Revisiting Immune-Based Therapies for Aggressive Follicular Cell

Abstract

Background:

With our growing understanding of the immune system and mechanisms employed by tumors to evade destruction, the field of cancer immunotherapy has been revitalized. Concurrent inflammation has long been associated with follicular cell–derived thyroid cancer (FDTC). In the last decade, much research has focused on characterizing the tumor-associated immune response in patients with FDTC.

Summary:

Mast cells, natural killer cells, macrophages, dendritic cells, B cells, and T cells have been identified within FDTC-associated immune infiltrate. Collectively, these findings suggest that the immune response to FDTC is compromised and may even promote tumor progression. A more thorough characterization of the tumor-associated immune response in FDTC may lead to the development of immune-based adjuvant therapies for patients with aggressive disease.

Conclusions:

Immune-based therapies could provide essential alternatives to patients that cannot be treated surgically, those with recurrent or persistent lymph node metastases, and those with anaplastic thyroid cancer.

Introduction

I n 1975, Amino et al. (1) published the first and only immunotherapy study in patients with follicular cell–derived thyroid cancer (FDTC). Three patients (L.G., G.S., and R.W.) with aggressive papillary thyroid cancer (PTC) were vaccinated with autologous tumor homogenate in combination with pertussis adjuvant. Patients L.G. and G.S. presented with both locoregional metastases to the lymph nodes (LNs) and distant metastatic lesions. Patient R.W. had aggressive locoregional metastases but no distant metastases. Both L.G. and G.S. showed suppressed delay-typed hypersensitivity responses to common antigens prior to tumor vaccination, suggesting a general immune-compromised state. Following vaccination, these patients showed infiltration of neutrophils (L.G.) or lymphocytes (G.S.) at the site of injection, but tumor load was not affected. In contrast, patient R.W. displayed a normal delay-typed hypersensitivity response to common antigens prior to vaccination, and thyroid tumor mass was reduced to one third its original size 4 months after treatment.

Despite this promising study, little has been done in the past three decades to pursue immune-based therapies for patients with FDTC. Failure to pursue this research may be explained in part by the misconception that FDTC is a “good” cancer. For most patients with FDTC, 5- and 10-year survival rates are excellent, greater than 97% and 95%, respectively (2). Surgery and radioiodine therapy are generally successful in treating patients with localized or regional disease. However, 5% of patients present with distant metastases. The 5-year survival rate for patients with distant metastatic disease is reduced to 53.9% (2). Furthermore, 20%–30% of patients develop persistent or recurrent disease, most commonly in the locoregional LNs, requiring additional surgical intervention with increased morbidity and expense (3). These statistics refer largely to differentiated thyroid cancer (DTC), which constitutes 93% of all cases (86.6% PTC and 6.2% follicular thyroid carcinoma [FTC]) (2). Approximately 1% of FDTC cases are attributed to anaplastic thyroid cancer (ATC). ATC is largely refractory to current treatments, with 1-year and 10-year survival rates estimated at 10%–20% and <5%, respectively (4). Patients with aggressive DTC and ATC that are resistant to standard treatments may be excellent candidates for innovative adjuvant therapies, including immune-modulating approaches.

The failure to develop immune-based therapies for thyroid cancer may also be explained by waning enthusiasm for cancer immunotherapy, which has been largely unsuccessful. However, in the last decade tumor immunotherapy has been revitalized with our increasing understanding of tumor–host interactions. Although a connection between the immune system and cancer was first described by Paul Erlich in 1909 (5), the complexity of this interaction has precluded successful application of immunotherapy for cancer patients. In 1970, Burnet and Thomas (6) formally proposed the Cancer Immunosurveillance Hypothesis, which speculated that the immune system is capable of recognizing and eliminating transformed cells as they arise, protecting us from tumor development. In 2002, Dunn and Schrieber (7) proposed the Cancer Immunoediting Hypothesis, which incorporates the concept of immune surveillance into an expanded model that attempts to explain why clinically evident tumors develop in immune-competent individuals. This model divides tumor–immune system interactions into three stages: (i) elimination, (ii) equilibrium, and (iii) escape. The elimination phase is equivalent to immunosurveillance, in which the immune system is successful in destroying newly transformed cells. During the equilibrium phase, the selective pressure of the immune system is thought to promote the generation of tumor cell variants with an increased capacity to evade the immune response. In the escape phase, these variants are no longer subject to immune regulation, and the tumor progresses. Generally, by the time a tumor is clinically evident, it has escaped the host immune response and may even manipulate the immune system to promote tumor progression. The importance of immune escape in cancer is highlighted by the recent addition of “avoiding immune destruction” as a hallmark of cancer (8). Here, we review the growing body of literature that describes the tumor-associated immune response in patients with FDTC, the mechanisms employed by the tumor to escape this response, and the potential for immune-targeted therapies in patients with advanced disease.

Discussion

Mechanisms of immune escape

Tumors are capable of escaping the immune response through a number of mechanisms. The mechanisms of escape that are relevant to the current literature in FDTC are introduced in this discussion and summarized in Figure 1.

FIG. 1.

Immune response in the tumor microenvironment. Cell types and soluble factors involved in tumor elimination, promotion, tumor-induced immune suppression and T-cell dysfunction. Th1, Th2, and Th17 are CD4⁺ helper T-cell subsets. CTLA-4, cytotoxic T lymphocyte antigen-4; DC, dendritic cell; IDO, indoleamine 2,3-dioxygenase; iDC, immature DC; IFN, interferon; IL, interleukin; LAG-3, lymphocyte activation gene-3; M1 and M2, macrophage subtypes; MC, mast cell; NK, natural killer cell; PC, plasma cell; PD-1, programmed death-1; TGF, transforming growth factor; TIM-3, T-cell immunoglobulin and mucin domain-containing protein-3; TNF, tumor necrosis factor; Treg, regulatory T cells; VEGF, vascular endothelial growth factor.

T-lymphocyte dysfunction

T lymphocytes (CD4⁺ helper T cells and CD8⁺ cytotoxic T cells) are commonly found surrounding and infiltrating tumors. The majority of these cells are naïve cells that are ignorant to the tumor, while a portion of T cells show signs of activation but are functionally anergic or tolerant of the tumor (9). Tumor-induced T-cell tolerance was first described in tumors that express tumor-associated antigens. These molecules are considered “self,” thus T cells specific for these antigens should be deleted during development (i.e., central tolerance) or rendered dysfunctional in peripheral tissues (i.e., peripheral tolerance) (10,11). Tumor-associated T-cell tolerance was also evident in models in which nonself tumor antigens (e.g., SV40 T antigen) were expressed, suggesting that nonclassical mechanisms of tolerance were at play in the tumor microenvironment (12). Tumor-tolerized CD4⁺ T cells were characterized by reduced proliferative capacity and cytokine production in response to cognate antigen (13). CD8⁺ T cells have also been shown to be tolerant. Specifically, tumor antigen-specific T cells isolated from melanoma patients showed poor cytolytic and proliferative capacities and reduced cytokine production in vitro (14). More recent studies have identified a subset of dysfunctional tumor-associated T cells that express the inhibitory receptor programmed death-1 (PD-1) in combination with other inhibitory molecules such as T-cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) and lymphocyte activation gene-3 (LAG-3) (15 –18). Constitutive expression of these inhibitory molecules impairs T-cell function and impedes tumor elimination. Similar to anergic T cells, T cells with an exhausted phenotype are defective in their ability to proliferate, kill, and produce interleukin (IL)-2, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) (19). T-cell exhaustion is driven by chronic antigen exposure. Other extrinsic factors thought to contribute to T-cell exhaustion include tolerogenic antigen-presenting cells (APCs), suppressive cytokines, and regulatory T cells (20). These topics are discussed in more detail below.

Evading immune detection

Tumors are capable of actively evading recognition by cytotoxic CD8⁺ T cells and natural killer (NK) cells. Elimination of the tumor cells by previously activated CD8⁺ T cells requires efficient processing and presentation of tumor antigen by major histocompatibility class-I (MHC-I) molecules. Tumors are known to down-regulate key components of the antigen-processing machinery (e.g., peptide transport and proteasome components, MHC-I, and β2 microglobulin) (21). MHC-I down-regulation exposes the tumor to attack by NK cells, which interpret low MHC-I as a sign of an unhealthy cell. NK cell cytotoxicity is regulated by both activating and inhibitory receptors. Inhibitory receptors (i.e., KIRs and CD94/NKG2A) recognize the expression of MHC-I molecules by healthy normal cells. The activating receptor, NKG2D, recognizes stress-induced molecules (e.g., MHC-I–related chain [MIC]) on the target cell. The balance between these positive and negative signals directs the action of the NK cell. Tumor cells are known to shed MIC, generating a soluble form of MIC that can induce NKG2D internalization upon engagement, thus evading recognition and destruction by tumor infiltration NK cells (22).

Tolerogenic APCs

T-cell tolerance was originally thought to occur in the tumor microenvironment as a result of inefficient antigen presentation by the tumor cells, which lack the appropriate costimulatory molecules (i.e., CD80, CD86). However, early studies revealed that bone marrow–derived professional APCs (pAPCs) were required for tolerance induction in tumor models (23,24). Studies have now described numerous types of abnormal dendritic cell (DC) and macrophage populations that contribute to T-cell dysfunction in patients with cancer (21,25). Immature DCs (iDCs) are commonly found in tumors. These cells express low levels or lack expression of CD80 and CD86, which are normally up-regulated during maturation in an inflammatory environment. Of note, these costimulatory molecules are not inducible ex vivo, suggesting that these cells are not newly migrated iDCs, but rather they are dysfunctional in their ability to undergo maturation (26). In addition, plasmacytoid DCs (pDCs) are commonly found in tumor tissue and produce indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme. Immature myeloid cells, including macrophages, granulocytes, and myeloid-lineage DCs, are commonly found in tumors and defined by expression of the myeloid marker CD33 in the absence of maturation markers, including MHC-II, which is essential for antigen presentation to CD4⁺ T cells. Tumor-associated macrophages (TAMs) are well-studied in many types of cancer (27). TAMs are generally thought to carry an M2 phenotype. Unlikely M1 macrophages, which are associated with the classical inflammatory response and tumor resistance, M2-polarized macrophages produce IL-10 and transforming growth factor beta (TGFβ) and contribute both directly and indirectly to disease progression.

Tumor-induced immunosuppression and regulatory T cells

Tumor cells and stromal cells within the tumor microenvironment are known to produce a number of factors that contribute to immune dysfunction, including TGFβ, IL-10, and vascular endothelial growth factor (VEGF). TGFβ expression is associated with poor prognosis in a number of cancers. TGFβ specifically inhibits CD8⁺ T-cell cytotoxicity through repression of perforin, granzymes, Fas ligand, and IFNγ (28). IL-10 induces down-regulation of transporter associated with antigen processing (TAP) molecules, thus reducing antigen loading and presentation by the tumor (29). TGFβ, IL-10, and VEGF suppress DC differentiation resulting in the development of tolerogenic DCs (25). Dysfunctional DCs are capable of inducing differentiation and driving proliferation of immune suppressive regulatory CD4⁺ T cells (Tregs) (30). In addition, high levels of TGFβ in the tumor microenvironment can directly drive CD4⁺ T cell differentiation into the Treg phenotype (30). Tregs are commonly characterized by expression of the transcription factor FoxP3, elevated expression of the IL-2 binding receptor subunit CD25, and reduced expression of the IL-7 receptor CD127. Furthermore, Tregs express cytotoxic T lymphocyte antigen-4 (CTLA-4) and PD-1, inhibitory molecules central to the development of immune tolerance. Tregs mediate immune suppression by a number of mechanisms (30). CTLA-4 on Tregs is known to induce IDO production through ligation of CD80 or CD86 on DCs. Increased IDO production results in reduced levels of tryptophan and, consequently, T-cell anergy or death. Tregs produce IL-10 and TGFβ, which can lead to APC dysfuction and T-cell anergy. Furthermore, activated Tregs express perforin and granzymes, allowing direct killing of APCs or other T cells. Increased Treg frequencies in peripheral blood, tumors, and LNs have been associated with poor prognosis in many types of cancer (31 –35).

Immune checkpoint molecules

A number of inhibitory molecules have been identified that act as “immune checkpoints.” These checkpoints are essential for maintaining immune tolerance and regulating the duration and amplitude of the immune response (36). The best characterized checkpoint molecule is CTLA-4. CTLA-4 is expressed transiently by T cells following antigen recognition and competes with CD28, an essential costimulatory molecule during naïve T-cell activation, for ligation with CD80 and CD86 on pAPCs. Due to its higher affinity for CD80/CD86, CTLA-4 outcompetes CD28 resulting in cessation of the T-cell response. CTLA-4 knockout mice develop severe lymphoadenopathy and splenomegaly and die a few weeks after birth, suggesting that CTLA-4 plays an essential role in maintaining immune homeostasis (37,38). More recently, PD-1 has emerged as a key checkpoint molecule in peripheral tissues (36). PD-1 is expressed following T-cell activation and limits T-cell activation when ligated by PD-1 ligand 1 (PD-L1) (39,40). Constitutive expression of PD-1 is well established as a marker of T-cell exhaustion in both chronic viral infections and cancer (15,17,41). PD-L1 expression by tumor cells has been associated with poor prognosis in some epithelial cancers (42,43). PD-L1 expression in the tumor microenvironment may lead to T-cell exhaustion, thus promoting tumor progression. Both PD-1 and CTLA-4 are expressed by Tregs and, in contrast to their effects on conventional T cells, ligation of these checkpoint molecules has been shown to enhance their proliferation and immunosuppressive abilities (44,45). Blockade of the CTLA-4 and PD-1 interactions with their ligands is known to reverse T-cell dysfunction in vitro and support tumor elimination in mouse models through effects on both conventional T cells and Tregs (46 –48).

Tumor-promoting immune cells

The role of infiltrating immune cells in tumor promotion was recently added as an “enabling characteristic” in Hanahan and Weinberg's most recent description of the hallmarks of cancer (8). Both myeloid and lymphoid lineage leukocytes have been implicated in supporting tumor cell growth and proliferation, resisting cell death, and promoting angiogenesis, invasion, and metastasis (49). Mast cells and macrophages secrete proteolytic enzymes that cleave growth-limiting interactions between adhesion molecules, release key growth factors from the extracellular matrix, and remodel the extracellular matrix to encourage tumor cell invasion and metastasis. Myeloid cells, including mast cells, TAMs, and iDCs, produce angiogenic factors such as VEGF, angiopoietin-1, IL-8, histamine, heparin, TGFβ, TNFα, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF). In contrast to the T-helper 1 (Th1)-polarized CD4⁺ T cells, which support a productive antitumor response mediated through IFNγ, Th2, and Th17-polarized CD4⁺ T cells are associated with tumor progression. Th2 cells produce IL-4 and IL-10, which support polarization of TAMs to the M2 phenotype. A Th2 response is also associated with IgG-producing plasma cells and mast cells. Tumor antigen-specific IgG generated during a Th2-driven humoral response may form immune complexes in the tumor microenvironment, leading to complement fixation and subsequent mast cell activation (50). Th17 cells produce IL-17, which is well-established as an angiogenic factor (51). However, a number of studies have reported antitumor effects of Th17 cells, and this topic remains controversial (52).

Inflammation and FDTC

The association between inflammation and thyroid cancer has long been a topic of inquiry. Studies have focused on defining the role of autoimmune thyroiditis in both oncogenesis and established disease. This work has been reviewed recently and, thus, will not be covered in detail here (53,54). A growing body of literature focuses on understanding the tumor-associated immune response in PTC. Earlier studies supported the hypothesis that a tumor-associated immune response was protective. Matsubayashi et al. (55) showed that the presence of tumor-associated lymphocytes (TAL) either surrounding the tumor (peritumoral) or infiltrating the tumor (intratumoral) was associated with a higher 10-year survival rate. Of note, four patients (11%) in this study had concurrent Graves' disease or autoimmune thyroiditis (55). In a large study by Lundgren et al. (56), lymphocyte infiltration in the tumor corresponded to increased survival. This correlation held true when thyroiditis patients were excluded; however, their designation of thyroiditis relied upon retrospective review of pathology reports (56). Evidence of thyroiditis, especially mild to moderate inflammation, is often overlooked in standard pathological analyses of thyroid cancer specimens. Importantly, in the majority of studies analyzing the clinical effects of concurrent thyroiditis on thyroid cancer, concurrent thyroiditis is associated with reduced disease severity (57 –59). Thus, inclusion of patients with concurrent thyroiditis may mitigate any negative correlation between the tumor-associated response and disease.

Studies that have purposefully characterized both tumor-associated and autoimmune inflammation reveal that concurrent thyroiditis is present in more than one-third of all PTC samples. Ugolini et al. (60) characterized PTC samples for evidence of mononuclear cells, specifically noting their localization as intratumoral, peritumoral, and/or in the unaffected controlateral lobe. Of these PTC samples, 55.4% and 70.6% showed evidence of intratumoral and peritumoral mononuclear cells, respectively. A total of 440 samples were analyzed, and 218 (49.5%) had “moderate” to “massive” mononuclear cell infiltrates in the unaffected lobe. The frequency of patients with tumor-associated infiltrates in the absence of concurrent thyroiditis was not reported. Our study, analyzing 100 patients with PTC, found 37% showed histological evidence of thyroiditis in normal thyroid tissue and 24% displayed a tumor-associated response in the absence of concurrent thyroiditis (61). Comparison of these two groups revealed more severe disease in patients with tumor-associated lymphocytic infiltration. Specifically, tumor size, tumor invasiveness, and incidence of LN metastases were reduced in patients with concurrent thyroiditis compared to those with tumor-associated lymphocytes (61). This data suggests that the tumor-associated immune response is functionally distinct from an autoimmune response in the thyroid gland.

Of interest, Ugolini et al. (60) also showed that while tumor-associated mononuclear cells were present in greater than 70% of patients with PTC, an immune response was not found in any of the poorly-differentiated (n=21) or undifferentiated (n=6) samples. These data were interpreted to suggest that the immune response plays an active role in slowing or stopping disease progression (60). In contrast, our studies revealed that the presence of tumor-associated inflammation was associated with a higher incidence of LN metastasis, suggesting that the immune response is inefficient in eliminating the tumor and may even promote disease progression (61). These conflicting data may be reconciled if we consider that, in each analysis, we are seeing only a snapshot of the interaction between the tumor and the immune system. If these patients were followed over time without surgical intervention, we would likely find that, in a majority of cases, the tumor is successfully contained by the immune response and never progresses to a more aggressive form of thyroid cancer. In a subset of patients, however, the tumor would escape immune recognition leading to more aggressive disease (i.e., persistent or recurrent LN metastases and/or dedifferentiation).

To fully understand the role of the immune response in thyroid cancer, we must define the types of immune cells found in association with thyroid tumors, characterize their phenotype, and determine their functional capacity. Furthermore, we must understand the mechanisms of escape that are employed by thyroid cancers cells to evade immune destruction.

Characterization of the tumor-associated immune response in FDTC

Innate immune response in FDTC

Mast cells, proinflammatory granulocytes, have been found at increased levels in PTC samples compared to normal thyroid tissue and adenomas (62,63). High levels of mast cell infiltration in primary PTC samples were associated with extrathyroidal extension. Of interest, thyroid cancer cell lines were shown to produce VEGF-A, which acts as a chemoattractant for mast cells (62). In turn, mast cell–derived histamine supported thyroid cell line proliferation, and CXCL1 and CXCL10 chemokines enhanced both proliferation and invasion (62). Thus, in the established tumor, mast cells are thought to be recruited to the tumor microenvironment and promote disease progression. Of note, antibody–antigen complexes are known to lead to complement activation, initiation of the inflammatory cascade, and recruitment of mast cells and other innate immune cells (64). Additional studies may reveal that the pro-tumor activity of mast cells in PTC is associated with the presence of antithyroid antibodies.

NK cells have been found at low frequency in thyroid tumors (65, 66). CD3⁻CD16⁺CD56⁺ NK cells were recently shown to be increased in benign lesions compared with PTC nodules and were found at higher levels in patients with stage I PTC compared with those classified as stages II, III, or IV (65). Although the cytotoxic capacity of NK cells has not been assessed in thyroid cancer, a subset of PTC and ATC samples express MICA/B (67). Thus, infiltrating NK cells in thyroid tumors may receive an activation signal through MICA/B ligation of NKG2D. It remains to be determined whether thyroid cancer cells down-regulate MHCI molecules, thus predisposing themselves to NK cell–mediated destruction, and whether tumor-infiltrating NK cells are functional. Of note, Boros et al. (68) showed that NK cytotoxicity in peripheral blood was significantly reduced in patients with ATC compared with those with DTC and normal controls. Furthermore, those patients with metastatic or recurrent disease showed markedly reduced peripheral NK cell cytotoxicity compared to patients with no active tumor (68).

Macrophages and DCs have been identified in primary thyroid tumors. Increased frequencies of TAMs are associated with LN metastasis in patients with PTC (69). TAMs were found at increased levels in more aggressive, poorly differentiated thyroid tumors compared with differentiated thyroid tumors (70). Caillou et al. (71) performed a detailed analysis of TAMs in 27 ATC samples and found that TAMs constituted greater the 57% of the total cells in the tumor mass. These cells were termed “ramified” due to their long cytoplasmic processes connecting multiple TAMs in a network throughout the tissue. TAMs in ATC expressed CD163 and displayed no or very few intracytoplasmic phagocytic materials, suggesting an M2 phenotype (71). Similarly, TAMs in PTC express CD163 and IL-10 and likely promote tumor progression and/or inhibit tumor elimination (69). iDCs, identified by CD1a or S100 expression, were found in a higher frequency of PTC samples compared to poorly differentiated thyroid cancers (60,72). While macrophages were identified infiltrating and surrounding PTC lesions, iDCs (CD1a^hi, CD11c⁺, CD40⁺, CD86⁻, HLA-DR⁻) were primarily peritumoral (73). It remains to be determined whether these iDCs are tolerogenic; however, the presence of M2-polarized TAMs and iDCs in thyroid cancers suggests that suppressive mechanisms within the tumor microenvironment have compromised antigen presentation in these patients.

Acquired humoral response in FDTC

B lymphocytes are often found associated with primary thyroid tumors (61,66,74). Our studies revealed that B cells commonly accumulate in peritumoral regions, in some cases, forming follicular structures (61). Semiquantitative analysis of B-cell frequency revealed no correlation with disease severity (61). Future studies defining the phenotype of B cells in FDTC may uncover a role for these cells in tumor elimination or promotion. Of note, antithyroid antibodies are present in the serum of patients with benign nodules (38.7%) and PTC (35.6%) (75). In a large study assessing the contribution of thyroid antibodies to disease, serum antithyroglobulin and anti-thyroid peroxidase antibodies were present in 23.9% of patients with PTC. Antibody titers became negative in 54.6% of patients with no residual disease after surgery and increased over time in 50% of patients with persistent disease, suggesting that the presence of tumor is necessary to perpetuate antibody production. The presence of autoantibodies had no affect on disease severity or survival (76). Thus, although a humoral immune response is commonly generated to FDTC, it is inefficient in preventing disease progression. This finding is not surprising, given our growing knowledge of the immune suppressive environment in aggressive thyroid cancers. Future studies may reveal that patients with existing antithyroid antibodies will respond better to immune-modulating therapies that overcome local immune suppression.

Acquired immune response of T lymphocytes in FDTC

A number of studies have shown T lymphocytes in association with and infiltrating FDTC (61,66,74). Modi et al. (66) found CD4⁺ T cells and CD8⁺ T cells in PTC samples from children and young adults. The presence of these lymphocyte subsets showed no correlation with disease severity; however, no attempt was made to quantify each subset. Our analysis of a small set of patients with evident tumor-associated lymphocytic infiltration confirmed that CD4⁺ T cells and CD8⁺ T cells are present in peritumoral and intratumoral lymphocyte aggregates (61). Semiquantitative analysis revealed that an increased frequency of tumor-associated CD4⁺ T cells among the total T-cell population was associated with increased tumor size (61). CD4⁺CD25⁺FoxP3⁺ Tregs were present in all samples tested and higher frequencies of Tregs correlated with more extensive LN metastases (61). Although increased CD8⁺ T-cell frequencies were not associated with less severe disease, the ratio of CD8 to Tregs was inversely associated with tumor size (61).

In a larger study, Cunha et al. (74) assessed the presence of lymphocyte subsets in patients with DTC (253 PTC and 13 FTC). In contrast to our findings, tumor-infiltrating Tregs were associated with a favorable prognosis. It is important to note that 32% (84/266) of the patients included in this study exhibited concurrent thyroiditis. As already described, we showed that disease severity is markedly reduced in patients with concurrent thyroiditis compared with those with only a tumor-associated immune response (61). Similarly, Cunha et al. (74) showed that concurrent thyroiditis was associated with a more favorable prognosis. Of interest, they showed a positive association with disease for all lymphocytes subsets analyzed (B cells, CD4⁺ T cells, CD8⁺ T cells, and Th17 cells) (74). Their conclusions should be interpreted with caution, however, since the ensuing autoimmune response in nearly one third of the patient samples may overshadow the effects of tumor-infiltrating lymphocytes.

In another recent study, Gogali et al. (65) assessed Treg frequency in benign and PTC nodules using flow cytometry (CD4⁺CD25⁺CD127⁻) and immunohistochemistry (FoxP3⁺). Tregs were increased in PTC nodules compared to benign tissues, and increased levels were associated with higher disease stage. The authors reported that higher Treg frequencies were associated with tumor invasiveness and LN metastasis, confirming our initial finding, although these data were not shown. Patients with concurrent thyroiditis were identified in this study (21/65; 32%) and the average frequency of Tregs in tumor-associated lymphocyte aggregates was comparable in patients with and without concurrent thyroiditis. Disease severity was reported to be similar in patients with and without concurrent thyroiditis, although these data were not shown, and concurrent thyroiditis patients were grouped together in their analysis.

Two of three studies analyzing the role of Tregs in DTC suggest that Tregs are a sign of more aggressive disease. In addition to discrepancies in concurrent thyroiditis definition and inclusion, differences in sample size and method of Treg quantification may explain conflicting results. Importantly, in our studies and work by Gogali et al. (65), Treg frequency was determined from counting 360–1600 total CD4⁺ T cells or a minimum of 300–4500 total lymphocytes, respectively, within the tumor section (61,65). In contrast, Cunha et al. (74) relied upon microarray analysis of three core samples. FoxP3⁺ lymphocyte frequency was scored as 0 (absent), 1 (<10 Tregs/core), or 2 (>10 Tregs/core). This method likely omits areas of peritumoral inflammation, which is often the most populated site of lymphocyte accumulation. While no range was reported for the number of cells counted in these core samples, this method would assess only 3–27 (score=1) or >30 FoxP3⁺ cells per patient sample (score=2). Furthermore, analysis of FoxP3⁺ cells alone without reference to the total number of lymphocytes or CD4⁺ T cells may not accurately portray the immunological relevance of Tregs. For example, 10 Tregs in an area where 100 lymphocytes (10%) are present may have a very different clinical implication than 10 Tregs among 1000 lymphocytes (1%).

Tumor-associated T lymphocytes in locoregional LNs

LN metastasis and persistent or recurrent disease in locoregional LNs is common in patients with PTC. Despite the prevalence of LN metastasis, very few patients develop distant metastasis. This observation could be explained, in part, by the immune response in tumor-involved (TILNs) and nearby uninvolved (UILNs) lymph nodes. In many patients the immune response may be successful in discouraging further growth and metastases. Conversely, in patients with aggressive LN metastastes, the tumor has likely escaped the immune response and invaded through the LN capsule and/or vasculature to seed additional regional or distant metastases. In our recent study, we analyzed CD4⁺ T cell polarization and markers of T-cell exhaustion in UILNs and TILNs from patients with PTC (77). Fresh tissue samples were acquired by ex vivo fine-needle biopsy aspiration, and lymphocytes were analyzed by flow cytometry. These studies revealed that Tregs are found at a higher frequency on average in TILNs compared with UILNs. Tregs were further enriched in TILNs from patients with recurrent disease. CD4⁺ T cells polarized to Th17, Th2, or Th1 phenotypes were present in both UILNs and TILNs. The frequency of Th2 cells was not significantly altered in TILNs compared to UILNs. While Th1 and Th17 cells were enriched in a portion of TILNs, the clinical relevance of these subsets remains to be determined.

To further investigate the T-cell response in metastatic disease, we assessed both CD4⁺ and CD8⁺ T cells for expression of PD-1 and markers of activation. The frequency of PD-1⁺ T cells was increased on average in both CD4⁺ and CD8⁺ T-cell subsets in TILNs compared to UILNs. PD-1⁺ cells were CD45RA⁻, suggesting recent antigen exposure (78). As noted above, PD-1 is expressed transiently following T-cell activation but expressed constitutively as a sign of T-cell exhaustion following chronic antigen exposure (39,41). In contrast to activated T cells, exhausted T cells show reduced proliferative capacity and an altered molecular activation profile (i.e., CD27^hi, CD127^lo, CD69^hi) (41). In TILNs from PTC patients, a majority of PD-1⁺ T cells fail to down-regulate CD27, and less than 20% of PD-1⁺ cells showed evidence of recent or active proliferation as measured by Ki67 expression (77). Although additional studies are necessary to determine the functional capacity of PD-1⁺ T cells in PTC, our findings suggest that T-cell exhaustion contributes to disease progression in patients with aggressive PTC. In support of this hypothesis, the frequency of PD-1⁺ CD4⁺ and CD8⁺ T cells correlated with the frequency of Tregs in TILNs, and high levels of PD-1⁺ T cells were found in TILNs that exhibited extranodal invasion (77).

Immunosuppressive characteristics of FDTC

Cytokine and chemokine production by thyroid cancer cells has been review recently (54). While thyroid cell lines and tumor samples have been shown to express a number of pro-inflammatory cytokines and chemokines, IL-10, TGFβ, and VEGF are also expressed by differentiated and undifferentiated thyroid cancers (61,79 –82). These immunosuppressive cytokines likely contribute to immune dysfunction in the tumor microenvironment. Follicular thyroid cancers are known to express Fas ligand, which could induce apoptosis of Fas⁺ infiltrating lymphocytes (83). PD-L1 expression by thyroid cancers has been documented previously; however, the data were not shown in either publication (84,85). Preliminary studies in our laboratory suggest that metastatic PTC express PD-L1. A more thorough analysis of PD-L1 expression in DTC and ATC samples is necessary. Expression of PD-L1 by thyroid tumors may contribute to T-cell exhaustion in patients with aggressive disease.

T-cell recognition of FDTC

Little is known about tumor antigens, antigen processing, and antigen presentation in FDTC. Many potential tumor antigens have been identified; however, it remains to be determined whether thyroid cancer patients generate an immune response against these antigens. Thyroid-specific proteins (e.g., thyroglobulin, thyrotropin receptor [TSHR], and thyroid peroxidase) are expressed by many thyroid tumors and could be viable targets for the immune response. MUC1, MAGEA3, and cMet are known to be overexpressed in thyroid cancer and could provide a source of tumor-associated antigens (86 –89). In addition, common mutations in BRAF (i.e., BRAF^V600E) and p21 RAS (codon 61) and the RET/PTC translocation could provide antigenic peptides that would be recognized by the immune system as nonself tumor-specific antigens (90). In support of this theory, Gedde-Dahl et al. (91) identified a patient with FTC that harbored peripheral blood memory CD4⁺ T cells specific for mutant p21 RAS (glutamine to leucine at position 61 [G61L]).

An accurate analysis of T-cell function in thyroid cancer will require identification and characterization of tumor-specific T cells. A recent clinical trial assessing the effects of the BRAF^V600E-specific inhibitor, GSK2118436, on immune function included two patients with PTC (92). Using MHC-I multimers loaded with peptides from well-characterized tumor antigens, Hong et al. (92) identified a small population of CD8⁺ T cells in peripheral blood of one patient following treatment with GSK2118436 that were reactive to NY-ESO1. Although NY-ESO1 has been identified as a tumor antigen in medullary thyroid cancer, its expression pattern in follicular thyroid cancers is not known (93). Identification of NY-ESO1–reactive cells in the blood of this patient suggests that tumor-specific T cells will be found, likely at a much higher frequency, in association with primary thyroid tumor or metastatic lesions.

As already noted, MHCI down-regulation is a common mechanism of immune evasion, allowing escape from infiltrating CD8⁺ T cells. Given that dysfunctional antigen presentation is a common mechanism of immune evasion in cancer, one would predict that thyroid tumors acquire the ability to evade immune detection during disease progression. Of interest, however, DTC are known to aberrantly express MHC-II molecules (47%–53%) (94). Furthermore, CD80 and CD86 are expressed by a majority of thyroid tumors (78%–88% of PTC and 88%–100% of FTC). Thus, if tumor antigens are successfully processed and presented by MHC-I and MHC-II, thyroid cancer cells could bypass the need for pAPCs in the activation of naïve T cells, rendering them susceptible to elimination by the ensuing immune response.

Potential for immune-based therapy in aggressive FDTC

In general, immunotherapy has been thought of as a “last resort” for patients with aggressive forms of cancer due to previously poor response rates and autoimmune side effects. With our growing understanding of tumor–immune interactions, immunotherapy has been reborn. Current strategies are focused on driving DC maturation and tumor antigen presentation, eliminating TAMs, and overriding the suppressive effects of Tregs and immune checkpoint molecules. The potential of these therapies for patients with aggressive thyroid cancer should be evaluated (Table 1).

Table 1.

Potential Immune-Based Therapies for Aggressive Follicular Cell-Derived Thyroid Cancer

Immune-based therapy	Source of antigen	Patient material	Cellular target(s)	Biological outcomes
GM-CSF-engineered tumor cells (e.g., GVAX)	Whole tumor	Autologous or allogeneic tumor	DCs, CD4⁺ and CD8⁺ T cells	Enhanced DC maturation and antigen presentation to T cells
Antigen–GM-CSF fusion protein (e.g., Sipuleucel-T)	Defined thyroid tumor antigen	PBMC-derived DCs	CD4⁺ and CD8⁺ T cells	Enhanced DC maturation and antigen presentation to T cells
Opsonizing antibodies (e.g., cetuximab)	EGFR, HER2, TSHR, NIS	None	Tumor cells, NK cells and neutrophils, macrophages	Immune-mediated destruction of antibody-labeled tumor cells, enhanced antigen processing
Radioisotope or toxin-conjugated antibodies (e.g., ⁹⁰Y-ibritumomab)	EGFR, HER2, TSHR, NIS	None	Tumor cells	Direct destruction of tumor cells
Bisphosphonate clodronate and trabectedin	N/A	None	TAMs	Elimination of TAMs and release from immune suppression
Anti-CCL2 antagonist (e.g., CNTO 888)	N/A	None	TAMs and tumor cells	Reduced migration of macrophages to tumor, direct antitumor effects
Anti-CD40 agonist	N/A	None	Macrophages	Activation of M1 macrophages
PD-1/PD-L1 blockade (e.g., BMS-936558)	N/A	None	PD-1⁺ T cells and PD-L1⁺ tumor cells and leukocytes	Release from inhibitory checkpoint resulting in enhanced T-cell function. Treg inhibition?
CTLA-4 blockade (e.g., ipilimumab)	N/A	None	Activated T cells, Tregs	Release from inhibitory checkpoint resulting in enhanced T-cell function, Treg inhibition
CCR4 antagonist	N/A	None	Tregs	Reduced Treg migration to tumor

The source of antigen, requirement for patient-derived cells, cellular targets, and expected biological outcome is noted for each category. Examples of model drugs currently approved by the U.S. Food and Drug Administration or in clinical trials are listed where applicable.

GM-CSF, granulocyte-macrophage colony-stimulating factor; PBMC, peripheral blood mononuclear cell; HER2, human epidermal growth factor receptor 2; TSHR, thyrotropin receptor; NIS, Na⁺/I⁺ symporter; TAM, tumor-associated macrophage; CCL2, CC chemokine ligand 2; PD-L1, PD-1 ligand 1.

DC maturation and DC-based vaccines

DC maturation and antigen presentation is central to successful activation of the anti-tumor T-cell response. Among the many vaccine strategies, we will highlight GVAX and Sipuleucel-T as well-studied approaches that may be applicable to thyroid cancer. GVAX, irradiated autologous or allogeneic tumor cells engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF), have shown some success in combination therapies. This approach provides a source of multiple tumor antigens and localized production of GM-CSF, which induces recruitment and activation of APCs, thus, enhancing antigen presentation. GVAX used alone and in combination with other treatments has shown some promise in prostate cancer and hematologic malignancies (95). Sipuleucel-T, the first DC-based vaccine to obtain U.S. Food and Drug Administration (FDA) approval, uses autologous peripheral mononuclear cells exposed to an engineered antigen–cytokine fusion protein composed of prostatic acid phosphatase (PAP)-GM-CSF. In patients with castrate-resistant prostate cancer, Sipuleucel-T treatment resulted in a 4-month benefit (96). Such antigen-specific DC-based therapies require identification of relevant tumor antigens in each patient. DC-vaccine strategies have shown some initial success in medullary thyroid cancer using calcitonin, carcinoembryonic antigen, and tumor lysates (97,98). Further characterization of tumor antigens in thyroid cancers may support the pursuit of similar DC-based therapies in patients with aggressive DTC or ATC. While previous vaccine trials have shown only minimal success, current and future studies combining improved vaccine strategies with treatments that enhance T-cell stimulation or inhibit immune suppression (i.e., checkpoints and Tregs) are expected to achieve greater success.

Tumor-specific monoclonal antibodies

There is growing support for the use of tumor-specific monoclonal antibodies in targeting immunotherapies (99). In addition to any direct effects on tumor cells themselves, tumor-specific antibodies can be used as opsonization agents that prime tumor cells for recognition by cells the innate immune system. Opsonization leads to direct destruction by NK cells and neutrophils. Opsonized tumor cells may be phagocytosed by macrophages providing a source of tumor antigen for presentation to tumor-specific T cells. Alternatively, such antibodies can be conjugated with radioisotopes or other toxins, facilitating delivery of chemotherapy directly to the tumor cell. Radioimmunotherapy using anti-CD20 antibodies has been successful in patients with non-Hodgkin lymphoma (i.e., ⁹⁰Y-ibritumomab and ¹³¹I-tositumomab) (100). Current antibody-mediated therapies targeting epidermal growth factor receptor (cetuximab, panitumumab) and human epidermal growth factor receptor 2 (HER2) (trastuzumab), which are commonly expressed by FDTC, may be viable therapies for advanced thyroid cancer in combination with standard chemotherapies or other immune-targeted strategies (101 –103). Furthermore, therapeutic antibodies specific for thyroid surface proteins (i.e., TSHR, Na⁺/I⁺ symporter [NIS]) could be useful in the treatment of well-differentiated thyroid cancers.

Macrophage-targeted therapies

With growing understanding of TAMs in cancer progression, much effort has been focused on eliminating these immunosuppressive cells. Selective depletion of TAMs has been achieved in murine and canine models with the bisphosphonate clodronate encapsulated in liposomes (104,105). The potential for clodronate in elimination of TAMs in human cancers remains to be determined; however, bisphosphonates are commonly used in the treatment of bone metastases and are associated with reduced tumor burden. Trabectedin, a marine-derived anti-tumor agent, is highly cytotoxic to mononuclear phagocytes and is currently used in Europe for treatment of sarcoma and ovarian cancer (106,107). Carlumab (CNTO 888) an anti-CC chemokine ligand 2 (CCL2) antibody that blocks macrophage migration, in addition to its affects on tumor angiogenesis, invasion, and metastasis, is in Phase I/II trials (108,109). A number of strategies have been proposed to convert TAMs into M1 macrophages with antitumor activity. Histidine-rich glycoprotein (HRG), an anti-angiogenesis factor, and IL-12 promote M1 polarization in murine models (110,111). In human pancreatic cancer, treatment with agonistic anti-CD40 antibody resulted in tumor regression in a subset of patients. Using a mouse model of pancreatic cancer in parallel, the authors showed that the effects of anti-CD40 were dependent on macrophages and that CD40 ligation resulted in the activation and infiltration of tumoricidal macrophages (112). Macrophage-targeted therapies would be applicable in aggressive thyroid cancers, especially in the case of ATC in which TAMs are most evident.

Checkpoint-targeted therapies

Much recent excitement has been generated for immune-based therapies that target the immune checkpoint molecules, specifically CTLA-4 and PD-1 (36). Ipilimumab, a CTLA-4 blocking antibody, is FDA approved as a first- or second-line treatment for patients with malignant melanoma and is also in development for treatment of prostate cancer and non–small cell lung cancer (NSCLC). Recent phase III trials in metastatic melanoma revealed that patients treated with ipilimumab alone or in combination with a gp100 peptide vaccine had a median survival rate of 10.1 or 10.0 months, respectively, compared with 6.4 months in those treated the gp100 alone. An objective response was observed in 60% of patients (9/15) treated with ipilimumab and maintained for at least 2 years (113). Tremelimumab, another CTLA-4 blocking antibody with a longer half-life than ipilimumab, has shown similar efficacy in melanoma (114, 115).

Inhibitory antibodies targeting PD-1 and PD-L1 are currently in clinical development. In a phase I trial, BMS-936558, the most studied anti-PD-1 blocking antibody, was given in escalating doses to 39 patients with solid tumors (116). One patient with metastatic colon cancer showed a complete response, and two patients (melanoma and renal cell cancer) achieved partial responses. This study was expanded in a recent larger phase I trial (296 patients) to include patients with NSCLC and prostate cancer in addition to melanoma, renal, and colon cancers (117). Response rates of 18% (14/76), 28% (26/94), and 27% (9/33) were seen in patients with NSCLC, melanoma, and renal cell cancer, respectively. Responses were maintained over 1 year in 20 of 31 (65%) patients. In a phase I trial, the anti-PD-L1 antibody BMS-936559 was given in increasing doses to patients with NSCLC, melanoma, colorectal, renal cell, ovarian, pancreatic, gastric, and breast cancers (118). An objective response was observed in 17% (9/52), 12% (2/17), 10% (5/49), and 6% (1/17) patients with melanoma, renal, NSCLC, and ovarian cancers, respectively. In 8/16 patients, the response lasted over 1 year. Of note, these therapies achieved significant success without the need for vaccination with tumor antigen. It remains to be determined whether the objective response rate will improve when combined with tumor vaccines or standard chemotherapies. Our initial characterization of PD-1 and T-cell exhaustion in metastatic PTC suggests that patients with aggressive thyroid cancer could benefit from immune therapies targeted at overcoming the PD-1 checkpoint.

Treg-targeted therapies

The general failure of past immune-based therapies is thought to be due, largely, to the domineering immune suppressive environment in the tumor. Tregs are a key player in local immune suppression and much study has been focused on eliminating these cells from the tumor microenvironment. Current approaches focus on depleting Tregs, inhibiting Treg function, and blocking Treg migration to the tumor. Depletion of Tregs using an anti-CD25 antibody (daclizumab) or an IL-2 diphtheria toxin conjugate (denileukin diftitox, also known as ONTAK), have shown only limited success (119 –121). Importantly, Tregs are phenotypically diverse with both CD25⁺ and CD25⁻ subsets and activated T cells express CD25 transiently. Thus, targeting CD25 may deplete both Tregs and tumor-specific activated T cells. As described above, Tregs express the inhibitory receptor CTLA-4. Ipilimumab has been shown inhibit Treg-mediated suppression in addition to enhancing the effector T-cell response (48,122). Antibodies that block the PD-1/PD-L1 checkpoint may be successful in blocking Treg function based on in vitro data; however, the effects of these antibodies on Tregs in cancer patients remain to be determined (44). Early investigations with a small-molecule antagonist of CCR4, a chemokine receptor essential for Treg migration to the tumor and draining LNs, showed enhanced tumor vaccine responses when Treg migration is inhibited (123,124). Of interest, many kinase inhibitors (i.e., sunitinib, imatinib meyslate, dasatinib, and temozolomide) have been shown to inhibit Treg function and/or frequency in peripheral blood and may be valuable in combination with immune-based therapies (125 –129).

Side effects of immune-based therapies

The common goal of the therapies described above is to enhance the dysfunctional anti-tumor immune response. An unfortunate but inherent consequence of such therapies is the loss of tolerance leading to tissue-specific autoimmune disorders and, in extreme cases, the development of acute systemic immune activation. Early studies that achieved systemic immune activation resulted in serious toxicities and even death (130 –133). Systemic therapies using IL-2 and agonistic anti-CD28 to enhance T-cell activation or IL-12 and agonistic anti-CD40 to encourage DC activation and maturation, although successful in principle, must be used cautiously to avoid devastating side effects. It is somewhat surprising that more recent strategies that target the immune checkpoint molecules CTLA-4 and PD-1 have not shown more severe side effects, given their central role in the maintenance of tolerance. While 20%–30% of patients developed immune-related toxicities, most commonly dermatitis and colitis, only 10%–15% of patients treated with the FDA-approved short course of ipilimumab developed severe adverse toxicities (113). In the most recent BMS-936558 trial, 11% of patients developed severe adverse events. Immune-related toxicities from ipilimumab and BMS-936558 were generally resolved with systemic steroids. Long-term studies of cancer survivors from these trials are necessary to determine whether immune-modulation results in the development of persistent autoimmune disease.

In some cases, localized or regional delivery of immune-modulating therapies would be feasible, potentially avoiding the side effects of systemic therapies (134). In 1988, IL-2 was injected perilymphatically in patients with squamous cell carcinoma of the head and neck (135). More recently this technique was used to treat squamous cell carcinoma of the oral cavity and oropharnyx and resulted in improved survival (136). In melanoma trials, CpG was injected either directly into the tumor or intradermally near the surgical scar, resulting in an enhanced immune response in the sentinel LNs (137,138). Furthermore, in a preclinical mouse model, agonistic anti-CD40 antibodies injected locally near the tumor or tumor-draining LNs resulted in local DC activation and elimination of local and distant tumors. Side effects were reduced compared to systemic delivery of anti-CD40 (139).

Candidates for immune-based therapies

Patients with aggressive forms of FDTCs can be divided into three categories: (i) ATC, (ii) DTC (PTC or FTC) that cannot be cured with surgery and radioactive iodine, and (iii) DTC that persists in locoregional LNs. The low frequency of ATC cases renders this gravely aggressive form of thyroid cancer difficult to study; however, the presence of TAMs and iDCs in ATC samples suggests that immune suppression is an active component of tumor progression. Patients with nonresectable and radio-resistant DTC have not been studied specifically, but data from primary thyroid tumor surgical specimens suggest that immune modulation is evident even in less aggressive tumors. Local or systemic immune-based therapies may be beneficial in these cases. Persistent DTC that is confined to locoregional LNs may be eliminated with local therapy injected directly, under ultrasound guidance, into the affected LNs.

Given the indolent nature of DTC compared to ATC and other types of cancer, patients in the latter two categories may be readily responsive to immune-based therapies. These slow-growing tumors may be a more evenly matched opponent for the enhanced immune response. Furthermore, these patients have likely retained systemic immune function and, thus, their antitumor response could be more readily enhanced by immune-based therapies. In support of this hypothesis, Liaw et al. (140) reported that peripheral blood lymphocytes displayed reduced response to phytohemagglutinin in patients with distant metastasis compared with those with disease localized to the neck. Moreover, patient R.W. in the immunotherapy study by Amino et al. (1), who had aggressive locoregional disease, showed no evidence of peripheral immune suppression and achieved a partial response to the tumor vaccine.

Conclusion

With our growing understanding of the immune response in thyroid and other cancers, the future of immune-based therapies is promising. Immune-based adjuvant therapies should be considered for those patients with nonclassical aggressive thyroid cancer.

Footnotes

Acknowledgments

Thanks to Drs. Bryan R. Haugen, Jennifer A. Morrison, and Joshua P. Klopper for their input during the writing of this review.

Disclosure Statement

No competing financial interests exist.

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Revisiting Immune-Based Therapies for Aggressive Follicular Cell–Derived Thyroid Cancers

Abstract

Background:

Summary:

Conclusions:

Introduction

Discussion

Mechanisms of immune escape

T-lymphocyte dysfunction

Evading immune detection

Tolerogenic APCs

Tumor-induced immunosuppression and regulatory T cells

Immune checkpoint molecules

Tumor-promoting immune cells

Inflammation and FDTC

Characterization of the tumor-associated immune response in FDTC

Innate immune response in FDTC

Acquired humoral response in FDTC

Acquired immune response of T lymphocytes in FDTC

Tumor-associated T lymphocytes in locoregional LNs

Immunosuppressive characteristics of FDTC

T-cell recognition of FDTC

Potential for immune-based therapy in aggressive FDTC

DC maturation and DC-based vaccines

Tumor-specific monoclonal antibodies

Macrophage-targeted therapies

Checkpoint-targeted therapies

Treg-targeted therapies

Side effects of immune-based therapies

Candidates for immune-based therapies

Conclusion

Footnotes

Acknowledgments

Disclosure Statement

References