation of immune stimulatory cytokines has approaches are being investigated in preclinical studies in also been used. GM-CSF, a potent immunostimulatory sarcoma models but have yet been advanced to clinical trials. cytokine that recruits and activates antigen-presenting cells, €e intratumoral immunotherapy approaches that have has been included in several constructs, including ONCOS- been tested clinically or are in the process of being tested 102 [68], Ad5/3-D24-GMCSF [72], and JX-594 (pex- clinically are summarized below. astimogene devacirepvec), an oncolytic vaccinia virus [73]. TNFerade is a replication-deŠcient adenovirus that ex- 4.1. Viral Vectors. Viruses engineered to exploit their in- presses TNFα, which not only inhibits tumors directly but herent antigenicity/immunogenicity as well as to express also has multiple immune e‰ects, including activation of Tcells and DC [74, 75]. €e TNF-α in TNFerade is expressed immunostimulatory molecules are emerging as clinically relevant cancer therapeutics. While direct killing of infected downstream of the radiation-inducible Egr-1 promoter tumor cells is central to their antitumor e‰ect, their ability to gene, which provides spatial and temporal control of the enhance immune responses generated to the tumor antigens cytotoxicity provided by TNF-α when administered intra- released through that process is also considered key. tumorally with radiation [76]. HSV-1 M002 is also engi- Intratumoral therapy with viruses has been shown to induce neered to expresses interleukin- (IL-) 12, a major activator of immune inŠltrates not only in the injected tumor but also in T and NK cells [71]. distant tumor [59]. Adenoviral vectors, which can accom- Several factors can be limiting in viral therapies for modate relatively large segments of DNA, have a broad host cancer. Neutralizing antibodies are highly prevalent and range and lack pathogenicity, and herpes viruses, which can can reduce the e¤cacy of repeat injections although how also accommodate relatively large segments of DNA and are antivector immunity in¥uences the clinical or biologic strongly cytolytic in human cancer cells, have been the best response of intratumoral virotherapy is not known. Ex- studied (Table 1). tracellular matrix and areas of tissue necrosis, which can be Several strategies have been used to enhance the tumor seen in high-grade tumors, may impair the spread of vi- speciŠcity. ONYX-015 is an E1B-55K gene-deleted adeno- ruses [77]. Hypoxia, a key regulator of the tumor micro- virus. ONYX-015 was originally designed to selectively environment, has been shown to decrease infectivity and replicate in and lyse p53-deŠcient cancer cells [64, 65]. cytotoxicity of HSV [78]. Macrophages can either support Sarcomas manifest a high frequency of p53 mutations and oncolytic virus therapy through proin¥ammatory stimu- functional p53 inactivation [66]. However, ONYX-015 was lation of the antitumor response at the cost of hindering later found to be e‰ective regardless of p53 status, indicating direct oncolysis or through immunosuppressive protection that other mechanisms are responsible for its tumor spec- of virus replication at the cost of hindering the antitumor iŠcity [67]. ONCOS-102 is an adenovirus with an engineered immune response [79]. 6 Sarcoma Table 1: Clinical trials of intratumoral viral vector therapy in soft parvum, an approach also tested in the 1970s [61]. (e tissue sarcoma. antitumor activity observed was not considered to be suf- ficient to warrant regulatory approval. Species of Clostridium Agent N Results Ref. bacteria are notable for their ability to lyse tumor cells Of 13 evaluable patients, 11 (85%) growing in hypoxic environments. More recently, spores TNFerade 14 objective or pathological tumor [60] from an attenuated strain of Clostridium novyi (C. novyi- responses (2 CR and 9 PR), 1 SD NT) have been developed as a therapeutic. A patient with ONYX-015 ONYX-015 viral DNA detected in 2 advanced leiomyosarcoma has been reported to respond to (+mitomycin-C, 6 patient biopsies and 5 patient plasma [61] an intratumoral injection of C. novyi-NT spores [62]. doxorubicin, after treatment cisplatin) Pathogen-associated molecular patterns (PAMPs), including 2 minor responses, 6 SD, and 4 PD in synthetic constructs that mimic compounds expressed by Ad5/3-D24- 12 evaluable patients; median survival several types of microbes, are potent immunomodulators. 15 [62] GMCSF time after treatment was 170 days. (e sequential intratumoral and intramuscular injections of One patient was alive at 1459 days the synthetic PAMP, polyinosinic-polycytidylic acid- Four of 5 patients evaluated at day polylysine-carboxymethylcellulose (Poly-ICLC), has been +14 had stable disease by cross- reported to be active in sarcoma [63]. sectional imaging. (ree of 7 patients HSV1716 9 evaluated at day +28 had stable [63] disease, and one of these patients had 5. Future Prospects a decrease in PETstandardized uptake values (e rationale as well as the feasibility and safety for the intratumoral injection of immunotherapeutics into sarcoma tumors has been established in several studies. Several 4.2. Immune Cells. Administration of DCs intratumorally clinical trials are in progress (Table 2). has been tested clinically in several cancers [80]. A Phase I Further clinical investigation is needed to better define study was conducted in 18 patients with high-risk localized how the expression and presentation of intratumoral anti- soft tissue sarcoma of the intratumoral injection of DCs gens are regulated in soft tissue sarcomas. (ere remain combined with radiation [81]. An encouraging 11 of 18 many unanswered questions about which immune cells may (61%) patients were alive with no systemic recurrence over dictate prognosis, and no available data to date correlating a period of 2 to 8 years. Ten out of 18 (56%) demonstrated immune infiltration with response to modern immuno- evidence of a systemic immune response to either tumor cell therapy in sarcoma. (e immune checkpoints operational in lysates or to survivin, a sarcoma-associated antigen. A soft tissue sarcoma progression are not established. A better freeze-stored allogeneic DC preparation, known as INTO- assessment of particular T-cell phenotypes, activation status, VAX, in which DC derived from blood of healthy donors and the presence of other suppressive immune cells and with GM-CSF and IL-4 are 17 activated with toll-like re- factors is needed to optimize intratumoral immunotherapy. ceptor (TLR) 7/8 agonist R848and TLR3 agonist Poly I:C, Although generating systemic antitumor immunity is and human recombinant interferon gamma is under in- a major goal of intratumoral immunotherapy, very few vestigation [82]. studies have examined systemic immune responses. Neo- adjuvant approaches in patients with high-risk localized soft tissue sarcomas considered candidates for surgery would be 4.3. Cytokines. Cytokines are in common use to treat many an ideal system to examine these. malignancies. As noted, viral vectors have been developed to (ere is little to suggest that intratumoral immuno- express several cytokines intratumorally, including GM-CSF, therapy that focuses only on one aspect of immune effector TNF, and IL-12. (e intratumoral administration of IL-2, activation will be highly effective either locally or systemi- a major activator of lymphocyte cytotoxicity, has also been cally. Single agent therapy is usually not completely effective, recommended as treatment options for patients with in- even in preclinical models. Studies of treatments in which transit melanoma metastases. Although tumor regressions mechanistically distinct immunotherapeutics are combined were not observed, 6 of fifteen sarcoma patients that were need to continue. Systemic chemotherapy has been shown to included in a study of the intratumoral administration of increase the antitumor activity of intratumoral immuno- direct gene transfer of an IL-2 DNA/DMRIE/DOPE lipid therapy [85, 86]. Radiation has also been effectively applied complex had stable disease lasting from 3 to 18 months and to enhance intratumoral immunotherapy [87]. (ere are continuing [83]. One of the three patients with soft tissue many questions that need to be addressed regarding the sarcoma-treated IL-2-transfected xenogeneic cells (Vero-IL-2) immune effects of chemotherapy and radiation in patients showed durable reduction of two distant, noninjected me- with soft tissue sarcoma. Other combination approaches the tastases [84]. merit study. Efferocytosis may be modifiable [56]. Ap- proaches to disrupt the sarcoma stroma by targeting, for 4.4. Microbial Products. Intratumoral injections of BCG example, matrix metalloproteinases, angiogenic factors, and were tested in patients with sarcoma in several trials con- hyaluronic acid, and to improve tumor oxygenation are ducted in the 1970s [60]. Tumor regressions were also re- under investigation [88]. (ese approaches may have ap- ported with the intratumoral injections of Corynebacterium plication to immunotherapeutics. Sarcoma 7 Table 2: Currently recruiting clinical trials of intratumoral im- have failed to demonstrate significant clinical activity due to munotherapy in soft tissue sarcoma. a tumor microenvironment characterized by low tumor antigenicity, limited infiltration of effector cells, and several ClinicalTrials.gov Treatment Phase processes that suppress immune cell function. Intratumoral identifier immunotherapy can enhance tumor antigenicity, promote HSV-1 M002 (IT or IV) I NCT00931931 TILs, and generate a systemic antitumor immune response. HSV-1 M002+concurrent radiation I/II NCT02453191 Because tumor is often easily accessible and immune HSV-1 M002+pembrolizumab II NCT03069378 mechanisms are implicated in regulating its progression, the TVEC+preoperative radiation I/II NCT02453191 JX-594+cyclophosphamide I/II NCT02630368 intratumoral application of immune modulators has been an TTI-621 I NCT02890368 attractive treatment for sarcoma. Although intratumoral INTUVAX I NCT02686944 immunotherapy has not yet been established as a standard Poly-ICLC II NCT01984892 option in the treatment in sarcoma, its feasibility, safety, and Clostridium novyi-NT spores I NCT01924689 biologic activity have been proven in clinical trials. (e ra- tionale design of immunotherapy strategies will require im- proved understanding of the regulation of immune responses Since conventional computed tomography (CT) or in soft tissue microenvironment. Given Coley’s early foray, it magnetic resonance imaging (MRI) alone may not be ade- has been disappointing that research into the immune biology quate to determine immunotherapy response, the utility of of soft tissue sarcoma has been slower than in other cancers. more functional imaging, such as positron emission to- Progress has recently been made. Novel, more effective im- mography, diffusion-weighted imaging, dynamic contrast- munotherapeutic strategies should be forthcoming. enhanced magnetic resonance imaging, and perfusion com- puted tomography, should be investigated. Furthermore, fine- Conflicts of Interest needle injection expertise is not uniformly available, even among cancer centers. Development of specific devices, in- (e authors declare that there are no conflicts of interest cluding needles suited for the administration of immunologic regarding the publication of this paper. agents, is another issue to be addressed. Newer systems, such as three-dimensional ultrasound-CTdual imaging, should be Acknowledgments tested to plan and to monitor immune drug delivery. Finally, novel animal models are needed. Most studies (e authors thank Dr. Douglas Lyles for helpful suggestions have been performed using chemically induced tumors in on the manuscript. mice. (e immune system of rodents, however, has well recognized differences from that of humans. It should be noted that most studies applied intratumoral treatments to References subcutaneous and not orthotopic tumors. It is not clear that [1] A. J. Jacobs, R. Michels, J. Stein, and A. S. Levin, “Im- the microenvironment of even orthotopic rodent sarcoma provement in overall survival from extremity soft tissue mimics the human situation. Immunotherapy studies require sarcoma over twenty years,” Sarcoma, vol. 2015, Article ID mouse models with an intact immune system. 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