Abstract
Cancers of the central nervous system (CNS) are unique both in the immunopathology underlying their development and in the challenges they present to designing effective immune-based therapeutic strategies. The immune response in the CNS has fundamental differences from that seen elsewhere in the organism. Moreover, a series of evasion mechanisms have been described for CNS tumors, which limit effective recognition and effective antitumoral cytotoxic responses by the immune system. A series of therapeutic approaches are currently being developed for enhancing and focusing the immune system to elicit and enhance a therapeutic antitumoral response. In this chapter, an overview of the intricacies of the immune system in the CNS within the context of tumor immunology is presented. In addition, some of the most salient immunotherapeutic advances for treatment of CNS tumors are explored.
18.1 Introduction
The immunopathology of central nervous system (CNS) cancers is unique; hence, they pose various challenges to designing effective immune-based therapeutic strategies. Considering the modest efficacy of current CNS cancer treatment, developments in preclinical and early clinical investigations of immune-based therapies appear promising. Cancers of the brain, spinal cord, and surrounding structures are diagnosed in approximately 9–11 per 100,000 people in the United States per year and effect an age-adjusted mortality rate of 4.3 per 100,000 per year [1], underscoring the general lethality of these tumors. Current treatments are rarely curative and often aimed primarily at reducing short-term mortality while minimizing neurological morbidity. Generally, these tumors are treated through a combination of cytoreductive surgery, chemotherapy, and radiation therapy, although exceptions exist for particular tumors. These treatments are largely nonspecific to cancer cells and therefore may be damaging to bystander neurological tissue while achieving only modest therapeutic benefits. For instance, in a recent series, treatment of glioblastoma multiforme (GBM) with surgery, radiation therapy, and chemotherapy with temozolomide led to an overall 2-year survival in only 27 % [2]. The vital functions and poor resiliency of neurologic tissue and the often diffusely infiltrating nature of CNS cancers pose a significant challenge to current therapies.
Antineoplastic properties of the immune system are well documented and known to be dysregulated in many human cancers, including those of the CNS [3]. Understanding the mechanisms by which immune cells may prevent CNS tumor development or by which these cells may contribute to tumor-mediated immune evasion is of key importance to combat cancer on a cell-specific level. Appreciation of the distinct features of immune activation and modulation within the CNS will be fundamental to the development of any immune-based therapy for brain tumors.
This chapter provides an overview of the intricacies of the immune system in the context of the CNS. The potential interactions between the immune system and a developing CNS tumor will be discussed. Additionally, some interesting immunotherapeutic approaches currently under development in the setting of CNS cancer will be discussed.
18.2 Antitumor Mechanisms of the Immune System
Generally, antitumor immune surveillance is thought to occur in three different circumstances. First, eradication of pathogens that cause chronic inflammation is believed to prevent the development of some cancers. The proposed explanation for this principle involves the inflammatory milieu, which contains free radicals and genotoxic agents that act as carcinogenic stimuli. Barricaded as a sterile space, immune mediators of the CNS are not routinely exposed to pathogens. Nevertheless, evidence from parenchymal infection or infarction and from autoimmune disease such as multiple sclerosis demonstrates the capacity to initiate classical inflammatory cascades within the CNS upon exposure to pathogens [4, 5]. Second, control of oncogenic viral infections through Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cell immunity is key to prevention of viral induced transformation. Examples of viral induced cancers include some lymphomas caused by Epstein-Barr virus [6] and cervical carcinoma caused by papilloma virus [7]. Regarding CNS tumors, this principle may be relevant in the context of cytomegalovirus (CMV), which is hypothesized to underlie the development of gliomas. This highly controversial hypothesis is mainly supported by association studies, none of which have provided scientific evidence of causality. One of the most notable reports founding this hypothesis consists of a study in which CMV antigens were detected in glioma tissue specimen [8]. Lastly, CTLs and NK cells are capable of recognizing and eliminating tumor cells that overexpress developmental or tumor-specific antigens derived from cancer-related genetic alterations. Accumulating evidence suggests that immune mediators are capable of eliciting this mechanism for combating CNS cancers. Yet, additional evidence suggests that these effector immune cells are limited in doing so, that there is a scarcity of tumor antigens capable of being specifically recognized, and that these effector cells are overcome by tumor-derived immunosuppressive influences. A focus of ongoing CNS cancer immunotherapeutic research is the investigation of the limitations to antitumoral immunity and the design of strategies to enhance it.
18.3 Immune Compartment of the CNS
In the past, the CNS was perceived to possess little immunologic potential to resist tumor development [9].
This idea was based on a perceived lack of specialized antigen-presenting cells (APCs), restriction from circulating lymphocytes and other immune mediators by the blood-brain barrier (BBB), and absence of lymphatic drainage in the CNS. However, evidence accumulated over the last 20 years has largely debunked this view of the CNS by demonstrating distinct immune activation cascades in response to cerebral ischemia, traumatic brain injury, and autoimmune diseases such as multiple sclerosis [5, 10]. In each of these pathological states, immune competence is contingent upon the activation of resident microglia and infiltrating macrophages capable of effective antigen presentation and lymphocyte activation, all permissible through inducible permeability of the BBB to leukocytes and immune mediators [4, 11]. Activated microglia have been shown to phenotypically resemble both macrophages and dendritic cells (DCs), capable of presenting antigens and activating T-cell lymphocytes [12]. Following activation, CNS APCs are capable of returning to the systemic circulation through drainage via perivascular Virchow-Robin spaces and the nasal mucosa as conduits to cervical lymph nodes [13–15]. Subsequently, both activated and naive T cells responding to chemotactic signals have been shown to traverse the BBB and engraft into sites of inflammation [16]. Activated T cells remain in the CNS, as demonstrated in tumor extracts from multiple CNS cancer types which display tumor antigen-specific CTLs and helper T cells capable of tumoricidal immune function in vitro [17–19]. Additionally, circulating CNS antigen-specific antibodies and CTLs have been isolated from the peripheral blood of patients with CNS cancer, further indicating the potential for competent tumor-specific responses within the CNS [20, 21].
On the other hand, opposing immunosuppressive phenomena have been described in the setting of CNS cancer. A series of reports have demonstrated anergy and apoptosis following TCR stimulation in CNS cancer-infiltrating T cells (reviewed in [18]), as well as an overwhelming presence of suppressive regulatory T cells (Tregs) within high-grade CNS tumors (reviewed in [22]). Furthermore, tumor-infiltrating macrophages have been shown to possess immunosuppressive and tumorigenic phenotypes in the setting of glioma [23, 24]. Understanding the forces driving lymphocyte activation vs. suppression following stimulation with tumor antigens within the CNS is imperative to the success of CNS cancer immune-based therapies.
18.4 CNS Tumor-Derived Immunosuppression
Suppression of both CNS immune surveillance and activated tumoricidal immune cells by tumor cells is a fundamental feature of tumor development. Unfolding evidence implicates many cellular participants in this process, including resident microglia, peripherally invading macrophages, and lymphocytes, most notably Tregs. Interactions among these players are believed to underlie the state of generalized immunosuppression observed in many patients with CNS cancers, likely extending systemically from the potently immunosuppressive local tumor microenvironment at the interface of tumor and immune cells. A brief overview of the main cellular players for CNS tumor-induced immunosuppression is provided here.
18.4.1 Tumor Cells
Transformed cells are clear targets for CNS immune sentinels responding to the expression of aberrant or mutated antigens, as well as to cellular stress antigens which are associated with cancer-induced cell proliferation and stromal remodeling. These antigens activate immune sentinel cells through stimulation of major histocompatibility complex (MHC) class I and II molecules, in coordination with co-stimulatory signals including B7 isoforms 1 and 2 (CD80/86) [25, 26]. As a principal means of evading tumoricidal immune activation, CNS tumor cells markedly downregulate expression of both MHC I and II proteins. In malignant glioma, the most extensively studied CNS cancer, an inverse correlation has been observed between the extent of MHC expression and tumor lymphocyte infiltration. MHC expression demonstrated an inverse correlation with tumor grade [27], suggesting its downregulation as an immune-evasion mechanism for tumor cells. Additionally, through a potent cocktail of secreted mediators, glioma cells induce the downregulation of co-stimulatory molecules B7-1 and B7-2 on both tumor cells and surrounding APCs, most notably tumor-associated macrophages (TAM), removing a necessary signal for proper T-cell activation [28–30]. Furthermore, glioma cells can express immunosuppressive molecules such as the co-stimulatory molecule homologue B7-H1 [31], the expression of which is normally limited to Gemcitabine and carboplatin at the end of immune responses. B7-H1 expression has been demonstrated both on glioma cells themselves and on TAMs and functions to induce apoptosis in activated T cells [31, 32].
Upregulation of secreted molecules and cell surface proteins by glioma cells also contributes to potent immunosuppression and tumor propagation. Among the most extensively documented are transforming growth factor beta (TGF-β), prostaglandin E2 (PGE2), Fas ligand (FasL), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and the immunomodulatory cytokines IL-4, IL-6, and IL-10 (reviewed in [33]). TGF-β is also known to inhibit the development and activation of APCs, repress activation of NKs, and prevent the activation and differentiation of CTL [34]. PGE2 is associated with suppression of T-cell activation and proliferation and has been demonstrated to induce the production of Tregs [35]. Among the main pathways mediating programmed cell death in a variety of effector immune cell types is the cell surface protein FasL, which has been detected on the surface of tumor cells isolated from gliomas, as well as in multiple CNS cancer cell lines [36]. Both microglia and T cells express its receptor, Fas, and therefore may be susceptible to the death signal provided by FasL expressed on CNS tumor cells. Indeed, multiple studies have demonstrated that FasL was responsible for the death of T lymphocytes when cocultured with glioma cells in vitro and that the downregulation of FasL on tumor cells enhances tumor infiltration by T cells, reducing tumor growth in vivo [37]. Increased expression of the immunomodulatory cytokines IL-4, IL-6, and IL-10 has been demonstrated in high-grade gliomas, most notably GBM [38]; these cytokines limit inflammation, reduce immune activation, and drive the expression of immunosuppressive mediators such as TGF-β and PGE2 [39].
Recently, expression of indoleamine 2,3-dioxygenase (IDO) in gliomas has been implicated in the recruitment of immunosuppressive CD4+CD25+FOX-P3 Tregs and the subsequent ablation of antitumoral immunity. A series of in vivo experiments showed that IDO-derived Treg tumor infiltration led to a decrease of CD8+ cytotoxic T-cell tumor infiltration and, in contrast, IDO silencing on tumor cells led to an increase in CD8+ tumor infiltration and an increase in overall survival for mice bearing glioma xenografts. Interestingly, Wainwright et al. demonstrated that tumor cell-specific expression of IDO, rather than peripheral expression of this enzyme, is critical for maintaining this immunosuppressive state [40]. IDO might have a clinical and translational therapeutic potential, as its expression correlates with tumor grade and has a negative impact on overall survival for patients with gliomas [40, 41].
In addition to the mechanisms discussed above, cell-cell interactions might play a role in the complex local microenvironment involving tumor and immune cells, which are both potently immunosuppressive and tumorigenic [18].
18.4.2 Glioma Cancer Stem Cells
Cancer stem cells (CSCs) are a heterogeneous group of undifferentiated tumor cells which posses an enhanced capacity for self-renewal, multipotency, and tumorigenicity at low cell numbers and during isolation [42].
Some evidence suggests the implication of gCSC in tumor-mediated immunosuppression; gCSCs isolated from human glioma specimens and grown in vitro were shown to have reduced expression of MHC and co-stimulatory molecule expression but demonstrated high levels of expression of immune-inhibitory molecules [43]. Additionally, coculture experiments have shown that gCSCs induced apoptosis of both naïve and activated T cells through secretion of galactin-3. In addition, gCSCs also inhibited phagocytosis and expression of tumor necrosis factor alpha (TNF-α) in macrophages through secretion of macrophage inhibitory cytokine 1 (MIC-1) [44]. Finally, gCSCs are believed to confer radiation and chemotherapeutic resistance [44, 45]. The near inevitability of glioma recurrence following standard treatments may result from recalcitrant gCSCs, which escape the therapeutic targeting and regenerate the parent tumor. Any therapeutic strategy designed to affect a lasting tumor remission should therefore target gCSCs.
18.4.3 Tumor-Associated Macrophages/Microglia
Tumor-associated macrophages/microglia (TAMs) are the predominant infiltrating immune cells in malignant glioma and can account for up to 40 % of the tumor cell mass [23]. Though phenotypically indistinguishable following activation, TAMs are derived both from resident CNS microglia and from bone marrow mononuclear cells that colonize the CNS under pathological conditions [36]. A series of studies have shown that in the case of gliomas, TAMs under the influence of tumor cells can acquire a phenotype that contributes to the immunosuppressive and tumor-promoting local tumor microenvironment [24].
Characterization of TAMs in glioma has led to delineation between classically activated inflammatory M1-type TAMs with tumoricidal potential and alternatively activated immunosuppressive M2-type TAMs, which are predominant in the CNS tumor microenvironment. Classically activated M1-type TAMs participate in the coordinated response to immunogenic antigens primarily through production of proinflammatory and tumoricidal mediators such as NO, TNF-α, IL-1Β, and IL-12, upregulation of MHC and co-stimulatory molecules necessary for antigen presentation, and an overall enhanced ability to phagocytose pathogenic material (reviewed in [18]). Conversely, M2-type TAMs exert immune modulation through secretion of potent immunosuppressive mediators including IL-10, IL-6, and TGF-β. In addition to this cytokine cocktail, M2-type TAMs downregulate MHC and co-stimulatory molecules, show a decreased phagocytic capability, and upregulate the cell surface antigens FASL and B7-H1. The upregulation of these two molecules leads to the induction of anergy and apoptosis in effector T cells, which express Fas ligand. Thus, M2-type TAMs appear to play a role in the immunosuppressive environment seen on gliomas (Fig. 18.1).
Polarization of tumor-associated macrophages in glioma. Notice the distinct M1 and M2 phenotypes (Reprinted from Li and Graeber [18], with permission)
Alternatively activated M2-type TAMs are the predominant immune cell type in malignant glioma, and their presence has been shown to correlate with histological grade [46]. A recent investigation revealed increased expression of the M2 markers CD163 and CD204 by TAMs in WHO grade IV gliomas, compared to WHO grades II and III gliomas [47]. The perverse polarization of TAM precursors, both resident microglia and peripheral derived monocytes, to the alternative M2 state is generally believed to occur as these cells encounter the myriad cytokines, growth factors, and surface antigens of the tumor microenvironment. Among the factors implicated in the active recruitment and altered polarization of monocytes by CNS tumor cells, monocyte chemoattractant proteins 1 (MCP-1/CCL-2) and monocyte colony-stimulating factor (M-CSF) are believed to drive local recruitment and proliferation of TAM precursors, while TGF-β, IL-4, IL-10, and IL-13 together orchestrate polarization to the alternative M2 phenotype [47, 48]. Importantly, this polarization toward a M2 TAM phenotype takes place in the absence of IFN-γ, a potent driver of the classical M1 phenotype [49].
The absence of IFN-γ is likely due to the suppression of its principle source, activated type 1 T-helper cells (discussed below).
18.4.4 Myeloid-Derived Suppressor Cells
Recent refinements of the M1/M2 TAM characterization scheme describe a more heterogeneous population of systemically distributed M2 TAM-like myeloid-derived immunosuppressive cells at intermediate stages of maturation, which are able to suppress multiple phases of the immune response [50]. These myeloid-derived suppressor cells (MDSCs) have been shown both to perpetuate tumor-promoting microenvironments and to distribute peripherally to hinder lymphocyte activation in immune organs. MDSCs are therefore implicated in the general systemic immunosuppression observed in patients with malignant gliomas [48]. Recent evidence suggests that MDSC precursors must be exposed to the concentrated cocktail of immunomodulatory mediators and cell-cell interactions in the tumor microenvironment to become MDSCs [48]. These observations suggest that naïve monocyte traffic to the tumor microenvironment, mature into immunosuppressive MDSCs, and then redistribute systemically [48]. Systemically circulating MDSCs present a poorly understood hurdle to remediating CNS cancer immune suppression. Their heterogeneous expression profile and systemic distribution allow a potentially broad and widespread armament of immunosuppressive functions. If indeed these cells are generated by local tumor-derived factors of the microenvironment, as in the more clearly defined M2 TAM phenotype, then disabling the local “monocyte-educating” mechanisms of tumor cells may reduce the generation of MDSC.
18.4.5 Lymphocytes and Regulatory T cells
Lymphocyte effector cells are major players in antineoplastic immunity, yet lymphocytes which traffic to cancers of the CNS are disabled, reprogrammed to immunosuppressive phenotypes, and subsequently permitted to remain within tumor through failure of natural anergic cell deletion. As discussed above, the process of T-cell activation by APCs is severely hindered in CNS cancers by reductions in MHC and co-stimulatory molecules on both tumor cells and surrounding APCs and by the milieu of T-cell-deactivating mediators within the tumor microenvironment. NK cells are known to initiate deletion of T cells with reduced expression of MHC or co-stimulatory molecules, releasing TNF-α and IFN-γ (reviewed in [50]). This fail-safe mechanism is believed to be disabled by the immunosuppressive milieu of the local tumor microenvironment, most notably by IL-10, and by activation of the NK cell inhibitory receptor KIR2DL through the ligand HLA-G, which is expressed on Tregs [51]. Through these mechanisms, T lymphocytes that are polarized to immunosuppressive phenotypes are permitted to remain within CNS tumors.
Ongoing research implicates Tregs as a major lymphocyte player in CNS tumor immune biology. An increased systemic prevalence of Tregs among T cells has been observed in malignant glioma, consistent with their role in suppressing the immune rejection of neoplastic cells [52]. In addition, Treg infiltration of brain tumors has also been demonstrated, and in the case of gliomas, the fraction of Treg correlates with tumor grade [52, 53]. These observations reflect the significant role Tregs play as a negative immune modulator of lymphocytes both within the tumor and peripherally in lymphoid organs, leading to immune evasion by tumor cells.
Investigation of the origin, recruitment, expansion, and immunomodulatory effect of Tregs in malignant gliomas is an active effort within the tumor immunology field. Recent evidence shows that T cells may be converted to CD4+/Foxp3+-induced Tregs (iTregs) peripherally through exposure to APCs or suboptimal TCR stimulation in the presence of high levels of TGF-β, as is present in the tumor microenvironment [40].
Both iTregs and thymus-derived natural Tregs (nTregs) have been shown to infiltrate and proliferate within CNS tumors. These cells migrate in response to tumor-secreted MCP-1, which binds CCR4, a receptor highly expressed on Tregs and their precursors [51]. The mechanisms by which Tregs elicit immunosuppression involve Foxp3-mediated expression of the immunosuppressive cell surface ligands glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T-lymphocyte antigen (CTLA-4), and human leukocyte antigen G (HLA-G), as well as through the contribution of immunosuppressive cytokines TGF-β and IL-10 into the microenvironment [54]. This inhibitory signal replaces the stimulatory interaction between T-cell protein CD28 and APC co-stimulatory molecules B7-1 and B7-2 to prevent activation. HLA-G on placental cells has been shown to contribute immune tolerance in pregnancy by binding the KIR2DL receptor of NK cells, blocking activation in the presence of cells lacking MHC or co-stimulatory molecules. By this mechanism, Tregs are hypothesized to disable NK cell surveillance.
18.5 STAT3 Pathway
As discussed, many soluble mediators and cell surface molecules expressed by tumor cells, TAMs, and Tregs participate to establish a potently immune-disabling microenvironment. Expression profiles across these various cellular players are similar, raising suspicion for unifying mediators of signal transduction or gene expression common to these shared phenotypes. Signal transducer and activator of transcription protein 3 (STAT3), a transcription factor active in both glioma cells and TAMs, has been shown to influence multiple immunosuppressive signaling pathways implicated in CNS tumor-induced immunosuppression [55].
Furthermore, considering the myriad targets of STAT3 modulation, activation of this intracellular mediator may also augment CNS tumor angiogenesis and stromal remodeling [56]. STAT3 activation in glioma TAMs is induced downstream of many mediators known to constitute the local microenvironment such as IL-10, IL-6, EGF, and FGF [57]. In both tumor cells and TAMs, STAT3 decreases the expression of surface molecules necessary for antigen presentation such as MHC II, B7-1, and B7-2 and upregulates M2-specific immunomodulatory mediators including IL-10, EGF, VEGF, and various matrix metalloproteinases (MMPs) (reviewed in [18]). Experiments blocking the activation of STAT3 in gCSCs cocultured with allogeneic T-cell precursors demonstrate reduced Treg differentiation and reduced overall T-cell apoptosis [58]. Therefore, STAT3 may serve as a critical “molecular hub” linking multiple immunosuppressive pathways in CNS tumor cells and M2 TAMs. STAT3 target molecules such as IL-10 and IL-6 have been shown to subsequently trigger STAT3 activation [59], leading authors to propose a feedforward mechanism of reinforced STAT3 activation, which may account for its constitutive activation in both glioma cells and glioma-infiltrating TAMs.
18.6 Cytomegalovirus in Glioma
Accumulating evidence demonstrating an association between active human CMV infection and malignant glioma has inspired exciting innovations to current treatment strategies. A recent investigation reported the presence of CMV-associated nucleic acids and proteins in over 90 % of ex vivo GBM specimens analyzed. Neither HCMV-associated nucleic acids nor proteins were present in surrounding normal brain specimens, and over 80 % of recently diagnosed GBM patients also demonstrated CMV DNA in peripheral blood samples [60]. Though CMV is known to infect 50–80 % of the American population, effective immune control typically limits active disease to the immunosuppressed [61]. It remains unclear if the high prevalence of active CMV infection in glioma patients plays any role in tumor pathogenesis or if tumor growth simply provides an environment permissive of local reactivation and propagation of the virus. Regardless, the presence of CMV in these tumors may be important considering its known potential to modulate growth, invasiveness, and immunological recognition of infected cells (reviewed in [62]). Indeed, active CMV infection has been shown in astrocytes to reduce expression of molecules necessary for antigen presentation, increase the expression of TGF-β and IL-10, and limit the susceptibility of infected cells to apoptotic pathways [63, 64]. Elucidation of the impact CMV virus has on the immunosuppressive phenotypes of CNS tumor cells will require extensive investigation. The presence of viral antigens specifically in tumor cells may allow for tumor cell-specific targeting through the use of CMV antigens in CNS tumor vaccines. If in fact active CMV activation contributes to cellular transformation or malignant behavior, then vaccination strategies against its antigens could additionally provide a functionally disabling therapy toward preventing recurrence.
18.7 Immunoediting in CNS Cancer
As most human CNS tumor analysis is conducted on ex vivo specimens acquired from surgical excision following presentation of clinical deficits, the data and conclusions may not be representative of earlier stages of immune system and tumor interaction. Thus, whereas it is possible to study the immunosuppressive environment present in a malignant tumor, the sequence of events that leads to this state remains obscure. The theory of tumor immunoediting has emerged as a paradigm for understanding the dynamics of tumor progression and immunosuppression. The hypothesis proposes three distinct phases: an initial elimination, a period of equilibrium, and, finally, cancer cell immune escape [65] (tumor immunoediting is summarized in Fig. 18.2). Due to genetic instability and rapid proliferation, tumor cells are generated with different immunogenic antigens in a developing tumor. In the initial elimination phase, cytotoxic immune cells target and eliminate those cancer cells that are highly recognizable and lack immune-evasion mechanisms, leading to the selection of poorly immunogenic and/or immunosuppressive tumor cells. Elimination is limited, and some tumor cells are not eradicated, either due to their antigenic or immunosuppressive-related gene expression profile, allowing these cells to survive the initial immune surveillance and enter an equilibrium phase. In this phase, there is a dynamic balance between the antitumoral immunity and tumor cell expansion. During this long phase, there is no clinical tumor burden. The prolonged latency period during equilibrium is thought to constitute an editing state in which neoplastic cells that are susceptible to the host immunity are eradicated, and those that are not recognized are selected to survive. Finally, the escape phase occurs when those tumor cells that are not detectable or have developed mechanisms to avoid immune recognition are selected and grow into a symptomatic lesion (Fig. 18.2).
Cancer immunoediting paradigm, highlighting the three proposed phases of immunoediting: elimination, equilibrium, and escape (Reprinted from Schreiber et al. [65], with permission of AAAS)
Considering the competence of immune surveillance and activation within the CNS, the principles of tumor immunoediting are believed to apply to CNS cancers. Support for the paradigm of immunoediting in CNS cancers comes from few transplant studies, citing the transmission of glioma tumors from liver and kidney organ donors to transplant recipients and from observations in ongoing immunotherapy trials. The first report of this phenomenon involved a 44-year-old woman with primary biliary cirrhosis who received an orthotopic liver transplant from a 14-year-old brain-dead donor with a glial tumor that had infiltrated the pons, pituitary, and spinal cord. Following 9 months of immunosuppression, the recipient developed several liver lesions that appeared histopathologically similar to that of the donor’s glial tumor, suggesting immune escape of glioma cells maintained in quiescent immune equilibrium prior to transplantation [65].
A similar report documented two recipients who each received a kidney from a deceased donor with GBM. Both recipients developed renal masses after approximately 18 months, which upon organ removal were pathologically consistent with GBM [66]. Further evidence comes from current GBM vaccine trials (detailed below). Analysis of recurrent GBM specimens following use of a vaccine targeting the highly expressed variant EGFRvIII in GBM demonstrated a paucity of EGFRvIII expression, suggesting successful elimination of the EGFRvIII-expressing cells, followed by equilibrium and subsequent escape of cancer cell subpopulations which did not express EGFRvIII [67]. Ongoing investigation of the dynamic interactions between immune cells and tumor cells throughout the multiphasic progression of CNS tumors will test this theory of immunoediting in CNS cancers and potentially elucidate opportunities to enhance elimination and redirect the eventual failure of equilibrium.
18.8 Immunotherapy
In general terms, the CNS tumor immunotherapy strategies are focused on two goals: to direct the recognition of CNS cancer cells by immune effector cells necessary for a tumoricidal response and to counteract tumor-derived immunosuppression, thus leading to an effective antitumor activation state. A growing appreciation of the necessity for multimodal immune modulation in achieving durable control of CNS tumors through immune-based therapy has led to the combination of both strategies in preclinical and early clinical trials.
In efforts to enhance tumor detection by the immune system, antigen-specific vaccinations and primed dendritic cell-based infusions have both demonstrated promising results. With regard to efforts aimed at disabling immunosuppressive mechanisms, those targeting Tregs and immunomodulatory cytokines have shown preliminary success.
Some authors argue that surgery offers a means for disabling tumor-related immunosuppression by removing the bulk of immunosuppressive cells and mediators within the tumor [44]. Additionally, elimination of the mass effect and edema caused by a large tumor allows for discontinuation of steroids, which confer an iatrogenic immunosuppressive state to the patient. An example of the benefit of resection in the context of immunotherapy has been shown in post-resection GBM patients who, without a significant tumor mass and actively progressing disease, responded better to dendritic cell-based vaccines than did those who had received biopsy alone [68]. For this reason, many recent clinical trials of immune-based therapy in GBM patients are focused on patients who first receive a surgical resection of their tumor.
18.8.1 Adoptive Therapy
Considering the potent tumoricidal properties of activated lymphocyte effector cells, an obvious strategy toward overcoming the in vivo hindrances to adaptive immune activation utilizes infusion of in vitro activated autologous lymphocytes back into patients. Lymphokine-activated killer (LAK) cells are populations of autologous peripheral lymphocytes that can be reinfused into tumor-bearing hosts either peripherally or intraoperatively into post-resection surgical cavities following in vitro culture in the presence of IL-2 [69]. Multiple phase I clinical trials have investigated LAKs in patients with high-grade gliomas and medulloblastomas (reviewed in [70, 71]. The most promising of these trials included 40 GBM patients treated with intratumoral LAKs and demonstrated a slight but significant increase in median survival in the absence of any toxicity [69]. Unfortunately, additional trials could not reproduce these effects and were further hindered by variant levels of toxicity as the reinfused LAKs demonstrated cytotoxic properties that were not specific to tumor cells. Lower cellular doses of intralesional LAK are under continued investigation as adjuvant treatment of GBM [69].
An extension of LAK strategies to direct more tumor-specific targeting involved the collection of lymphocytes from the lymph nodes or peripheral blood of patients with CNS tumors after peripheral injection of irradiated autologous tumor cells (ATCs) and granulocyte/macrophage colony-stimulating factor (GM-CSF). The harvested lymphocytes were then stimulated in vitro with IL-2 and subsequently reintroduced into the tumor-bearing host [72]. Variations in this scheme include additional ex vivo exposure to ATCs [72] or tumor-infiltrating lymphocytes isolated from resection specimens [19] during in vitro stimulation. Despite reduced toxicity and more objective tumor-specific targeting as compared to LAKs stimulated with IL-2 alone, effects on clinical outcome were minimal across these trials [19, 73].
18.8.2 Vaccination Strategies
Cancer vaccination strategies utilize tumor antigen-driven stimulation of host immune processes to target transformed cells. Cancer vaccines are designed to direct tumor-specific cellular immunity by stimulating the proliferation of high-avidity T cells capable of homing to and selectively attacking transformed cells within a tumor. Some of the major challenges to this strategy include failure of the delivered stimulus to adequately activate T cells, relative lack of tumor-specific antigens that are expressed by a large fraction of tumor cells, nonspecific targeting by stimulated T cells of healthy bystander cells resulting in toxic autoimmunity, and disabling of activated tumor cell-specific T cells by the local microenvironment. To overcome these issues, some vaccination strategies utilize reinfusion of autologous tumor material following ex vivo manipulation [74], as well as the use of non-antigen-specific tumor lysate preparations [75]. More recently, purified tumor antigen formulations have also been attempted as direct peptide infusions and as a priming stimulus to DCs prior to their infusion.
18.8.2.1 Autologous Tumor Material
ATCs may be harvested from ex vivo tumor resection specimens and used to generate direct CNS vaccination formulations. Subcutaneous or intradermal injection of autologous tumor material is believed to circumvent the immune-disabling tumor microenvironment by providing specific immune-stimulating material to peripheral DCs. Prior to their use in vaccination strategies, this tissue is processed to isolate whole tumor cells, parts of cells, or simply protein extracts and often inactivated by radiation or genetic modification. Eight trials have employed such strategies to treat GBM, including one phase I clinical trial [74], two case reports [76, 77], and five pilot vaccination studies (reviewed in [70]). In three of the pilot studies, processed cells were delivered concomitantly with adjuvant compounds, including IL-2 [76], IL-4 [77], and B7-2 plus GM-CSF infusions [78]; the amount of cells delivered varied across trials.
The induction of an immune response was demonstrated in over half the patients enrolled in each trial, with evidence both in peripheral blood [79] and at the tumor site [80]. Toxicity was minimal and no patient demonstrated severe adverse effects. Furthermore, clinical benefit was demonstrated with nearly 50 % overall survival across five studies, which recorded three complete responses, four partial responses, two minor responses, and six cases of stable disease in 48 total GBM patients [74, 76–78]. The phase I clinical trial of ATC vaccination included a concomitant infusion of GM-CSF through a programmable pump and effected a significant increase in survival in three of the five patients who demonstrated a postvaccination immune response, out of a total of nine who were treated [74].
18.8.2.2 Dendritic Cell-Based Vaccination Strategies
As discussed, activation of T cells that specifically target brain tumor cells is limited by a reduction in the expression of molecules necessary for effective antigen presentation, including MHC class I/II and co-stimulatory molecules [28]. To overcome this limitation, DCs from patients with malignant brain tumors may be extracted, activated in vitro with tumor-derived antigens favoring APC maturation, and reintroduced as potent activators of tumor-specific T cells. This approach can lead to the generation of tumor-specific T-helper cells (Th) capable of altering the composition of the microenvironment through expression of immune-activating mediators and, subsequently, the activation of CTLs and NK cells capable of selectively eliminating tumor cells. Furthermore, the generation of memory T cells following introduction of tumor antigen-primed DCs presents the potential for lasting immunity to counter the recurrent proliferation of residual cancer cells. Indeed, coculture of glioma-associated antigen-primed DCs with undifferentiated lymphocytes has been shown to induce activation of T cells and subsequently T-cell cytotoxicity when autologous glioma cells were introduced [81, 82]. Furthermore, a robust cytotoxic (CTL) and memory T-cell lymphocyte infiltration into intracranial tumors was observed in murine models of glioma following vaccination and peripheral infusion of tumor antigen-primed DCs, favoring a Th1 lymphocyte activation state, capable of homing to and expanding within tumor tissue [83].
Though many investigative protocols for DC-based vaccination of malignant glioma differ with regard to protocol specifics, most involve extraction of DC precursors in the form of peripheral blood mononuclear cells (PBMCs); exposure to tumor-associated formulation in the presence of GM-CSF and IL-4, both known to direct APC maturation; and reintroduction though subcutaneous, intradermal, intranodal, or intratumoral injection.
The mechanism by which tumor-associated antigens are loaded in vitro into DCs is of critical importance. Multiple DC-loading strategies have been employed, including the use of autologous tumor lysates, formulations of apoptotic material following ATC irradiation, and purified or synthetic tumor-associated peptide antigens [84–86].
A potential advantage of loading strategies which do not isolate individual antigens, such as the use of autologous tumor lysates, is the induction of an immune response against multiple tumor epitopes, though likely at the expense of non-tumor-specific cross-reactivity and subsequent autoimmune toxicity. Those strategies using distinct and tumor-specific antigens, either purified or synthetic, limit the activation of cross-reactive lymphocytes, allowing for the escape of non-expressing clonal populations.
To date, 15 clinical trials including 316 total patients have evaluated the use of DC-based vaccination in the treatment of malignant gliomas including primary and recurrent GBM, anaplastic astrocytoma (AA), anaplastic oligoastrocytoma (AOA), and anaplastic oligodendroglioma (AO) (reviewed in [70]): eight phase I trials, six phase I/II trials, and one phase II trial. Table 18.1 summarizes the vaccination details and clinical results of these trials. Across all included in these trials, only one patient suffered grade IV neurotoxicity resulting from a large residual tumor and perilesional edema [90], highlighting the safety and feasibility of antigen-primed DC vaccinations for CNS cancers. Immune response was largely evaluated by delayed-type hypersensitivity (DTH); increased proportions of CTLs, NKs, and memory T cells both in peripheral blood and as infiltrating lymphocytes in subsequent tumor resections; increased tumor cell reactivity of postvaccination extracted PBMCs exposed to ATC in vitro; and increased presence of IFN-γ both peripherally and within the tumor-infiltrating lymphocytes. Over half of the patients enrolled in these trials demonstrated some evidence of an immune response following vaccination, and all 15 studies reported a survival benefit following vaccination (Table 18.1). Moreover, two of these trials focusing on patients with GBM demonstrated an improved response to chemotherapy delivered in a second phase following DC-based vaccination, suggesting an exciting potential for synergy with these treatments [85, 96].
Despite the variability across these trials, salient insights include the safety of DC-based approaches to CNS tumor vaccination and the feasibility of these immune strategies as some features of an elicited immune response were demonstrated in over half of all patients enrolled. Other interesting results include the improved success of matured DC vaccinations generated by combining antigen priming with maturation factors such as TNF-α, Toll-like receptor (TLR) ligands, or IFN-γ and the potentially synergistic effect of DC-based vaccination and chemotherapy in treating brain tumors. Details regarding precise protocols for loading of DCs, amount and site of injection, and composition of accompanying adjuvants remain to be optimized.
An additional trial evaluated the use of DC-based immunotherapy in 45 pediatric patients with high-grade glioma, medulloblastoma, primitive neuroectodermal tumors (PNETs), ependymoma, and atypical teratoid-rhabdoid tumors (ATRTs) [97]. The authors utilized autologous tumor lysates to load PBMC-derived DCs and delivered these by intradermal injections followed by two subsequent boost vaccinations of tumor lysate. No severe adverse effects occurred in those patients with high-grade gliomas and ATRTs, and additionally, overall survival was increased compared to historical controls in those two tumor types. In those patients with PNET and medulloblastoma tumors, vaccinations were discontinued due to adverse effects. These findings show the potential of the DC-based immunotherapy to pediatric brain tumors, but data regarding efficacy remains preliminary and poorly controlled.
The optimization of tumor-associated antigen-loading strategies is under active exploration. A recent study compared specific antigenic peptide-loaded vs. autologous tumor lysate-loaded DC vaccines for treating malignant glioma [98].
Twenty-eight patients were treated with autologous tumor lysate-pulsed DC vaccines, whereas six patients were treated with glioma-associated antigen peptide-pulsed DCs, utilizing a synthetic formulation of four epitopes known to be expressed on malignant gliomas. These antigens included survivin, HER-2/neu, gp100, and TRP-2, which are present in approximately 60, 80, 60, and 50 % of malignant gliomas specimens, respectively [27]. No adverse events were reported in either study group. The median survival of patients on the autologous tumor lysate-DC trial was 34.4 months, whereas that of patients on the synthetic glioma-associated antigen-DC group was significantly different with a median survival of 14.5 months [27]. Though limited to small cohorts under individual protocols, these results support the use of autologous tumor lysate preparation in priming DCs for vaccination in CNS cancer. The authors also noted a significant correlation between decreased Treg ratios (pre- vs. postvaccination) and overall survival, evident in both study groups.
18.8.2.3 Antigen-Specific Peptide Strategies
In contrast to the DC-based techniques discussed above, direct peptide vaccines rely on the ability of host APCs in the periphery to process, migrate, and present the introduced antigens. Extensive preclinical analysis has demonstrated the ability of peripheral APCs to activate T cells within lymph nodes regional to the site of injection in animal models of brain tumors [70, 99]. Refinements to direct antigen vaccination strategies have demonstrated the utility of adjuvant compounds such as keyhole limpet hemocyanin (KHL) as an immunogenic peptide carrier protein [100] and GM-CSF as a mitogenic stimulus for APCs [101], both of which ultimately augment antigen presentation.
The selection of tumor-associated peptides to enable selective tumor cell targeting with minimal secondary autoimmunity is critical to the success of any vaccination utilizing target peptide sequences, both in direct peptide injection and in specific antigen-primed DC infusion. Considerable effort has been expended in identifying antigens differentially or exclusively expressed in CNS tumors, including genes only normally expressed during embryological development, differently spliced or mutated genes, and genes giving rise to fusion proteins, which result from the general genetic instability of transformed cells, as well as housekeeping or metabolic pathway antigens which may be exclusive to tumor cells [90]. Nevertheless, an increasing appreciation of intratumor clonal heterogeneity [102] complicates effective targeting of a clinically significant proportion of tumor cells through a specific antigen vaccination strategy.
The National Cancer Institute (NCI) recently performed an in-depth review of 75 general tumor-associated antigens to evaluate their potential as targets for immunotherapy [103]. The potential of tumor antigens to serve as targets for immunotherapy was graded according to the following criteria: therapeutic function, immunogenicity, oncogenic function, specificity, expression level in tumors, in cancer stem cells, percentage of tumors that express it and cellular localization of the protein. Based on this, the antigens that showed the most potential for immunotherapy where. The highest-ranked antigens included WT-1, MUC1, LMP2, HPV E6/E7, HER2/neu, EGFRvIII, melanoma antigen-encoding (MAGE)-A3, and NY-ESO-1. While expression of some of these antigens in CNS cancer is well established, such as the expression of EGFRvIII in GBM [104], the presence or absence of others in CNS cancers warrants future investigation. Additional insight into CNS cancer-specific antigen targets for immune-based therapy has come from tumor antigen investigations in melanoma [105]. The genes MAGE-1 [106], MAGE-E1 [107], MAGE-3, and glycoprotein-240 (a cell surface glycoprotein of 240,000 molecular weight present in most melanomas) [108] were expressed in many different glioma subtypes but never in normal brain tissue and therefore present as potential targets for CNS tumor-specific immunotherapy. Many additional CNS cancer-associated antigens have been described as potential tumor-selective targets for immunotherapy in a variety of CNS tumor types; these include but are not limited to tenascin, homo sapiens testis (HOM-TES)-14 (also known as stromal cell-derived protein (SCP)-1), HOM-TES-85, synovial sarcoma X chromosome breakpoint (SSX)-1, SSX-2, GAGE-1, SRY-related high-mobility group (HMG)-box-containing gene (SOX)-5, cancer testis antigen 6, IL-13 receptor a2, ephrin (Eph) A2, antigen isolated from immunoselected melanoma (AIM)-2, squamous cell carcinoma antigen recognized by T cells (SART)1, SART3, and kinesin superfamily protein (KIF)1C and KIF3C [70]. See Table 18.2 for a list of GBM-associated antigens under preclinical or clinical investigation in tumor vaccines. Ongoing effort to characterize these many relevant antigens in various CNS cancer subtypes will hopefully yield firm footing of which to launch future antigen-specific immune-based therapies.
Following promising preclinical results, a number of clinical trials utilizing direct vaccination with some of the aforementioned peptides are currently underway for cancers of the CNS. Among those under most active vaccine developments are the IL-13 receptor a2 (IL-13Ra) [73, 113] and EphA2 [109, 142] though disproportionate attention and success have come from vaccination strategies against EGFRvIII. A potent mitogenic signaling motif, stimulation of the EGF receptor, is believed to play a significant role in the development of malignant glioma; approximately 50–60 % of glial tumors overexpress EGFR, and 24–67 % express the most commonly mutated form, EGFRvIII [143]. The functional relevance of EGFRvIII for malignant gliomas is also suggested by the fact that presence of EGFRvIII is associated with reduced survival on multivariate analysis [144] and also may confer malignant cells with resistance to radiation and chemotherapy [145]. The amino acid sequence obtained from the fusion of the remaining exons 1 and 8 includes the addition of a glycine residue at the junctional site (LEEKKGNYVVTDH), rendering a totally novel peptide [143]. Consequently, the resultant protein is unique to glioma cells and therefore may allow for the generation of an immune response which does not cross-react with wild-type EGFR protein. See Fig. 18.3 for a synopsis of a vaccination strategy developed to target EGFRvIII.
Schematic of EGFRvIII-targeted vaccination. Notice the formation of EGFRvIII-specific antibodies, which selectively target tumor cells (Reprinted from Sonabend et al. [54], with permission)
An early phase I trial to evaluate the safety of GBM vaccination against EGFRvIII was performed in which treatment with an intradermal KLH-conjugated EGFRvIII-based peptide (PEPvIII) was utilized. No serious adverse effects were reported, and immunological responses were detected ex vivo [12]. Subsequently, a multicenter phase II trial, entitled “A Complimentary Trial of an Immunotherapy Against Tumor Specific EGFRvIII” (ACTIVATE), was performed, in which 19 patients with GBM were treated with PEPvIII and adjuvant GM-CSF following tumor resection and standard radiation plus chemotherapy [146]. Importantly, EGFR amplification and EGFRvIII expression were not criteria for enrollment. No patient experienced adverse effects aside from local injection site reactions, and both humoral and delayed-type hypersensitivity immune responses specific to EGFRvIII were observed in the majority [147]. Furthermore, in the patients who demonstrated an immune response to vaccination, median time to tumor progression (TTP) and overall survival were significantly increased when compared to historical controls (TTP 12 months vs. 7.1 months, p < 0.05, with a median survival of over 32 months vs. 14 months in historical controls p < 0.01). Interestingly, histological analysis of recurrent tumor specimens revealed complete absence of EGFRvIII expression in all patients demonstrating an immune response. Though pretreatment EGFRvIII expression was not published, this finding may suggest successful targeting of antigen-bearing tumor cells.
The promising results of this trial were further extended in a subsequent phase II trial which enrolled 21 GBM patients in a similar KLH-conjugated PEPvIII plus GM-CSF vaccination schedule, concurrent with two different temozolomide (TMZ) chemotherapeutic dosing schedules [148].
Although grade II TMZ-associated lymphopenia was observed in nearly all treated patients, immune responses specific to EGFRvIII were documented in the majority of patients. Unexpectedly, antigen-specific immune responses were observed to be either sustained or enhanced with successive TMZ treatments. Follow-up investigations, including an ongoing randomized phase III clinical trial, hope to validate this observation and to further elucidate optimal vaccination regimens. The possibility of synergy between immunotherapy and chemotherapy in CNS cancer is promising. Together, these trials suggest that vaccination with a peptide containing an EGFRvIII tumor epitope safely elicits a specific immune response against EGFRvIII and that this approach might be effective against cancers bearing the variant antigen.
18.8.2.4 Heat Shock Protein Peptide Complex 96
An exciting new frontier of immune-based therapy for CNS cancers involves the use of heat shock protein peptide complexes (HSPPCs). HSPs are known to lead to “chaperone” protein folding and protein-protein interactions and are unregulated in states of cellular stress [149]. Certain HSPs have been shown to play instrumental roles in the delivery and intracellular processing of antigens in APCs and are therefore an attractive target for exploitation in immunotherapy [150]. HSPPC-96 is composed of HSP gp-96 and a wide array of bound chaperoned proteins, including antigenic peptides. This protein complex can be easily purified from solid tumor specimens of patients with a variety of solid tumor types [151]. Immunization strategies with HSPPC-96 work by interacting with APCs via specific receptors, including CD91 [152]. After binding to CD91, the HSPPC-96 complex is internalized, and the chaperoned peptides are presented by class I and class II MHC molecules. The highly specific nature of the interaction between HSPPC-96 and APCs may present an advantage over the aforementioned vaccine approaches and has been shown to facilitate robust T-helper cell and CTL immune responses [153].
Vaccination with HSPPC-96 was recently extended to patients with CNS tumors: 12 patients with recurrent high-grade gliomas were treated with autologous HSPPC-96 vaccines derived from resected tumor tissue [154]. No toxicity attributable to HSPPC-96 was observed in any of the 12 patients treated. In 11 of the 12 patients, a significant immune response was demonstrated, as indicated by robust activation of peripheral blood leukocytes isolated postvaccination, when exposed to antigenic peptides carried on the HSPPC-96 complex (gp-96). Vaccination led to a significant increase in IFN-γ expression as compared to peripheral blood leukocytes isolated prior to vaccination. Furthermore, an increase in IFN-γ-positive T-helper cells, CTL, and NK cells accompanied a decrease in Tregs in biopsy specimens from all 11 patients who responded, whereas these findings were not observed in the one patient who did not respond. The 11 responders had an overall survival of 47 weeks compared with 16 weeks in the one nonresponder. Collectively, these results suggest the safety, feasibility, and potential therapeutic benefit of autologous HSPPC-96 vaccination in patients with high-grade gliomas.
18.8.3 Immunotherapy Targeting CNS Cancer-Induced Immunosuppression
In addition to directed activation of immune mediators against tumor-specific antigens, parallel efforts to counteract CNS cancer-induced immunosuppression have gained attention. A comprehensive inventory of ongoing efforts is beyond the scope of this chapter, but cytokine therapy and antibody-mediated neutralization of Tregs will be discussed as two notable examples of this therapeutic principle.
As key transmitters of cellular communication, cytokines are known to play predominant roles both in proper immune cell activation schemes and in the irregular immunosuppressive milieu of the CNS tumor microenvironment. Cytokines direct the phenotypic fate of stimulated monocytes and lymphocytes and are involved in signaling exchanges upon encountering pathogenic or neoplastic stimuli. Thought to precipitate escape from immune equilibrium, the aberrant cytokine expression profile of transformed CNS cells eventually acts to alter the cytokine expression profiles of resident (microglia) and infiltrating (monocytes) myeloid cells, and subsequently lymphocytes, which thereafter collude to construct potent local immunosuppression. The therapeutic introduction or inhibition of immune-modulating cytokines is hypothesized to reorient M2 TAMs back to tumoricidal effector phenotypes [24]. Among the cytokines identified for such efforts, TGF-β, IL-2, IL-4 [41, 155], IL-12 [156, 157], and IFN-γ [158, 159] have received the most attention, both as principal treatment and as adjuvants in combination with the antigen-based strategies discussed earlier.
As discussed above, TGF-β plays a prominent role in the multiple pathways implicated in CNS tumor-induced immunosuppression, proliferation, angiogenesis, and invasion-permitting stromal remodeling. TGF-β expression is observed to increase following radiation treatment in both in vitro [160] and in vivo [161], raising the possibility of a therapeutic benefit derived from TGF-β modulation in conjunction with radiation treatments in patients with GBM. Trabedersen (AP12009) is an antisense molecule consisting of 18 DNA oligonucleotides which specifically targets TGF-β2 mRNA, inhibiting its protein synthesis [162]. Trabedersen’s utility in AA was recently investigated in a phase IIb clinical trial which reported significantly improved 14-month tumor control rates evaluated by the presence of recurrent tumor on MRI (p < 0.05) when compared to standard chemotherapy [152]. Overall, patients with GBM in this trial did not demonstrate the same tumor control benefit, though a subgroup analysis of young GBM patients with good performance status did suggest a trend toward improved 2- and 3-year survival (p = 0.08). Trabedersen is currently in phase III clinical trials for treatment of AA [155].
IL-2 is known to be an essential stimulus for the proliferation and differentiation of both Th type 1 cells and CTL following TCR antigen recognition, and it has been shown to abrogate the immunosuppressive effects of TGF-β [163]. Commonly used to stimulate the expansion and maturation of PBMCs in the development of LAK, multiple investigators have attempted to use IL-2 as an immunotherapeutic agent in CNS cancer. Early clinical trials with high-dose IL-2 delivered intratumorally or intraventricularly were discontinued on account of significant adverse effects resulting from local edema [164]. An IL-2 transgene was delivered into the tumors of 12 patients with recurrent GBM, followed by systemic treatment with acyclovir. In this trial, a retroviral vector was used as a vehicle for IL-2 and herpes simplex virus thymidine kinase (HSV-tk), which helped the selective elimination of infected cells with acyclovir [164]. None of these patients demonstrated adverse effects to treatment, and although no complete response was recorded, two experienced a partial response, four a minor response, and four stable disease. Additionally, expression analysis on posttreatment biopsies in three of the patients with a partial or minor response demonstrated increased expression of TNF-α, IFN-γ, IL-2, IL-1B, and IL-10, suggesting the induction of a local Th1 immune response. Together, these findings suggest that local IL-2 transgene delivery may be a safe and at least a modestly effective therapeutic strategy for further development in CNS cancer. Efforts to integrate IL-2 into combination strategies for the treatment of CNS cancers are ongoing [165].
The immunotherapeutic potential of IL-2 manipulation extends further through its impact on the potently immunosuppressive Tregs. Defined by a set of constitutively expressed antigens which include the high-affinity IL-2 receptor alpha chain (IL-2Rα/CD25), Tregs may be selectively targeted and functionally impaired by interventions specific for this component of the IL-2 receptor complex. Indeed, blockage of the IL-2Rα in murine models of glioma was observed to deactivate Treg-induced suppression through functional inhibition as well as depletion [165, 166]. Still, as discussed above, IL-2-mediated stimulation of the IL-2 receptor (heterotrimer of α, β, γ chains) is necessary for the proliferation and differentiation of Th1 cells and CTLs, particularly following the administration of therapeutic vaccines designed to stimulate a tumor antigen-specific lymphocyte response. Thus, blockage of IL-2 signaling, though it may hinder the expansion of immunosuppressive Treg, may also limit the production of tumoricidal immune effector cells and therefore be of limited benefit.
Initial investigations into the use of the humanized anti-IL-2Rα monoclonal antibody (mAb) daclizumab for the treatment of malignant melanoma demonstrated this suspicion. Though tumor infiltration and peripheral Treg populations were effectively depleted, the functionality of vaccine-induced tumor-specific T cells was impaired, and the formation of vaccine-induced humoral immunity was minimal [167]. Still, attempts to use daclizumab in the treatment of malignant cancers were not abandoned, largely as a result of the observation of different IL-2 signaling responses in Tregs as compared to mature effector T cells during times of lymphopenia, such as is induced by chemotherapy. A preclinical investigation attempting to exploit this discrepancy delivered daclizumab during TMZ-induced lymphopenia in a murine model of glioma and demonstrated an effective depletion of Treg populations while sparing tumor-specific vaccine-activated effector cells [60]. The authors additionally reported an increased reduction in tumor growth in the vaccinated mice given daclizumab after TMZ as compared to those treated with TMZ followed by vaccination alone. These encouraging findings were extended in a pilot clinical trial of six patients with recently diagnosed EGFRvIII-expressing GBM, undergoing standard TMZ treatment followed by a single-dose infusion of daclizumab concurrent with a course of PEPvIII peptide EGFRvIII-targeting vaccination [168]. No adverse events were reported beyond minor irritation at the vaccine injection site. Peripheral lymphocyte analysis demonstrated a significant reduction in circulating Tregs in the group treated with daclizumab without a corresponding depletion of overall CD4+ or CD8+ T cells, suggesting a Treg-specific inhibition of proliferation under lymphopenic circumstances. Furthermore, increases in vaccination-induced anti-PEPvIII antibodies directly correlated with reductions in Tregs and with increases in effector cell to Treg ratios. Together, these results suggest that mAb blockade of IL-2Rα in TMZ-treated malignant glioma may create an environment conducive to further immunotherapeutic intervention. Moreover, these encouraging findings underscore the potential benefit of multimodal combinations of immune-modulating therapies in treating CNS cancers.
18.9 Concluding Remarks
A comprehensive understanding of the dynamic balance between tumoricidal immunity and tumor-derived immunosuppression that take place during CNS cancer development is essential for successful immunotherapy for this disease. As this chapter highlights, ever-unfolding insight into CNS-distinct immune mechanisms and their derailment by transformed tumor cells has already allowed for innovative, safe, and therapeutically promising techniques. The feasibility of individual immune-altering therapies is leading to a combination of strategies for achieving the ultimate goal of synergistic tumoricidal immunity. Such efforts might rely on use of DC- or peptide-based vaccines with either chemotherapy or biological therapy. The final goal of these interventions is augmenting lymphocyte and NK cell activation or disabling tumor-derived immunosuppressive barriers. Additionally, recent insight into the clonal heterogeneity of CNS tumors, the presence of recalcitrant gCSCs, and the expression of CMV antigens in a majority of transformed cells in some CNS cancers have spurred new and innovative strategies that are being evaluated to further enhance antitumoral immunity.
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Sonabend, A.M., Showers, C.R., Anderson, R.C.E. (2015). Immunopathology and Immunotherapy of Central Nervous System Cancer. In: Rezaei, N. (eds) Cancer Immunology. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-46410-6_18
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