Immunotherapy in Sarcoma: A Brief Review

    

    

    Figure 1:  Anti-CTLA-4 and Anti-PD-1 antibodies are used to target T cell immune check points inhibitors in cancer immunotherapy.

(A). CD8 Tcell activation by antigen presenting cells (APC) requires two signals. The first signal is the presentation of specific tumor associated antigenic
peptides by the major histocompatibility complex class I molecules (MHC I) on APCs to the T-cell receptors (TCR) on CD8 T cells. The second signal is the
interaction of CD86 molecules on the APCs with CD28 molecules on the CD8 T cells. After these two activation signals CTLA-4 is up-regulated on the CD8 T
cells and starts to compete with CD28 for binding to CD86. Binding of CD86 on the APCs to CTLA-4 on the CD8 T cells leads to inactivation of CD8 T cells. An
anti-CTLA-4 antibody blocks this interaction and keeps the CD8 T cells activated.

(B). Programmed death ligand 1 and 2 (PDL-1/2) molecules are expressed on the surface of sarcoma cells. Interaction between sarcoma associated PDL1/
2 and T cell programmed death 1 (PD-1) on the CD8 T cells leads to inactivation of tumor-directed CD8 T-cells, allowing the sarcoma to evade CD8 T
cell mediated killing. Use of anti-PD-1 antibodies blocks this T cell inactivation and allows the tumor-directed CD8 T-cells to remain active in destroying the
sarcoma cells.
    
    

A phase II clinical trial was conducted using ipilimumab in synovial sarcoma patients whose tumors expressed the NY-ESO-1 antigen [33]. In this study, 6 patients were treated with one course of ipilimumab given at 3 mg/kg every 3 weeks for 3 doses. There were no responses according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria observed and only one patient demonstrated an immune response. This study was terminated early for slow accrual, lack of activity, and lack of immune responses. It is noted that the doses in two positive melanoma trials were at 10 mg/kg for four doses instead of 3mg/kg for 3 doses as used in this synovial sarcoma trial. In addition standard RECIST criteria were used in the assessment of patients in the sarcoma trial instead of immune related response criteria (irRC). Though not reported in this study, pseudo-progression characterized by increases in measurable tumor and new lesions can complicate the interpretation of progressive disease in patients treated with immunotherapy. Therefore, part of the lack of response in this trial could be attributable to the unique pattern of responses seen in tumors subjected to immunotherapy which could be missed by standard RECIST criteria. In previous immunotherapy trials of melanoma patients, it was noted that many tumors may increase in size or develop new lesions prior to responding to treatment. By using immune related response criteria which allows for a 25% increase in tumor volume and new lesions, more accurate clinical assessment of immunotherapy effects could result [34].

The Programmed Death one (PD-1) and programmed death ligand one pathway (PD-L1) is another checkpoint pathway that can be exploited using anti-PD-1 approaches (Figure 1B). Early studies of anti PD-1 agents (MDX 1106) resulted in 3 of 39 responses. These responses in colon cancer, renal cancer, and melanoma led to a large study in melanoma [35]. The role of the PD-1/PD-L1 axis in STS and melanoma is presently being elucidated. In one retrospective study of tumor specimens from 105 cases, it was determined that the degree of PD-1 positivity in tumor infiltrating lymphocytes and PD-L1 overexpression in tumor specimens correlated with a poorer prognosis and more aggressive disease [8]. These studies suggest that PD-1 and PD-L1 staining may be viable biomarkers for prognosis. As a predictor for response using anti-PD-1 treatment strategies, PD-1 and PD-L1 staining may not be as predictive. In preclinical trials using implanted fibrosarcoma mouse models, activity of anti-PD-1 therapy was independent of PD-L1 staining [36]. Therefore, just as in melanoma, the role of PD-L1 staining as a biomarker is still largely unknown.

Other studies in fibrosarcoma rodent xenografts have shown modest activity using anti-PD-1 therapy alone, but significantly enhanced activity when combined with a dual checkpoint antibody directed at LAG-3 [37]. Therefore, although anti-PD1 therapy has not been tested in sarcoma patients, there is ample rationale for using anti-PD-1 treatment alone and possibly in combination with other checkpoint inhibitors in patients with advanced STS. There is an ongoing phase I clinical trial in solid tumors that is testing anti-LAG -3 antibody alone and combined with anti-PD-1 therapy. (NCT01968109)

(3) Vaccine (Active Immunization) Therapy, Dendritic Cell Approaches, and Generation of "Autovaccines" via the Abscopal Effect

The manipulation of tumor antigens to stimulate both humoral and cellular immune responses.

The sine qua non of an immune response is its specificity for the unique molecular target, also known as an epitope. Therefore, the search for unique molecules that would be recognized as "foreign" by the immune system is the cornerstone of development of specific immunotherapy. The identification of immune stimulating tumor antigens is therefore critically important in the development of tumor vaccines.

In studies examining the specificity of Tumor Infiltrating Lymphocytes (TILS) for melanoma antigens, surface receptors with specificity to over 50 different antigens have been identified [38]. These included both melanoma antigens as well as a wide variety of cancer testis antigens. The importance of this knowledge is that these antigens may serve as targets responsible for the specific recognition by cytotoxic T cells. The lists of such antigens in sarcomas include not only melanoma differentiation antigens and cancer testis antigens, but also mutated or other overexpressed antigens such as synovial sarcoma transcript proteins and gangliosides [39,40]. Since up to 30 percent of sarcomas may express unique fusion proteins, a variety of antigens can be exploited as targets for immunotherapy. The NY-ESO-1 antigen is a known cancer testis antigen recognized by cytotoxic T cells and is expressed in over 80% of synovial sarcomas, 100 percent of myxoid round cell liposarcomas, and in a high percentage of osteosarcomas and uterine leiomyosarcomas [41- 43]. MAGE-A3 is another cancer testis antigen that was originally discovered in melanoma and, interestingly, is expressed in some nonuterine leiomyosarcomas as well as uterine leiomyosarcomas [44].

Although molecular characterization of tumor antigens has been successful, there has been little evidence that active vaccination alone can lead to tumor regression. It appears that it may be necessary, but insufficient by itself. Use of tumor derived peptides, proteins, whole tumor cells, recombinant viruses, dendritic cells, and heat shock proteins have yielded few responses in any solid tumors. A review of over 1000 published vaccine treatment articles, showed an abysmal overall objective response rate of 3.3% [45]. Analyses of surrogate markers of benefit, such as the presence of circulating T cells and tumor infiltration by T cells, have taught us that detection of an immune response to these epitopes is clearly not linked to clinically meaningful responses. In sarcoma, one study used peptides from the unique and restricted SYT: SSX fusion gene, found in most synovial sarcomas, and demonstrated induction of peptide specific cytotoxic T cells in 4 patients, but only modest clinical benefit in one patient [40].

Resetting the balance towards immune stimulation underlies a series of studies in which vaccines were combined with a second immune stimulation signal. Augmenting the immune response after vaccination using a cytokine was attempted in a 21 patient pilot study with synovial sarcoma patients in whom an SYT-SSX fusion region peptide vaccine was given in conjunction with interferon-alpha [46]. These patients were injected subcutaneously with the vaccine and assessed for response. Although only one of 21 patients had a minor response, 6 of the patients demonstrated stable disease at 12 weeks. The duration of stable disease was not reported, so no conclusion about durability could be ascertained, however, the treatment was feasible and tolerable. The efficacy of GM-CSF based approaches in melanoma patients has been inconsistent when used as the sole therapy or combined with vaccines [47]. In one study using whole cell vaccine combined with genetically engineered tumor cells for GM-CSF expression, melanoma patients showed a significantly lower number of circulating CD8 positive cells and inferior rates of progression free survivals [48]. A phase 1 study in melanoma and sarcoma patients treated with autologous tumor vaccine that was genetically engineered to express GM-CSF showed only one patient with immunologic response to tumor vaccine and no responses in a group of 6 sarcoma patients [49]. Therefore, the effectiveness of cytokine enhanced vaccine approaches is inconclusive and future studies should include additional modifying techniques to alter the balance in favor of immune stimulation.

The Major Histocompatibility Complex (MHC), in humans called the Human Leukocyte Antigen (HLA) system is known to play a major role in tumor immunogenicity. By developing a vaccine that presents tumor antigens in an MHC restricted manner, better immune responses may be elicited. In one sarcoma study, multiple tumorassociated antigens associated with four HLA matched peptides were used to vaccinate 20 sarcoma patients in a personalized peptide vaccination approach. This personalized vaccine approach resulted in stable disease in 6 of 20 patients with the median stabilization period being 9.5 months and the longest duration of response being 35 months. This compared favorably with other studies where patients typically survived a median of 8 months and PFS was 23% at 6 months [50]. Patients that did not respond had elevated IL-6 levels during therapy. Interleukin-6 is reported to increase the concentration of Myeloid Derived Suppressor Cells (MDSC) and other immune suppressing cell subsets. Cytotoxic T-cells derived from peripheral blood mononuclear cells were noted to be enhanced in a high proportion of the patient cohort, as only 3 of 20 demonstrated peptide specific T cell responses pre-vaccination whereas 13 of 17 patients developed cytotoxic T cells post vaccination. Although it is difficult to draw conclusions from this study, it does re-emphasize the importance of MHC contributions to tumor recognition.

Other attempts at enhancing vaccine potency have centered on dendritic cell vaccines. By isolating dendritic cells and pulsing them with tumor antigens ex-vivo, enhanced immunogenic responses may be realized. The most recent example of this was performed in metastatic prostate cancer where dendritic cells were apheresed from patients and pulsed ex vivo with prostate acid phosphatase antigen linked to GM-CSF. This large study, which led to the FDA approval of Sipuleucel-T, demonstrated a low percentage of objective PSA and RECIST responses of 2.7% with no difference in PFS, however, the vaccine group had a median survival of 25.8 months vs 21.7 months for the placebo group. (p=.03) [51].

In sarcoma, dendritic cell vaccines have been used in small pilot studies. In a report from a study of solid tumors in children using pulsed dendritic cells with tumor cell lysates and Keyhole Limpet Hemocyanin (KLH), a dramatic tumor response was seen in a fibrosarcoma patient who had metastatic disease to his back and lungs. This lysate-pulsed dendritic vaccine showed no observable toxicity and an objective response in both tumor and bone [52]. Another application of dendritic cell vaccines is its use in vivo by direct injection of tumors after radiation therapy. Since local tumor irradiation had been shown to elicit immune responses when combined with direct tumor dendritic cell injection in preclinical models, a therapeutic strategy using standard fractionated radiation combined with intra-tumoral injection was completed. Finkelstein reported on the injection of dendritic cells in sarcoma patients that received radiation to their primary tumor. Immune responses were seen in 9 of 17 injected patients, and 12 of 17 remained progression free at one year [53]. It was not reported whether immune response correlated with progression free survival, however, a randomized study looking at locally advanced sarcoma patients with greater than 5 centimeter tumors is underway comparing intra-tumoral dendritic cell vaccine to no vaccine. (NCT01347034)

One of the theoretical issues with the use of the aforementioned approaches is that one is never sure that the correct epitope is being targeted. There are two approaches to this issue, one is to induce the up-regulation of tumor antigens and the other is to cause a release of these via cytolytic mechanisms such as tumor irradiation.

The use of epigenetic modifying compounds such as decitabine is an example of the first strategy. Preclinical studies show that cancer testis antigens; NY-ESO-1, LAGE-1, SSX, and MAGE-A10, can be up-regulated in sarcoma cell lines using decitabine. These in vitro cell line studies likewise show that in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma; MAGE-A1, MAGE-A3, and NY-ESO-1 can increase up to 2500 fold under the influence of decitabine [54]. Concomitant up-regulation of MHC molecules and antigen specific CTL recognition were also demonstrated. These findings raise the possibility of enhancing an antitumor effect by priming tumors to overexpress cancer antigens using systemic agents, in essence, increasing the target volume in the presence of the targeting cells. An ongoing Phase 1 study using decitabine followed by a dendritic cell vaccine pulsed with NY-ESO-1, MAGE-A1, and MAGE-A3 in young patients with neuroblastoma, synovial sarcoma, osteosarcoma, rhabdomyosarcoma, and Ewing's sarcoma is ongoing [55]. (NCT01241162)

The work by the Oregon group led by Drs. Urba and Curti is an example of using radiation to prime the immune system such that the application of a subsequent immune stimulus, in this case high dose IL-2, results in a significant percentage of patients obtaining a meaningful response. In their study, treatment naive patients with metastatic melanoma or renal cell carcinoma underwent Stereotactic Body Radiation Therapy (SBRT) followed by high dose IL-2 [56]. Eight of 12 patients (66.6%) achieved a complete (CR) or Partial Response (PR) in a setting where mature historical data showed response rates in the 10 to 20 percent range.

This is perhaps the most vivid example of an effect that is being observed post radiation, where untreated tumor deposits distant from an irradiated tumor can show tumor response. The so-called abscopal effect exists in the realm of case reports and cohort studies. The Oregon investigators hypothesized that radiation increases tumor antigen release and changes in the tumor microenvironment such that the immune effects of IL-2 are significantly more effective in melanoma and perhaps renal cell carcinoma. Whether these effects can be seen in sarcoma is not known.

(4) Adoptive Cell Therapy

Infusion of manipulated T-cells that have been activated and/or expanded ex-vivo.

Adoptive cellular immune therapy is an immunotherapy approach that infuses immune-manipulated lymphocytes into a cancer patient in order to elicit an antitumor response. One advantage of this strategy is that the ex vivo expansion of these manipulated T cells allows for the reintroduction of large numbers of these activated cells. In addition, the absence of physiologic counterbalancing inhibitory mechanisms which naturally occur in vivo can be avoided or manipulated to favor the immune balance in favor of these infused activated T-Cells. Oftentimes, this strategy is combined with host conditioning, such as non-myeloablative chemotherapy or body radiation, which further enhances the possibility of the desired immune response [57].

This strategy is best worked out in melanoma patients. In a study of 13 patients treated with a conditioning regimen of non-myeloablative therapy and IL-2 combined with adoptive autologous T cells directed at MART antigens, 6 of 13 patients had a partial response [58]. T-cell expression of peripheral lymphocytes showed evidence of viability of infused T-cells and evidence of in vivo expansion of these cells. Biopsy of tumor samples that showed increased tumor infiltration by CD8 positive lymphocytes was evidence of T cell receptor activity towards the MART antigen.

The demonstration of effective adoptive therapy in sarcoma patients was carried out in a study using autologous T cells engineered with T-cell receptor directed at NY-ESO-1 antigen in synovial sarcoma patients. Eighty percent of synovial cell sarcomas express this antigen, and its expression was an inclusion criterion for this study. Patients were initially lympho-depleted using cyclophosphamide and fludarabine; autologous T cells genetically engineered to recognize NY-ESO-1 and HLA-A*0201 antigens were then infused. Partial responses occurred in 4 of 6 sarcoma patients, with one response durable to 18 months. This therapy was also well tolerated with no off target effects [7].

Use of isolated human T cells with receptor specificity for unique molecular signature breakpoints has not been used in studies in synovial sarcoma patients. However, the feasibility of this adoptive approach directed at the unique synovial sarcoma x break point 2 (SSX2) in melanoma cells has been tested. These clonal T cells were observed to release gamma interferon and included cell lysis in an HLA restricted manner after culture of TCR engineered Peripheral Blood Lymphocytes (PBL) with relevant tumor cell lines [59]. Although these cells were originally derived from melanoma cells of two melanoma patients, application of this technology to synovial sarcoma patients who preferentially express the SSX2 antigen is an exciting possibility.

Other sarcoma translocation breakpoint studies using adoptive approaches directed at pediatric sarcomas have been completed. Dendritic cell vaccines pulsed with specific pediatric translocation breakpoint peptides along with a peptide known as E7 (which binds to HLA-A2) were used as priming antigens and combined with infusion of autologous T-cells in a study of Ewing's patients harboring the 11:22 translocation and alveolar rhabdomyosarcoma patients harboring the 2:13 translocation. There were 52 patients entered into this study with 30 patients getting immunotherapy as consolidation treatment after maximal cytoreduction of tumor by chemotherapy, radiation therapy, or surgery. These alveolar rhabdomyosarcoma and Ewing's sarcoma patients had a 43% 5 year overall survival despite recurrent or metastatic disease. Although these results could be related to selection bias, patients generally tolerated the therapy well. Immune responses as measured by serum gamma interferon production were inconsistent and not sustained by dendritic cell vaccination. This study serves as a sound framework to study future consolidative immune therapy approaches in the minimal disease setting [60]. With future improvements in antigen immunogenicity and dendritic cell maturation processes, better immunologic effects may be seen and applied to the low disease burden setting using combined approaches of enhanced target exposure combined with adoptive T-cell therapy.

Unfortunately the HLA restriction of the above adoptive T-cell therapies limits the proportion of patients that can be effectively treated. Furthermore, tumor cells can often times lose MHC antigens thereby mitigating recognition by cytotoxic T-cells. Engineering an adoptive T-cell therapy whose response is not HLA restricted could potentially include a larger proportion of patients and recognize tumor cells that may have lost MHC antigen. Chimeric Antigen Receptor (CAR) therapy consists of the introduction of an engineered T-cell receptor with specificity to a desired antigen without the need for MHC restriction. The prototype of this strategy was attempted in neuroblastoma patients [61]. The ganglioside GD-2 antigen is a highly expressed antigen on neuroectodermal derived tumors and sarcomas. It is often expressed in various sarcomas including: osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, liposarcoma, fibrosarcoma, and leiomyosarcoma [39,62]. In a preliminary report of a phase 1 study in neuroblastoma patients, persistence of the CAR modified T cells were evident and a tumor response was seen in half the patients. T Cell-CAR therapy directed at GD-2 has also been reported in a preclinical model of Ewing's Sarcoma [63]. In this mouse study, there was a noticeable delay in tumor growth in lungs compared to control mice. The efficacy of GD-2-directed therapy in this study, though modest, may be more applicable in the setting of a lower tumor burden such as in patients that had pulmonary metastatectomy. For bulky disease the authors concluded that combined approaches aimed at tumor cytoreduction before use of CAR therapy may be the most effective application of this therapy. There have been various molecular iterations of this strategy in the development of second generation CAR engineered T-cells. The latest development has been manipulation of the co-stimulatory domains of the CAR in effort to enhance in vivo T cell expansion and activity. An anti-GD-2 study using CAR engineered T cells is ongoing using a next generation CAR T cell approach aimed at GD-2 in combination with a vaccine for sarcomas. (NCT01953900).

Conclusion

The use of cytotoxic agents remains the dominant paradigm for the treatment of advanced or metastatic bone and soft tissue sarcomas. Aside from gastrointestinal stromal tumors, the recent introduction of targeted therapy to sarcomas has only provided marginal benefits. Unfortunately, only the occasional patient with advanced stage disease can be cured with present strategies. With melanoma and renal cell carcinoma leading the way, immunotherapy has consistently demonstrated the ability to give rise to durable longterm responses, some of which are complete, in a subset of patients. Because sarcomas possess unique genetic changes and immunogenic antigens, immunotherapeutic strategies may be particularly fruitful in these conditions. In addition, the biological heterogeneity of sarcomas will not likely yield a unified molecular pathway that could be exploited to treat a large percentage of sarcomas, thus emphasizing the need for a therapy that may be applicable to a higher proportion of sarcoma patients. At this time, the unique confluence of both checkpoint inhibitors along with the prevalence of cancer testis antigen expression in many adult sarcomas provides a gateway to test immunotherapy strategies in sarcoma. Other strategies that include cytokines, other checkpoint inhibitors, vaccination, and adoptive T cell transfer- all of which can be given singly or in combination are currently under study. One of the major impediments has been the plethora of small, under-powered clinical trials in this area. Through collaborative efforts of the entire sarcoma community, definitive answers can be realized that may change the paradigm of treatment of a disease that has witnessed few major breakthroughs.

Acknowledgement

WM Kast holds the Walter A. Richter Cancer Research Chair. Contributions from the Karl H. and Ruth M. Balz Trust are also gratefully acknowledged. This review has been made possible by support of the Norris Comprehensive Cancer Center NCI support grant 5 P30 CA 014089-39. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

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