Sarcoma

Volume 2014 (2014), Article ID 391967, 13 pages

http://dx.doi.org/10.1155/2014/391967

Review Article

Sarcoma Immunotherapy: Past Approaches and Future Directions

S. P. D'Angelo,1,2 W. D. Tap,1,2 G. K. Schwartz,1,2 and R. D. Carvajal1,2

¹Department of Medicine/Melanoma-Sarcoma Oncology Service, Memorial Sloan Kettering Cancer Center, 300 East 66th Street, New York, NY 10065, USA

²Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA

Received 23 October 2013; Revised 17 December 2013; Accepted 16 January 2014; Published 20 March 2014

Academic Editor: Eugenie Kleinerman

Copyright © 2014 S. P. D'Angelo 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.

Abstract.

Sarcomas are heterogeneous malignant tumors of mesenchymal origin characterized by more than 100 distinct subtypes. Unfortunately, 25–50% of patients treated with initial curative intent will develop metastatic disease. In the metastatic setting, chemotherapy rarely leads to complete and durable responses; therefore, there is a dire need for more effective therapies. Exploring immunotherapeutic strategies may be warranted. In the past, agents that stimulate the immune system such as interferon and interleukin-2 have been explored and there has been evidence of some clinical activity in selected patients. In addition, many cancer vaccines have been explored with suggestion of benefit in some patients. Building on the advancements made in other solid tumors as well as a better understanding of cancer immunology provides hope for the development of new and exciting therapies in the treatment of sarcoma. There remains promise with immunologic checkpoint blockade antibodies. Further, building on the success of autologous cell transfer in hematologic malignancies, designing chimeric antigen receptors that target antigens that are over-expressed in sarcoma provides a great deal of optimism. Exploring these avenues has the potential to make immunotherapy a real therapeutic option in this orphan disease.

1. Introduction/Overview

Sarcomas are a group of heterogenous malignant tumors of mesenchymal origin characterized by more than 100 distinct subtypes. Approximately 13,000 cases of soft tissue and bone sarcomas are diagnosed annually in the US [1]. Surgery, followed by adjuvant radiation for larger tumors, is the mainstay of treatment [2]. Perioperative chemotherapy is used in specific subtypes such as rhabdomyosarcoma, osteosarcoma, and Ewing’s sarcoma [2]. Dependent upon initial stage and subtype, 25–50% of patients develop recurrent and/or metastatic disease [3, 4]. Complete responses to chemotherapy for metastatic sarcoma are rare and the median survival is 10–15 months [5, 6]. The development of novel and effective therapies is desperately needed for the treatment of sarcoma.

The immune system is critical in cancer control and progression, and appropriate modulation of the immune system may provide an effective therapeutic option for sarcoma. Thus far, however, no effective immunological therapy for sarcoma has been identified. Nevertheless, building on the progress made in other solid tumors, as well as the expanding understanding of cancer immunology, provides optimism for the development of new immunologic therapies for sarcoma therapies.

Herein, we provide a review of previously investigated immunological therapies for sarcoma and discuss promising future directions. Previously investigated immunotherapies include interferon, interleukin-2, liposomal-muramyl tripeptide phosphatidylethanolamine, and vaccines. Promising future directions for the development of effective immunotherapies include immunologic checkpoint blockade with the targeting of the cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and the programmed cell death protein 1 (PD-1) axis, as well as therapies such as adoptive cell transfer.

1.1. History of Immunotherapy in Sarcoma

Immunotherapeutic strategies may be a promising approach to this disease. The role of the immune system as a mechanism of cancer therapy was first observed in sarcoma patients. Dating back to 1866, Wilhelm Busch in Germany observed tumor regressions in patients with sarcoma after postoperative wound infections [7]. Coley described a dramatic response in a patient with small cell sarcoma after an erysipelas infection, suggesting that that the body’s response to infection also had potential antitumor effects [8]. He attempted to test this theory by injecting patients with heat-inactivated bacteria to promote an immune response [8]. He treated a patient with recurrent head and neck sarcoma with local injections of streptococcal broth cultures and noted a near complete response which lasted close to eight years [9]. The data generated by Coley was not reproducible given its inconsistent nature and, ultimately, the American Cancer Society refuted the role of Coley’s toxin as an effective treatment [10].

The observation that the development of sarcoma is more common in patients that are immunosuppressed also supports the relevance of the immune system in this disease [11]. The development of sarcomas has been described in allograft transplant recipients. In a study of 8191 transplant patients, 8724 malignancies occurred and 7.4% of them were sarcomas [12]. While a majority of patients developed Kaposi sarcoma, 1.7% of patients developed other sarcomas including malignant fibrous histiocytoma (MFH), leiomyosarcoma (LMS), fibrosarcoma, rhabdomyosarcoma, hemangiosarcoma, and undifferentiated sarcoma which is nearly tripled compared to an incidence of 0.5% in the general population [12].

1.2. Innate and Adaptive Immunity in the Nonmalignant State

The immune system is comprised of the innate and adaptive arms [13]. The innate immune system includes dendritic cells (DC), natural killer cells (NK), macrophages, neutrophils, basophils, eosinophils, and mast cells [13]. Innate immune cells serve as the initial defense against foreign antigens. Once activated, macrophages and mast cells release cytokines that engage additional immune cells and initiate an inflammatory response [13]. Dendritic cells can serve as antigen presenting cells (APC). They uptake foreign antigens and present them to the adaptive immune cells, providing interaction between the 2 arms of the immune system. Natural killer cells can both activate DC and eliminate immature DC. Through their interaction with the DC, NK cells can also provide interplay between the innate and adaptive immune system.

The adaptive immune system includes B lymphocytes, CD4⁺ helper T lymphocytes, and CD8⁺ cytotoxic T lymphocytes (CTLs) and requires formal presentation by APC for its activation. The adaptive immune system generates T/B cell lymphocytes that are antigen specific. These pathways work together to eliminate invading pathogens and damaged cells [13].

1.3. Immune System and Tumorigenesis

Antitumor immunity requires antigen presentation by DC, production of T cell responses, and overcoming immunosuppression at the tumor bed [14]. T cell activation requires dual signaling [15, 16]. The binding of the T cell receptor to antigens presented by antigen presenting cells via major histocompatibility classes (MHC) I and II is the first required signal. The second signal is generated when the B7 ligand binds to CD28, a costimulatory receptor. This signaling leads to T cell proliferation, cytokine release, and upregulation of the immune response. After DC present tumor antigens on the MHC I or II, they migrate to nearby lymph nodes [14]. If the appropriate maturation signal is present, the DC elicit effector T cell response in the lymph node. Further, the interaction of other T cell stimulation molecules such as CD28 or OX40 with CD80/86 or OX40L will promote a protective T cell response (Figure 1).

Figure 1: Preventing tumorigenesis in sarcoma. The adaptive immune response initiates with presentation of antigens by DC. DC migrates to the lymph node. Antigens are presented to CD4⁺ and CD8⁺ T cells through MHC class I and II, respectively, and costimulatory molecules such as B7 bind to CD28 leading to activation of the lymphocytes. Once stimulated, these lymphocytes are now effector cells with the ability to migrate to the tissue and initiate an immune response against the developing sarcoma (abbreviations: CTL, cytotoxic T lymphocyte; DC, dendritic cell; TH, T helper lymphocyte). Reference: adapted from [25].

Tumor antagonizing immune cells such as CTLs and NK cells are believed to play a role in eradication of tumors [17]. It has been demonstrated that mice with impaired function of CD8⁺ CTLs, CD4⁺ Th1 helper T cells, or NK cells have increased incidence of tumors [18, 19]. As such, individuals that are immunocompromised have increased incidence of certain kinds of cancers [20].

Lack of the immunogenic maturation stimuli will lead to T-cell depletion and production of regulatory T cells (Tregs) [14]. Further, interaction of CTLA-4 with CD80/86 or PD-1 with PD-L1/PD-L2 will also suppress the T cell response. CTLA-4 competes with B7 for CD28 binding. Since, CTLA-4 has higher affinity for the CD28 receptor, the T cell response is downregulated. CTLA-4 is thus a negative regulator of T cell responses that prevents autoimmunity and allows tolerance to self-antigens [15].

PD-1 is a member of the CD28 family of T-cell costimulatory receptors that also includes CD28, CTLA-4, inducible T cell costimulator, and B and T lymphocyte attenuators [21]. PD-1 is expressed on activated T cells, B cells, and myeloid cells [22]. There are 2 ligands, programmed cell death ligand (PD-L1 and PD-L2) that are specific for PD-1. Once they bind to PD-1, downregulation of T-cell activation occurs [23, 24]. If this interaction is interrupted, the checkpoint is turned off and antitumor T-cell activation may be enhanced (Figure 2).

Figure 2: Immunoediting. The 3 phases of immunoediting include elimination, equilibrium and escape. (a) Elimination. Cancer cells are transformed (red) but are actively destroyed by the cells of the immune system. (b) Equilibrium. Cancer cells continue to transform (red and teal). Immune system cannot completely remove the transformed cells but controls their growth and there is a dynamic equilibrium that keeps the tumor in check. (c) Escape. Cancer cells continue to grow and transform (red, teal, and pink). These cells now grow unchecked and exhibit immunosuppressive mechanisms (CD4⁺CD25⁺ Treg) which ultimately lead to progressively growing tumors. Abbreviations: CD4⁺, CD8⁺, CD4⁺CD25⁺ Treg, and NKT cells are all types of T cells and NK cells are natural killer cells. Reference: figure modified from [21].

Ultimately, antigen primed T cells, B cells, and NK cells will exit the lymph node and enter the tumor bed. The tumor microenvironment can have additional defense mechanism which can also oppose the tumor response. Therefore, the tumor-associated inflammatory response can also have a paradoxical effect leading to tumor growth and progression [17]. Tumor promoting immune cells such as macrophage subtypes, mast cells, neutrophils, and certain T and B lymphocytes can prevent immune destruction of the tumor by secreting immunosuppressive factors. Suppression of the tumor immune response has been termed immune-evasion; this is emerging as the seventh hallmark of cancer [17].

1.4. Immunoediting

Immunoediting has been demonstrated preclinically in murine models of sarcoma [26]. Cellular immunity as a mechanism of protection from cancer was first described as the concept of immunosurveillance [27]. Immunosurveillance may in fact be the first phase of immunoediting. The immunoediting paradigm is a larger process describing how an individual is protected from cancer growth and develops tumor immunogenicity [27–30]. Immunoediting includes 3 phases: elimination, equilibrium, and escape [31, 32] (Figure 3). In the elimination phase, the immune system is capable of recognizing and destroying cancer cells. Examples include (1) lymphocytic infiltration of the tumor, which has been demonstrated in sarcomas such as Ewing’s sarcoma and GIST [33–35] and (2) spontaneous regression of primary tumors which have been seen in desmoid tumors and osteosarcomas [36, 37]. A case report described a 15-year-old girl that presented with a large desmoid tumor in the left iliac fossa. The patient was considered inoperable given the extent of disease and involvement of local structures. Ultimately, after 5 years, the tumor began to regress and, by 10 years, it was no longer palpable [36]. In another example, a 57-year-old woman underwent surgical resection of a proximal thigh extraskeletal osteosarcoma [37]. The pathology specimen noted tumor cells that had been replaced by fibrocollagenous tissue with lymphocytic infiltration. The prognostic implications of lymphocytes in soft tissue sarcomas have been reported [38, 39]. Tissue microarrays were constructed and immunohistochemistry was used to evaluate multiple white blood cells that play a role in the immune system these including CD3⁺ (T cell coreceptor), CD4⁺, CD8⁺, CD20⁺ (expressed on B cell surface, which plays a role in B cell activation), and CD45⁺ lymphocytes (lymphocyte common antigen, which plays a role in T cell activation) in tumors [39]. CD20⁺ lymphocytes in resected soft tissue sarcoma were independent positive prognostic factors associated with improved disease specific survival [39].

Figure 3: Mechanism of action of CTLA-4 and PDL1 blockade. (a) Activation of T cell requires interaction of MHC bearing tumor antigen with the TCR and interaction of the costimulatory molecule B7 with CD28. CTLA-4 is a negative regulator of the immune response that competes with CD28 binding with B7. Ipilimumab is a monoclonal antibody that binds CTLA-4 and promotes continued T cell activation. (b) The role of the PD-1 receptor is more significant in the peripheral tissue, once T cell activation has already occurred. After antigen exposure, PD-1 receptor is expressed on the T cells. When the PD-1 receptor interacts with its ligands PD-L1 and PD-L2, there is negative regulation of T cells in the tumor microenvironment. Blocking PD-1 or PD-L1 leads to activation of T cells. Reference: figure modified from [57].

In the equilibrium phase, cancer cells maintain a balance with the immune system where they are able to avoid immune-mediated destruction but are not able to progress [40, 41].

In the escape phase, cancer cells grow and metastasize due to loss of control by the immune system [32]. The loss of MHC I on sarcoma cells is an example of the escape phenomenon [42]. MHC 1 loss was demonstrated in 46/72 (62%) of bone and soft tissue sarcomas. In the 21 osteosarcoma patients, expression of MHC I was associated with improved overall and event-free survival [42].

2. Previously Investigated Immunotherapies

2.1. Cytokines

Stimulation of the immune system has been evaluated with cytokines such as interleukin-2 (IL-2) and interferon (IFN). Cytokines regulate the function of the immune system and are involved in antigen presentation as well as T cell activity [43].

IL-2 stimulates and upregulates T and NK cells and mediates lymphocyte proliferation [44–46]. IL-2, which typically binds to the type I cytokine receptors [43], has the ability to activate a population of lymphocytes into lymphokine-activated killer cells (LAK) [47]. LAK cells are lymphocytes that have the potential to eradicate tumor cells regardless of histocompatibility expression [48–50]. It has been demonstrated that lymphocytes that are stimulated with IL-2 can lyse malignant cells [51]. Responses to IL-2 have been described in ovarian, nonsmall cell lung, breast, melanoma, and renal cell cancers [51]. High dose infusional IL-2 is FDA approved for the treatment of metastatic melanoma and renal cell carcinoma [52, 53]. Limitations of this therapy include significant treatment associated toxicity.

In a small study of high dose IL-2 used in conjunction with LAK cells, none of the six sarcoma patients treated responded [54]. A more recent study of high dose IL-2 in 10 heavily pretreated pediatric patients with multiple malignancies included 4 patients with osteosarcoma and 2 patients with Ewing’s sarcoma [51]. Of the four osteosarcoma patients, two achieved complete responses that were durable, with a median followup of 28 months while the other two had progressive disease. Both patients with Ewing’s sarcoma had progressive disease. The responses observed with IL-2 suggest that this agent may have efficacy in a subset of patients with sarcoma [51]. The future of IL-2 as a single agent for the treatment of sarcoma patients is not known; there is, however, a study combining IL-2 with vaccine therapy (NCT00101309). The rationale is to potentially improve on the efficacy of either agent alone by enhancing the immune response.

2.2. Interferon

Interferons are a family of molecules which bind to either type I or type II IFN receptors, which are subsets of the type II cytokine receptors [43, 55]. IFN and IFN both activate the type I IFN receptors; IFN binds distinctly to the type II IFN receptors [55]. Since they bind to the same receptors, both IFN and IFN likely have similar biological effects; however the actual mechanism of antitumor activity is not clear [55]. IFN and IFN are capable of activating immune cells and increasing antigen presentation to T lymphocytes [55]. There are many different forms of IFN, including IFN-2a, IFN-2b, and IFN-2c all which vary by a few amino acids [55]. IFN is approved in the adjuvant setting for high-risk melanoma. In a cooperative group study, patients with stage II or III melanoma received 20 million units/m2/day IV 5 days per week, followed by 10 million units/m2/day SQ 3x per week for 48 weeks. This study demonstrated an initial survival benefit that subsequently was lost with longer followup [56].

There have been multiple studies with interferon in sarcoma. A case report of patients with metastatic disease described two of three patients with osteosarcoma who received interferon and achieved partial responses [58]. A phase II study demonstrated that three of twenty patients with metastatic bone sarcomas (2 with osteosarcoma and 1 with malignant fibrous histiocytoma) received recombinant interferon -2a and achieved short lasting partial responses [59]. In the adjuvant setting, there have been many studies investigating the role of adjuvant interferon. While some studies appeared promising, none reached statistical significance. There is an on-going randomized trial of the European and American Osteosarcoma Study group for patients with resectable osteosarcoma with favorable responses to preoperative chemotherapy. This study randomizes patients to two different combination chemotherapy regimens with or without PEG-INT -2b (NCT00134030) (Table 1).

Table 1: Adjuvant interferon studies.

Study name
Patients
Type of sarcoma
Type of interferon
Dose/schedule
Outcome

Karolinska Hospital series
After surgical resection, 89 patients
Osteosarcoma
Interferon-α
Cohort 1 (70 patients) 3 × 10⁶ IU daily ×1 month, followed by 3×/week, 17 months

Cohort 2 (19 patients) 3 × 10⁶ IU daily for 3–5 years
10-year metastatic free survival: 39% (95% CI 29–49%)

10-year sarcoma specific survival: 43% (95% CI 33–54%) [65, 66]

Median time to metastasis 8 months (range 1–60 months)

value not specified

COSS-80

100 patients after preoperative chemotherapy and surgical resection were randomized +/− interferon
Osteosarcoma
Interferon-β
100,000 U/kg for 22 weeks (2 injections weekly ×2 weeks, daily ×4 weeks, weekly ×16 weeks)
30-month continuous disease-free survival: 77% interferon arm versus 73% noninterferon arm [67]

Log rank test,

EURAMOS 1
715 patients that had good response to preoperative chemotherapy were randomized to postoperative chemotherapy +/− interferon
Osteosarcoma
Pegylated interferon-α2b
0.5–1.0 μg/kg/week for 2 years
Primary endpoint: event-free survival (EFS) [68]

77% who received INT were disease free at 3 years compared to 74%

Hazard ratio for EFS from adjusted Cox model was 0.82 (95% CI 0.61–1.11; ) in favor of chemotherapy + interferon

2.3. MTP

Liposomal-muramyl  tripeptide phosphatidyl-ethanolamine (L-MTP) has also been investigated to potentially stimulate the immune system. MTP is a synthetic analogue of a bacterial cell wall that is capable of activating monocytes and macrophages [60]. MTP can result in inflammation, release of antimicrobial peptides, fever, DC recruitment, and potential tumor cell death [60]. The rationale for using MTP in oncology is to trigger an inflammatory response to eradicate micrometastatic disease [60].

The Children’s Oncology Group Intergroup-0133 was a 4-arm study in which patients with osteosarcoma without clinically detectable metastatic disease were double randomized at the time of study entry. The first randomization was to receive adjuvant cisplatin, doxorubicin, and high-dose methotrexate with or without ifosfamide. The second randomization was to receive either 3-drug or 4-drug chemotherapy alone or 3-drug or 4-drug chemotherapy plus liposomal-MTP. In a pooled analysis the study demonstrated that the addition of ifosfamide to cisplatin, doxorubicin, and high-dose methotrexate did not improve either event-free or overall survival. The authors concluded that there was improved survival (both event-free and overall) in those patients that received chemotherapy with L-MTP with an increase in the 5-year overall survival rate from 70% to 78% (; relative risk, 0.73) [61]. In patients who presented with metastases, there was also a benefit in event-free and overall survival although the analysis was not powered to support a statistically significant benefit [62]. L-MTP is approved for use in the European Union, Mexico, Turkey, and Israel. It is not FDA approved. A compassionate study of L-MTP for patients with high-risk osteosarcoma was completed in December of 2012 which also demonstrated a survival advantage for the patients who received L-MTP [63].

2.4. Vaccines

There have been multiple clinical trials investigating vaccines targeting whole cells, lysates, proteins, and peptides in patients with sarcoma. Vaccines can be combined with costimulatory adjuvants as well as immunostimulants such as GM-CSF or IL-2 to potentially enhance the immune response. The goal of vaccination is to expose patients to tumor antigens with hope of inducing an antitumor immune response with the generation of tumor specific antibodies or T cells that ultimately translates to clinical benefit [64]. Many studies have yielded disappointing results, although there were some patients that derived benefit (Table 2).

Table 2: Vaccine studies in sarcoma.

Vaccine
Sarcoma histology
Number of patients
Immune response
Results

Irradiated autologous tumor cells [69]
Various pediatric
16
Not reported
16.6 m versus 8.2 m survival (skin test responders)

Dendritic cell pulsed with autologous tumor lysate [70]
Various pediatric
10
Not reported
One response in patient with fibrosarcoma

Dendritic cell pulsed with peptides from tumor specific translocation breakpoints and E7 [71]
Metastatic Ewing’s family of tumors or alveolar rhabdomyosarcoma
30/52 initiated vaccine after standard therapy
39% with immune response to translocation breakpoint

25% with E7-specific response
5-year OS: 31% for all patients versus 43% for patients initiating immunotherapy

105AD7 (CD55 target) [72]
Osteosarcoma
28 patients within 1–6 months of chemotherapy
20/28 (71%) showed T cell proliferation response in vitro to 105AD7

9/28 (32%) weak antibody response to CD55
2 patients with possible clinical responses, alive and disease free 5.8 and 6.5 years from time of diagnosis

Dendritic vaccine pulsed with synthetic tumor specific peptide [73]
Posttransplant, residual tumor (synovial, Ewing’s)
5
DTH response against tumor lysate in 1 patient
1 patient complete response, 77 months

Peptide encompassing SYT-SSX [74]
Synovial
6
Peptide specific CTLs generated in 4 patients
Suppression of tumor progression 1 patient

SYT-SSX derived peptide [75]
Synovial
20
9 showed twofold increase in CTLs in tetramer analysis
1/9 stable disease (received vaccine with peptide alone)

6/12 stable disease (received vaccine with incomplete Freund’s adjuvant)

Abbreviations: CTL.

Currently, there is a phase II trial of a trivalent peptide vaccine to the gangliosides GD2, GD3, and GM2 in patients with sarcoma that have had solitary metastases excised (NCT00597272). This study has been closed to accrual and results are pending. Perhaps the ideal study population for vaccines includes postoperative patients to minimize micrometastases.

3. Immunologic Checkpoint Blockade

3.1. CTLA-4 Blockade

Ipilimumab is a human monoclonal antibody that binds CTLA-4 that is FDA approved for the treatment of metastatic melanoma [76]. In a small phase II study, patients with synovial sarcoma were treated with ipilimumab 3 mg/kg every 3 weeks and restaged after 3 cycles [77]. The primary endpoint of the study was RECIST 1.0 response rate. Secondary endpoints included determination of the clinical benefit rate and evaluation of NY-ESO-1 specific immunity. Four patients completed 3 doses of ipilimumab, while 2 patients each received 1 and 2 doses due to clinical or radiologic progression. There were no documented responses, and the time to progression ranged from 0.47 months to 2.1 months. There was no evidence of serologic or delayed type hypersensitivity to NY-ESO-1.

Although ipilimumab has demonstrated an improvement in overall survival in metastatic melanoma, the response rate is only 10–20% [78]. Clinical responses can be delayed and some patients do not demonstrate disease regression or stabilization for many weeks after therapy is complete. There are patients who have initial progressive disease and subsequent disease stabilization. Taking into account what we learned from melanoma, selecting progression-free survival or overall survival as primary endpoints may lead to a better designed study. Incorporation of the immune-related response criteria (irRC) may be prudent. With standard cytotoxic chemotherapy, typical patterns can include an increase, decrease, or no change in tumor burden which can be effectively interpreted with the response evaluation criteria in solid tumors (RECIST) [79]. With immunotherapy, patients can sometimes have a response after an increase in tumor burden or a response in the presence of new lesions. Per RECIST, this would qualify as disease progression. Progression as judged by tumor size may be deceptive as an increase in immune cell infiltration into the tumor may appear on a radiographic study as an increase in tumor size. Immune-related response criteria are a set of novel response criteria designed to capture these unique response patterns. The irRC were devised using the data from the phase II clinical trials of patients with metastatic melanoma treated with ipilimumab [80]. With the irRC, progressive disease is defined as total disease growth up to 25% from baseline or total disease burden (new lesions plus target) greater than 25%.

Therefore, it may be best to consider ipilimumab in the early metastatic setting, when patients have less disease burden. A patient with rapidly progressive disease may not be the best candidate. If interval imaging studies are performed, they should be interpreted with caution, as disease progression early on may not necessarily translate to lack of efficacy.

3.2. Enhancing the Activity of CTLA-4 Blockade

Therapies that can induce cell death such as radiation therapy, traditional chemotherapies, and targeted therapies can result in the release of antigens. These antigens may serve as priming events for immune specific responses mediated by CTLA-4 blockade [81, 82].

Targeted therapies such as tyrosine kinase inhibitors (TKIs) have “off-target” effects on the immune system and suppressing and stimulating effects on immune cells, such as CD4⁺ and CD8⁺ T cells [83, 84], NK cells [85], and DC [86]. These immune effects have been associated with improved preclinical [84] and clinical outcomes [85, 87].

The effective treatment of human GIST tumors with imatinib is associated with an increased intratumoral CD8⁺ effector T cells (Teff)/regulatory T cell (Treg) ratio [88]. Regulatory T cells contribute to decreased immune responses to tumors [89–92]. Tumors that are pathologically resistant to imatinib are associated with a lower Teff/Treg ratio which has been correlated to the level of indolamine 2, 3-dioxygenase (IDO) [88]. IDO is a heme-containing enzyme that catalyzes the oxidative breakdown of the essential amino acid tryptophan, via the kynurenine pathway [93]. IDO has been shown to inhibit T-cell proliferation and blockade of cell cycle progression by tryptophan depletion. Consideration of IDO blockade warrants further study, either alone or in combination with other immunotherapeutic strategies.

In a KIT-mutant GIST mouse model there has been augmentation of the antitumor effect of KIT-targeting with CTLA-4 blockade leading to improved, more durable responses [88]. There is a phase Ib/II study of ipilimumab with dasatinib for patients with soft tissue sarcoma with an expansion of GIST (NCT01643278).

3.3. PD-1 Blockade

The PD-1 receptor is another promising potential immunological target. In a phase I study that enrolled and treated 296 patients with nivolumab, an antibody to PD-1, response rates were 18%, 28%, and 27% in patients with nonsmall cell lung cancer (NSCLC), melanoma, and renal cell carcinoma (RCC), respectively [94]. There was suggestion that PD-L1 expression by immunohistochemistry may correlate with clinical activity of PD-1 blockade. Pretreatment expression of PD-L1 was performed in 42 patients. There were 18 patients that lacked expression and all of them did not have any evidence of benefit to the drug. Of the 25 patients with PD-L1 expression, 9/25 (36%) had an objective response, [94]. A phase I study of nivolumab and ipilimumab in patients with advanced melanoma demonstrated impressive objective response rates of 40% [95]. Among patients that received combination therapy, responses were seen both in patients with PDL-1 expression (6/13) or those without PDL-1 expression (9/22).

Even though the role of PDL-1 expression as a biomarker remains debatable, evaluation of sarcoma tumor specimens for expression of PDL-1 may provide potential justification for a clinical trial.

4. Exploring Surface Antigens and Cancer Testis Antigen

Many sarcomas express specific epitopes due to the cell of origin or as a result of gene products providing targets for immune-mediated therapeutic approaches. Prospective targets include cancer testis antigens that are commonly expressed in many sarcomas [64, 96]. Gangliosides and cancer testis antigens may prove to be potential targets for adoptive T cell transfer as well as for vaccine development.

4.1. Gangliosides

Gangliosides are glycosphingolipids containing a lipid component and a carbohydrate chain that are found on the cell surface that are believed to play a role in cell attachment and cell-cell interactions. Several of these gangliosides, including GM2, disialoganglioside (GD2), and GD3, are expressed by tumors such as melanoma [97–100], sarcomas, and neuroblastoma [101–103].

Using immunohistochemistry, gangliosides and protein antigens were explored as potential targets for immunotherapy in sarcomas [100, 104, 105]. Fresh frozen human sarcoma tumor samples were evaluated for GD2 and GD3 (using the purified mAbs 3F8 and R24, resp.) and demonstrated that 93% of the tumors expressed GD2 and 88% expressed GD3 by immunohistochemical staining [99, 106]. Certain histologic types showed a greater extent of expression of GD2 and GD3, including liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, and spindle cell sarcoma. Monophasic synovial sarcoma, embryonal rhabdomyosarcoma, alveolar soft part sarcoma, and hemangiosarcoma demonstrated less staining.

4.2. Cancer Testis Antigens

The cancer testis antigens (CTA) are proteins that are typically found of tumor cells and lack expression on normal tissue [107]. Approximately 20 CTA have been identified [107]. These antigens are typically expressed on germline tissues, placental trophoblasts, as well as specific cancers but most importantly, they can be recognized by CTL [108]. In order to be recognized by CTL, there must be adequate expression of human leukocyte antigen (HLA) class I by tumor cells [109]. Every CTA has epitopes for a minimum of one HLA type, although many of these HLA types remain uncommon [64]. Since class I HLA A*02.01 is found in approximately half the Caucasian population, many immunotherapy trials target HLA A*02.01 associated antigens such as NY-ESO-1, LAGE-1, PRAME, MAGE-A3, MAGE-A4, MAGE-A9, and SSX-2. While exploration of these tumor antigens has historically been focused on tumors with known spontaneous immunogenicity there has been more interest in exploring expression of CTA in sarcoma. Studies have utilized RT-PCR and IHC methodology to explore known CTA expressed in other malignancies [109–112]. Results are demonstrating that CTA are potential targets in sarcoma. Studies to date have shown that antigen expression does vary by sarcoma subtype (Table 3).

Table 3: Cancer testis antigens expressed in sarcoma.

Antigen
Sarcoma type
Total expression

NY-ESO [110, 111, 113]
Synovial

Myxoid round cell liposarcoma

Uterine leiomyosarcoma

Osteosarcoma
80%

100%

50% (3/6)

89% (8/9)

LAGE [109, 112]
Myxoid round cell liposarcoma

Nonmyxoid liposarcoma

Osteosarcoma
70%

60%

89% (8/9)

PRAME [112]
Synovial sarcoma

Nonmyxoid liposarcoma

Myxoid liposarcoma
100% (4/4)

100% (4/4)

14% (1/7)

MAGE-A3 [109]
Uterine leiomyosarcoma

Nonuterine leiomyosarcoma
67% (4/6)

14% (1/7)

5. Adoptive Cell Transfer

Adoptive cell transfer (ACT) involves the transfer of immune cells with antitumor activity. The mechanisms by which this can occur include the expansion and infusion of tumor infiltrating lymphocytes, the use of genetically modified lymphocytes, or the use of chimeric antigen receptors (CAR).

By using gamma retroviruses or lentiviruses, lymphocytes can be genetically modified to encode T cell receptors (TCR) that recognize specific tumor antigens or encode molecules that can enhance their antitumor activity [114]. These lymphocytes then acquire antitumor activity. Historically, these are conventional TCR that have alpha and beta chains that form heterodimers to recognize cancer antigens that are presented on the surface of MHC molecules on tumor cells [114]. T cells genetically engineered to target NY-ESO-1 expressing synovial sarcoma have shown some promise [115]. In a small study, patients with NY-ESO-1 expressing synovial sarcomas were treated with a lymphodepleting chemotherapy regimen consisting of cyclophosphamide and fludarabine, followed by infusions of autologous T lymphocytes designed to recognize a specific NY-ESO-1 antigen. Four of 6 patients with synovial sarcoma had evidence of partial response lasting from 5–18 months. Limitations of conventional TCR are that they are restricted to antigen presentation on specific MHC molecules [114]. In addition, downregulation of class I MHC molecules has been described as a mechanism of avoiding TCR recognition [116].

CAR are composed of the antigen-combining regions of the heavy and light chains of antibodies with T-cell intracellular signaling molecules [117]. CAR provide the opportunity to recognize antigens based on antibody interaction [117]. T cells can express CAR targeting different tumor-associated antigens such as GD2 which is expressed in neuroblastoma, melanoma, and sarcoma [118] or CD19 B cell antigen which expressed in non-Hodgkin lymphomas as well as chronic lymphocytic leukemia [119]. There have been phase I clinical trials using CAR to treat ovarian [120], renal cell carcinoma [121], lymphoma [122], and neuroblastoma [123]. Results have been disappointing, with minimal clinical responses, possibly due to lack of persisting CAR-expressing T cells [124]. The first generation CAR lack costimulatory signals such as CD28, 4-1BB, and OX40 and are only composed of an intracellular signaling domain derived from the TCR CD3- chain [125–128]. Second and third generation CAR include a CD28 signaling domain as well as OX40 or 4-1BB, respectively [118, 129, 130].

Presumably, second and third generation CAR should lead to more effective responses and persistence of the CAR. Thus far, there has been exciting data in hematological malignancies showing promise of second generation CAR [131]. The hope is that this success can be translated to similar efficacy in solid tumor malignancies.

6. Future Directions

To date, the field of sarcoma immunotherapy has not yet matured to show robust antitumor effects, but there has been suggestion of clinical activity in some patients. Although there have been multiple clinical trials evaluating the role of stimulants of the immune system such as IL-2 and IFN, these agents have failed to improve overall survival [51, 54, 58, 59]. MTP has shown promise demonstrating improvement both in event and overall survival [61–63]. The study design may have limited FDA approval, although this agent is available in the European Union, Mexico, Turkey, and Israel. Nonetheless, these drugs appeared to have offered benefit only for selected patients. There are now improved therapies and the success of these therapies in diseases such as melanoma has offered a better understanding of immunology. Through the melanoma experience, we have learned that more appropriate patient selection can perhaps lead to more successful clinical trials. Melanoma and sarcoma are clearly two distinct malignancies; however, like melanoma, sarcoma does have infiltration of tumor associated lymphocytes, which can provide rationale for immunomodulation [38, 39]. Like patients with melanoma, there have been cases of spontaneous regression in patients with sarcoma [36, 37]. Sarcoma does have evidence of effective immunosurveillance as demonstrated by Coley [8] and Wiemann and Starnes [9]. As noted in patients with GIST, TKIs such as imatinib can have stimulating effects on multiple immune cells [88]. Therefore immunotherapies may be effective in this disease, if the appropriate drugs are used in the appropriate patients. Moving forward, more precise immune modulation, enhancing activity of immunomodulatory agents, and targeting sarcoma specific epitopes may all lead to a more successful approach in treating this disease.

Investigating immunomodulators such as ipilimumab may be promising. Although it was only previously investigated in synovial sarcoma, our knowledge regarding this agent may in fact lead to a better designed trial [77]. Previously, solid tumors such as NSCLC did not appear to be immunogenic tumors. Yet, we have since learned that anti-PD-1 antibodies have led to durable and effective responses in lung cancers [94].

Through small numbers, the data with NY-ESO-1 T cell therapy appeared to be efficacious in some patients, providing hope [115]. Perhaps utilizing a CAR to target an epitope that is overexpressed in sarcoma may broaden applicability of this intervention to other sarcoma subtypes.

The knowledge and current understanding of the immune system can build enthusiasm to create a niche for sarcoma therapy. Moving forward, it is important to model the development of immunological therapies in sarcoma after the successful development of such therapy in other solid tumors. While there may not be any biological parallels amongst these malignancies, a better understanding of antitumor immunity mechanism may be incorporated into designing more rationale clinical trials.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

References

M. F. Brennan, C. R. Antonescu, and R. G. Maki, Management of Soft Tissue Sarcoma, Springer, New York, NY, USA, 2012.

M. F. Brennan, S. Singer, E. Maki, and B. O'Sullivan, Eds., Cancer: Principles and Practice of Oncology, Lippincott, Williams & Wilkins, Philadelphia, Pa, USA, 2008.

R. D. Lindberg, R. G. Martin, M. M. Romsdahl, and H. T. Barkley Jr., “Conservative surgery and postoperative radiotherapy in 300 adults with soft-tissue sarcomas,” Cancer, vol. 47, no. 10, pp. 2391–2397, 1981.

J. Weitz, C. R. Antonescu, and M. F. Brennan, “Localized extremity soft tissue sarcoma: improved knowledge with unchanged survival over time,” Journal of Clinical Oncology, vol. 21, no. 14, pp. 2719–2725, 2003.

K. G. Billingsley, M. E. Burt, E. Jara et al., “Pulmonary metastases from soft tissue sarcoma: analysis of patterns of disease and postmetastasis survival,” Annals of Surgery, vol. 229, no. 5, pp. 602–610, 1999.

M. Van Glabbeke, A. T. Van Oosterom, J. W. Oosterhuis et al., “Prognostic factors for the outcome of chemotherapy in advanced soft tissue sarcoma: an analysis of 2,185 patients treated with anthracycline- containing first-line regimens—a European organization for research and treatment of cancer soft tissue and bone sarcoma group study,” Journal of Clinical Oncology, vol. 17, no. 1, pp. 150–157, 1999.

T. J. Curiel, “Historical perspectives and current trends in cancer immunotherapy,” in Cancer Immunotherapy: Paradigms, Practice and Promise, T. J. Curiel, Ed., Springer, New York, NY, USA, 2012.

W. B. Coley, “II. contribution to the knowledge of sarcoma,” Annals of Surgery, vol. 14, pp. 199–220, 1891.

B. Wiemann and C. O. Starnes, “Coley's toxins, tumor necrosis factor and cancer research: a historical perspective,” Pharmacology and Therapeutics, vol. 64, no. 3, pp. 529–564, 1994.

American Cancer Society's Guide to Complementary and Alternative Cancer Methods, American Cancer Society, 2000.

R. A. Gatti and R. A. Good, “Occurrence of malignancy in immunodeficiency diseases. A literature review,” Cancer, vol. 28, no. 1, pp. 89–98, 1971.

I. Penn, “Sarcomas in organ allograft recipients,” Transplantation, vol. 60, no. 12, pp. 1485–1491, 1995.

K. E. De Visser, A. Eichten, and L. M. Coussens, “Paradoxical roles of the immune system during cancer development,” Nature Reviews Cancer, vol. 6, no. 1, pp. 24–37, 2006.

I. Mellman, G. Coukos, and G. Dranoff, “Cancer immunotherapy comes of age,” Nature, vol. 480, no. 7378, pp. 480–489, 2011.

K. S. Peggs, S. A. Quezada, A. J. Korman, and J. P. Allison, “Principles and use of anti-CTLA4 antibody in human cancer immunotherapy,” Current Opinion in Immunology, vol. 18, no. 2, pp. 206–213, 2006.

C. Robert and F. Ghiringhelli, “What is the role of cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma?” Oncologist, vol. 14, no. 8, pp. 848–861, 2009.

D. Hanahan and R. A. Weinberg, “Hallmarks of cancer: the next generation,” Cell, vol. 144, no. 5, pp. 646–674, 2011.

R. Kim, M. Emi, and K. Tanabe, “Cancer immunoediting from immune surveillance to immune escape,” Immunology, vol. 121, no. 1, pp. 1–14, 2007.

M. W. L. Teng, J. B. Swann, C. M. Koebel, R. D. Schreiber, and M. J. Smyth, “Immune-mediated dormancy: an equilibrium with cancer,” Journal of Leukocyte Biology, vol. 84, no. 4, pp. 988–993, 2008.

C. M. Vajdic and M. T. Van Leeuwen, “Cancer incidence and risk factors after solid organ transplantation,” International Journal of Cancer, vol. 125, no. 8, pp. 1747–1754, 2009.

G. J. Freeman, A. J. Long, Y. Iwai et al., “Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation,” Journal of Experimental Medicine, vol. 192, no. 7, pp. 1027–1034, 2000.

H. Nishimura and T. Honjo, “PD-1: an inhibitory immunoreceptor involved in peripheral tolerance,” Trends in Immunology, vol. 22, no. 5, pp. 265–268, 2001.

L. Carter, L. A. Fouser, J. Jussif et al., “PD-1:PD-L inhibitory pathway affects both CD4⁺ and CD8⁺ T cells and is overcome by IL-2,” European Journal of Immunology, vol. 32, no. 3, pp. 634–643, 2002.

Y. Latchman, C. R. Wood, T. Chernova et al., “PD-L2 is a second ligand for PD-1 and inhibits T cell activation,” Nature Immunology, vol. 2, no. 3, pp. 261–268, 2001.

M. J. Smyth, D. I. Godfrey, and J. A. Trapani, “A fresh look at tumor immunosurveillance and immunotherapy,” Nature Immunology, vol. 2, no. 4, pp. 293–299, 2001.

J. B. Swann, M. D. Vesely, A. Silva et al., “Demonstration of inflammation-induced cancer and cancer immunoediting during primary tumorigenesis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 2, pp. 652–656, 2008.

M. Burnet, “Cancer, a biological approach. I. The processes of control,” British Medical Journal, vol. 1, pp. 779–786, 1957.

F. M. Burnet, “The concept of immunological surveillance,” Progress in Experimental Tumor Research, vol. 13, pp. 1–27, 1970.

M. Burnet, “Immunological factors in the process of carcinogenesis,” British Medical Bulletin, vol. 20, no. 2, pp. 154–158, 1964.

K. Tsung and J. A. Norton, “Lessons from Coley's Toxin,” Surgical Oncology, vol. 15, no. 1, pp. 25–28, 2006.

N. Bhardwaj, “Harnessing the immune system to treat cancer,” Journal of Clinical Investigation, vol. 117, no. 5, pp. 1130–1136, 2007.

G. P. Dunn, A. T. Bruce, H. Ikeda, L. J. Old, and R. D. Schreiber, “Cancer immunoediting: from immunosurveillance to tumor escape,” Nature Immunology, vol. 3, no. 11, pp. 991–998, 2002.

D. Berghuis, S. J. Santos, H. J. Baelde et al., “Pro-inflammatory chemokine-chemokine receptor interactions within the Ewing sarcoma microenvironment determine CD8⁺ T-lymphocyte infiltration and affect tumour progression,” Journal of Pathology, vol. 223, no. 3, pp. 347–357, 2011.

P. Brinkrolf, S. Landmeier, B. Altvater et al., “A high proportion of bone marrow T cells with regulatory phenotype (CD4+CD2FoxP3+) in Ewing sarcoma patients is associated with metastatic disease,” International Journal of Cancer, vol. 125, no. 4, pp. 879–886, 2009.

S. Rusakiewicz, M. Semeraro, M. Sarabi et al., “Immune infiltrates are prognostic factors in localized gastrointestinal stromal tumors,” Cancer Research, vol. 73, no. 12, pp. 3499–3510, 2013.

N. H. Jenkins, L. S. Freedman, and B. McKibbin, “Spontaneous regression of a desmoid tumour,” Journal of Bone and Joint Surgery B, vol. 68, no. 5, pp. 780–781, 1986.

T. Matsuo, S. Shimose, T. Kubo et al., “Extraskeletal osteosarcoma with partial spontaneous regression,” Anticancer Research, vol. 29, no. 12, pp. 5197–5201, 2009.

S. W. Sorbye, T. K. Kilvaer, A. Valkov et al., “Prognostic impact of peritumoral lymphocyte infiltration in soft tissue sarcomas,” BMC Clinical Pathology, vol. 12, article 5, 2012.

S. W. Sorbye, T. Kilvaer, A. Valkov et al., “Prognostic impact of lymphocytes in soft tissue sarcomas,” PLoS One, vol. 6, no. 1, Article ID e14611, 2011.

N. Y. Crowe, M. J. Smyth, and D. I. Godfrey, “A critical role for natural killer T cells in immunosurveillance of methylcholanthrene-induced sarcomas,” Journal of Experimental Medicine, vol. 196, no. 1, pp. 119–127, 2002.

V. Shankaran, H. Ikeda, A. T. Bruce et al., “IFNγ, and lymphocytes prevent primary tumour development and shape tumour immunogenicity,” Nature, vol. 410, no. 6832, pp. 1107–1111, 2001.

T. Tsukahara, S. Kawaguchi, T. Torigoe et al., “Prognostic significance of HLA class I expression in osteosarcoma defined by anti-pan HLA class I monoclonal antibody, EMR8-5,” Cancer Science, vol. 97, no. 12, pp. 1374–1380, 2006.

K. Margolin, M. Lazarus, and H. L. Kaufman, “Cytokines in the treatment of cancer,” in Cancer Immunotherapy: Paradigms, Practice and Promise, T. J. Curiel, Ed., Springer, New York, NY, USA, 2012.

B. E. Lippitz, “Cytokine patterns in patients with cancer: a systematic review,” The Lancet Oncology, vol. 14, pp. e218–e228, 2013.

B. H. Nelson, “IL-2, regulatory T cells, and tolerance,” Journal of Immunology, vol. 172, no. 7, pp. 3983–3988, 2004.

C. A. Thornton, J. W. Upham, M. E. Wikström et al., “Functional maturation of CD4⁺CD25⁺CTLA4⁺CD45RA⁺ T regulatory cells in human neonatal T cell responses to environmental antigens/allergens,” Journal of Immunology, vol. 173, no. 5, pp. 3084–3092, 2004.

E. A. Fagan and A. L. W. Feddleston, “Immunotherapy for cancer: the use of lymphokine activated killer (LAK) cells,” Gut, vol. 28, no. 2, pp. 113–116, 1987.

E. A. Grimm, A. Mazumder, H. Z. Zhang, and S. A. Rosenberg, “Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes,” Journal of Experimental Medicine, vol. 155, no. 6, pp. 1823–1841, 1982.

S. C. Yang, E. A. Grimm, D. R. Parkinson et al., “Clinical and immunomodulatory effects of combination immunotherapy with low-dose interleukin 2 and tumor necrosis factor α in patients with advanced non-small cell lung cancer: a phase I trial,” Cancer Research, vol. 51, no. 14, pp. 3669–3676, 1991.

S. C. Yang, L. Owen-Schaub, E. A. Grimm, and J. A. Roth, “Induction of lymphokine-activated killer cytotoxicity with interleukin-2 and tumor necrosis factor-α against primary lung cancer targets,” Cancer Immunology Immunotherapy, vol. 29, no. 3, pp. 193–198, 1989.

W. Schwinger, V. Klass, M. Benesch et al., “Feasibility of high-dose interleukin-2 in heavily pretreated pediatric cancer patients,” Annals of Oncology, vol. 16, no. 7, pp. 1199–1206, 2005.

M. B. Atkins, M. T. Lotze, J. P. Dutcher et al., “High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993,” Journal of Clinical Oncology, vol. 17, no. 7, pp. 2105–2116, 1999.

M. B. Atkins, M. Regan, D. McDermott et al., “Update on the role of interleukin 2 and other cytokines in the treatment of patients with stage IV renal carcinoma,” Clinical Cancer Research, vol. 10, no. 18, pp. 6342S–6346S, 2004.

S. A. Rosenberg, M. T. Lotze, J. C. Yang et al., “Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients,” Annals of Surgery, vol. 210, no. 4, pp. 474–485, 1989.

S. Pestka, “The interferons: 50 years after their discovery, there is much more to learn,” Journal of Biological Chemistry, vol. 282, no. 28, pp. 20047–20051, 2007.

J. M. Kirkwood, J. G. Ibrahim, J. A. Sosman et al., “High-dose interferon alfa-2b significantly prolongs relapse-free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with resected stage IIB-III melanoma: results of intergroup trial E1694/S9512/C509801,” Journal of Clinical Oncology, vol. 19, no. 9, pp. 2370–2380, 2001.

C. Kyi and M. A. Postow, “Checkpoint blocking antibodies in cancer immunotherapy,” FEBS Letters, vol. 588, pp. 368–376, 2014.

H. Ito, K. Murakami, and T. Yanagawa, “Effect of human leukocyte interferon on the metastatic lung tumor of osteosarcoma: case reports,” Cancer, vol. 46, no. 7, pp. 1562–1565, 1980.

J. H. Edmonson, H. J. Long, S. Frytak, W. A. Smithson, and L. M. Itri, “Phase II study of recombinant alfa-2a interferon in patients with advanced bone sarcomas,” Cancer Treatment Reports, vol. 71, no. 7-8, pp. 747–748, 1987.

L. Kager, U. Potschger, and S. Bielack, “Review of mifamurtide in the treatment of patients with osteosarcoma,” Therapeutics and Clinical Risk Management, vol. 6, pp. 279–286, 2010.

P. A. Meyers, C. L. Schwartz, M. D. Krailo et al., “Osteosarcoma: the addition of muramyl tripeptide to chemotherapy improves overall survival—a report from the children's oncology group,” Journal of Clinical Oncology, vol. 26, no. 4, pp. 633–638, 2008.

A. J. Chou, E. S. Kleinerman, M. D. Krailo et al., “Addition of muramyl tripeptide to chemotherapy for patients with newly diagnosed metastatic osteosarcoma: a report from the Children's Oncology Group,” Cancer, vol. 115, no. 22, pp. 5339–5348, 2009.

P. M. Anderson, P. Meyers, E. Kleinerman et al., “Mifamurtide in metastatic and recurrent osteosarcoma: a patient access study with pharmacokinetic, pharmacodynamic, and safety assessments,” Pediatric Blood & Cancer, vol. 61, no. 2, pp. 238–244, 2014.

S. M. Pollack, E. T. Loggers, E. T. Rodler, C. Yee, and R. L. Jones, “Immune-based therapies for sarcoma,” Sarcoma, vol. 2011, Article ID 438940, 7 pages, 2011.

C. R. Müller, S. Smeland, H. C. F. Bauer, G. Sæter, and H. Strander, “Interferon-α as the only adjuvant treatment in high-grade osteosarcoma: long term results of the Karolinska Hospital series,” Acta Oncologica, vol. 44, no. 5, pp. 475–480, 2005.

H. Strander, H. C. Bauer, O. Brosjo et al., “Long-term adjuvant interferon treatment of human osteosarcoma. A pilot study,” Acta Oncologica, vol. 34, no. 6, pp. 877–880, 1995.

K. Winkler, G. Beron, and R. Kotz, “Neoadjuvant chemotherapy for osteogenic sarcoma: results of a cooperative German/Austrian study,” Journal of Clinical Oncology, vol. 2, no. 6, pp. 617–624, 1984.

S. S. Bielack, J. Whelan, N. Marina et al., “MAP plus maintenance pegylated interferon α-2b (MAPIfn) versus MAP alone in patients with resectable high-grade osteosarcoma and good histologic response to preoperative MAP: first results of the EURAMOS-1 “good response” randomization,” Journal of Clinical Oncology, vol. 31, supplement, abstract LBA10504, 2013.

R. Dillman, N. Barth, S. Selvan et al., “Phase I/II trial of autologous tumor cell line-derived vaccines for recurrent or metastatic sarcomas,” Cancer Biotherapy and Radiopharmaceuticals, vol. 19, no. 5, pp. 581–588, 2004.

J. D. Geiger, R. J. Hutchinson, L. F. Hohenkirk et al., “Vaccination of pediatric solid tumor patients with tumor lysate-pulsed dendritic cells can expand specific T cells and mediate tumor regression,” Cancer Research, vol. 61, no. 23, pp. 8513–8519, 2001.

C. L. Mackall, E. H. Rhee, E. J. Read et al., “A pilot study of consolidative immunotherapy in patients with high-risk pediatric sarcomas,” Clinical Cancer Research, vol. 14, no. 15, pp. 4850–4858, 2008.

K. Pritchard-Jones, I. Spendlove, C. Wilton et al., “Immune responses to the 105AD7 human anti-idiotypic vaccine after intensive chemotherapy, for osteosarcoma,” British Journal of Cancer, vol. 92, no. 8, pp. 1358–1365, 2005.

A. Suminoe, A. Matsuzaki, H. Hattori, Y. Koga, and T. Hara, “Immunotherapy with autologous dendritic cells and tumor antigens for children with refractory malignant solid tumors,” Pediatric Transplantation, vol. 13, no. 6, pp. 746–753, 2009.

S. Kawaguchi, T. Wada, K. Ida et al., “Phase I vaccination trial of SYT-SSX junction peptide in patients with disseminated synovial sarcoma,” Journal of Translational Medicine, vol. 3, article 1, 2005.

S. Kawaguchi, T. Tsukahara, K. Ida et al., “SYT-SSX breakpoint peptide vaccines in patients with synovial sarcoma: a study from the Japanese Musculoskeletal Oncology Group,” Cancer Science, vol. 103, no. 9, pp. 1625–1630, 2012.

J. D. Wolchok, B. Neyns, G. Linette et al., “Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study,” The Lancet Oncology, vol. 11, no. 2, pp. 155–164, 2010.

R. G. Maki, A. A. Jungbluth, S. Gnjatic et al., “A pilot study of anti-CTLA4 antibody ipilimumab in patients with synovial sarcoma,” Sarcoma, vol. 2013, Article ID 168145, 8 pages, 2013.

C. Robert, L. Thomas, I. Bondarenko et al., “Ipilimumab plus dacarbazine for previously untreated metastatic melanoma,” The New England Journal of Medicine, vol. 364, no. 26, pp. 2517–2526, 2011.

E. A. Eisenhauer, P. Therasse, J. Bogaerts et al., “New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1),” European Journal of Cancer, vol. 45, no. 2, pp. 228–247, 2009.

J. D. Wolchok, A. Hoos, S. O'Day et al., “Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria,” Clinical Cancer Research, vol. 15, no. 23, pp. 7412–7420, 2009.

S. S. Agarwala, “Novel immunotherapies as potential therapeutic partners for traditional or targeted agents: cytotoxic T-lymphocyte antigen-4 blockade in advanced melanoma,” Melanoma Research, vol. 20, no. 1, pp. 1–10, 2010.

A. V. Maker, G. Q. Phan, P. Attia et al., “Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: a phase I/II study,” Annals of Surgical Oncology, vol. 12, no. 12, pp. 1005–1016, 2005.

K. C. Lee, I. Ouwehand, A. L. Giannini, N. S. Thomas, N. J. Dibb, and M. J. Bijlmakers, “Lck is a key target of imatinib and dasatinib in T-cell activation,” Leukemia, vol. 24, no. 4, pp. 896–900, 2010.

J. Ozao-Choy, M. Ge, J. Kao et al., “The novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune-based cancer therapies,” Cancer Research, vol. 69, no. 6, pp. 2514–2522, 2009.

A. Kreutzman, V. Juvonen, V. Kairisto et al., “Mono/oligoclonal T and NK cells are common in chronic myeloid leukemia patients at diagnosis and expand during dasatinib therapy,” Blood, vol. 116, no. 5, pp. 772–782, 2010.

P. Ray, N. Krishnamoorthy, T. B. Oriss, and A. Ray, “Signaling of c-kit in dendritic cells influences adaptive immunity,” Annals of the New York Academy of Sciences, vol. 1183, pp. 104–122, 2010.

S. Mustjoki, M. Ekblom, T. P. Arstila et al., “Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy,” Leukemia, vol. 23, no. 8, pp. 1398–1405, 2009.

V. P. Balachandran, M. J. Cavnar, S. Zeng et al., “Imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor through the inhibition of Ido,” Nature Medicine, vol. 17, no. 9, pp. 1094–1100, 2011.

N. Casares, L. Arribillaga, P. Sarobe et al., “CD4⁺/CD25⁺ regulatory cells inhibit activation of tumor-primed CD4⁺ T cells with IFN-γ-dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination,” Journal of Immunology, vol. 171, no. 11, pp. 5931–5939, 2003.

H. Nishikawa, E. Jäger, G. Ritter, L. J. Old, and S. Gnjatic, “CD4⁺ CD25⁺ regulatory T cells control the induction of antigen-specific CD4⁺ helper T cell responses in cancer patients,” Blood, vol. 106, no. 3, pp. 1008–1011, 2005.

S. J. Prasad, K. J. Farrand, S. A. Matthews, J. H. Chang, R. S. McHugh, and F. Ronchese, “Dendritic cells loaded with stressed tumor cells elicit long-lasting protective tumor immunity in mice depleted of CD4⁺CD25⁺ regulatory T cells,” Journal of Immunology, vol. 174, no. 1, pp. 90–98, 2005.

J. Steitz, J. Brück, J. Lenz, J. Knop, and T. Tüting, “Depletion of CD25⁺ CD4⁺ T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon α-induced, CD8⁺ T-cell-dependent immune defense of B16 melanoma,” Cancer Research, vol. 61, no. 24, pp. 8643–8646, 2001.

G. A. Rabinovich, D. Gabrilovich, and E. M. Sotomayor, “Immunosuppressive strategies that are mediated by tumor cells,” Annual Review of Immunology, vol. 25, pp. 267–296, 2007.

S. L. Topalian, F. S. Hodi, J. R. Brahmer et al., “Safety, activity, and immune correlates of anti-PD-1 antibody in cancer,” The New England Journal of Medicine, vol. 366, no. 26, pp. 2443–2454, 2012.

J. D. Wolchok, H. Kluger, M. K. Callahan et al., “Nivolumab plus Ipilimumab in Advanced Melanoma,” The New England Journal of Medicine, vol. 369, no. 2, pp. 122–133, 2013.

R. G. Maki, “Soft tissue sarcoma as a model disease to examine cancer immunotherapy,” Current Opinion in Oncology, vol. 13, no. 4, pp. 270–274, 2001.

C. S. Pukel, K. O. Lloyd, and L. R. Travassos, “, A prominent ganglioside of human melanoma. Detection and characterization by mouse monoclonal antibody,” Journal of Experimental Medicine, vol. 155, no. 4, pp. 1133–1147, 1982.

F. X. Real, A. N. Houghton, and A. P. Albino, “Surface antigens of melanomas and melanocytes defined by mouse monoclonal antibodies: specificity analysis and comparison of antigen expression in cultured cells and tissues,” Cancer Research, vol. 45, no. 9, pp. 4401–4411, 1985.

W. B. Hamilton, F. Helling, K. O. Lloyd, and P. O. Livingston, “Ganglioside expression on human malignant melanoma assessed by quantitative immune thin-layer chromatography,” International Journal of Cancer, vol. 53, no. 4, pp. 566–573, 1993.

S. Zhang, C. Cordon-Cardo, H. S. Zhang et al., “Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides,” International Journal of Cancer, vol. 73, pp. 42–49, 1997.

N.-K. V. Cheung, U. M. Saarinen, and J. E. Neely, “Monoclonal antibodies to a glycolipid antigen on human neuroblastoma cells,” Cancer Research, vol. 45, no. 6, pp. 2642–2649, 1985.

Z.-L. Wu, E. Schwartz, R. Seeger, and S. Ladisch, “Expression of ganglioside by untreated primary human neuroblastomas,” Cancer Research, vol. 46, no. 1, pp. 440–443, 1986.

G. Schulz, D. A. Cheresh, N. M. Varki, A. Yu, L. K. Staffileno, and R. A. Reisfeld, “Detection of ganglioside in tumor tissues and sera of neuroblastoma patients,” Cancer Research, vol. 44, no. 12, part 1, pp. 5914–5920, 1984.

S. Zhang, H. S. Zhang, C. Cordon-Cardo et al., “Selection of tumor antigens as targets for immune attack using immunohistochemistry: II. Blood group-related antigens,” International Journal of Cancer, vol. 73, pp. 50–56, 1997.

S. Zhang, H. S. Zhang, C. Cordon-Cardo, G. Ragupathi, and P. O. Livingston, “Selection of tumor antigens as targets for immune attack using immunohistochemistry: protein antigens,” Clinical Cancer Research, vol. 4, no. 11, pp. 2669–2676, 1998.

H. R. Chang, C. Cordon-Cardo, A. N. Houghton, N. K. Cheung, and M. F. Brennan, “Expression of disialogangliosides and on human soft tissue sarcomas,” Cancer, vol. 70, pp. 633–638, 1992.

M. J. Scanlan, A. O. Gure, A. A. Jungbluth, L. J. Old, and Y.-T. Chen, “Cancer/testis antigens: an expanding family of targets for cancer immunotherapy,” Immunological Reviews, vol. 188, pp. 22–32, 2002.

A. J. G. Simpson, O. L. Caballero, A. Jungbluth, Y.-T. Chen, and L. J. Old, “Cancer/testis antigens, gametogenesis and cancer,” Nature Reviews Cancer, vol. 5, no. 8, pp. 615–625, 2005.

M. Ayyoub, R. N. Taub, M.-L. Keohan et al., “The frequent expression of cancer/testis antigens provides opportunities for immunotherapeutic targeting of sarcoma,” Cancer Immunity, vol. 4, article 7, 2004.

A. A. Jungbluth, C. R. Antonescu, K. J. Busam et al., “Monophasic and biphasic synovial sarcomas abundantly express cancer/testis antigen NY-ESO-1 but not MAGE-A1 or CT7,” International Journal of Cancer, vol. 94, no. 2, pp. 252–256, 2001.

A. A. Jungbluth, Y. T. Chen, E. Stockert et al., “Immunohistochemical analysis of NY-ESO-1 antigen expression in normal and malignant human tissues,” International Journal of Cancer, vol. 92, no. 6, pp. 856–860, 2001.

K. M. Skubitz, S. Pambuccian, J. C. Carlos, and A. P. N. Skubitz, “Identification of heterogeneity among soft tissue sarcomas by gene expression profiles from different tumors,” Journal of Translational Medicine, vol. 6, article 23, 2008.

S. M. Pollack, A. A. Jungbluth, B. L. Hoch et al., “NY-ESO-1 is a ubiquitous immunotherapeutic target antigen for patients with myxoid/round cell liposarcoma,” Cancer, vol. 118, pp. 4564–4570, 2012.

S. A. Rosenberg, “Cell transfer immunotherapy for metastatic solid cancer-what clinicians need to know,” Nature Reviews Clinical Oncology, vol. 8, no. 10, pp. 577–585, 2011.

P. F. Robbins, R. A. Morgan, S. A. Feldman et al., “Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1,” Journal of Clinical Oncology, vol. 29, no. 7, pp. 917–924, 2011.

P. G. Natali, M. R. Nicotra, A. Bigotti et al., “Selective changes in expression of HLA class I polymorphic determinants in human solid tumors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 86, no. 17, pp. 6719–6723, 1989.

M. L. Davila, R. Brentjens, X. Wang, I. Rivière, and M. Sadelain, “How do CARs work? Early insights from recent clinical studies targeting CD19,” Oncoimmunology, vol. 1, pp. 1577–1583, 2012.

M. A. Pule, B. Savoldo, G. D. Myers et al., “Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma,” Nature Medicine, vol. 14, no. 11, pp. 1264–1270, 2008.

J. N. Kochenderfer, Z. Yu, D. Frasheri, N. P. Restifo, and S. A. Rosenberg, “Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells,” Blood, vol. 116, no. 19, pp. 3875–3886, 2010.

M. H. Kershaw, J. A. Westwood, L. L. Parker et al., “A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer,” Clinical Cancer Research, vol. 12, no. 20, pp. 6106–6115, 2006.

C. H. J. Lamers, S. Sleijfer, A. G. Vulto et al., “Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience,” Journal of Clinical Oncology, vol. 24, no. 13, pp. e20–22, 2006.

B. G. Till, M. C. Jensen, J. Wang et al., “Adoptive immunotherapy for indolent non-hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells,” Blood, vol. 112, no. 6, pp. 2261–2271, 2008.

J. R. Park, D. L. DiGiusto, M. Slovak et al., “Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma,” Molecular Therapy, vol. 15, no. 4, pp. 825–833, 2007.

D. R. Shaffer, C. R. Y. Cruz, and C. M. Rooney, “Adoptive T cell transfer,” in Cancer Immunotherapy, T. J. Curiel, Ed., pp. 47–70, Springer, New York, NY, USA, 2012.

Z. Eshhar, T. Waks, G. Gross, and D. G. Schindler, “Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the γ or ζ subunits of the immunoglobulin and T-cell receptors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 90, no. 2, pp. 720–724, 1993.

T. Kobata, K. Agematsu, J. Kameoka, S. F. Schlossman, and C. Morimoto, “CD27 is a signal-transducing molecule involved in CD45RA+ naive T cell costimulation,” Journal of Immunology, vol. 153, no. 12, pp. 5422–5432, 1994.

R. H. Schwartz, “Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy,” Cell, vol. 71, no. 7, pp. 1065–1068, 1992.

T. H. Watts, “TNF/TNFR family members in costimulation of T cell responses,” Annual Review of Immunology, vol. 23, pp. 23–68, 2005.

C. Carpenito, M. C. Milone, R. Hassan et al., “Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 9, pp. 3360–3365, 2009.

X.-S. Zhong, M. Matsushita, J. Plotkin, I. Riviere, and M. Sadelain, “Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI₃ kinase/AKT/Bcl- activation and CD8⁺ T cell-mediated tumor eradication,” Molecular Therapy, vol. 18, no. 2, pp. 413–420, 2010.

R. J. Brentjens, M. L. Davila, I. Riviere et al., “CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia,” Science Translational Medicine, vol. 5, no. 177, Article ID 177ra138, 2013.