The Role of the Tumour Microenvironment in Immunotherapy

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S Gasser et al. Tumour microenvironment 24:12 T283–T295 Thematic Review and therapeutic implications

The role of the tumour microenvironment in immunotherapy

Stephan Gasser1,2, Lina H K Lim3 and Florence S G Cheung3

1Roche Pharma Research and Early Development, Roche Innovation Center Zurich, Roche Glycart AG, Schlieren, Switzerland Correspondence 2 Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, NUS Immunology should be addressed Programme, Centre for Life Sciences, National University of Singapore, Singapore to S Gasser or F S G Cheung 3 Department of Physiology, Yong Loo Lin School of Medicine, NUS Immunology Programme, Centre for Email Life Sciences, National University of Singapore, Singapore [email protected] or [email protected]

Abstract

Recent success in immunomodulating strategies in lung cancer and melanoma has Key Words prompted much enthusiasm in their potential to treat other advanced solid malignancies. ff pancreatic cancer However, their applications have shown variable success and are even ineffective ff thyroid cancer against some tumours. The efficiency of immunotherapies relies on an immunogenic ff immunotherapy tumour microenvironment. The current field of cancer immunology has focused on ff tumour microenvironment understanding the interaction of cancer and host immune cells to break the state of ff endometrial cancer immune tolerance and explain how molecular patterns of cytokines and chemokines affect tumour progression. Here, we review our current knowledge of how inherent properties of tumours and their different tumour microenvironments affect therapeutic

Endocrine-Related Cancer Endocrine-Related outcome. We also discuss insights into recent multimodal therapeutic approaches that Endocrine-Related Cancer target tumour immune evasion and suppression to restore anti-tumour immunity. (2017) 24, T283–T295

Introduction

Recent advances in immunotherapy have presented prognostic significance of immune signatures that may opportunities to eliminate cancers that were not achieved guide immunotherapies for endocrine-related cancers. with previous standard of care therapies (Delitto et al. 2016). Immune checkpoint inhibitor treatment has shown to be a tolerable therapy delivering very promising results Factors that correlate with responses to in treating many cancer types including melanomas and cancer immunotherapies lung cancer (Delitto et al. 2016, Rafei et al. 2017). However, Tumour histology recurrence of cancers and variable success among different cancers are not uncommon. Even in responsive The immune profile of a patient is influenced by cancers, the immune checkpoint inhibitor success rate intrinsic factors including genetic composition of the is often below 50% of patients (Chen & Mellman 2017). tumour and the expression of proinflammatory factors These inefficiencies and variability emphasise our lack of (Chen & Mellman 2017). In addition, extrinsic factors understanding of the immunologic events involved in such as prior or concurrent infections, age, microbiota the tumour microenvironment. In this review, we will or other environmental factors are likely important summarise our knowledge of the immune responses in determinates in the response to immunotherapies endocrine cancers, with new insights in predictive and (Chen & Mellman 2017).

http://erc.endocrinology-journals.org © 2017 Society for Endocrinology This paper forms part of a special section on Immunotherapy and Cancer. DOI: 10.1530/ERC-17-0146 Published by Bioscientifica Ltd. The guest editors for this section were Joanne Y Y Ngeow and Laura S Ward. Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 06:28:41PM via free access

10.1530/ERC-17-0146 Thematic Review S Gasser et al. Tumour microenvironment 24:12 T284 and therapeutic implications

Recent clinical findings using checkpoint inhibitors tumours and many immune excluded tumours recruit suggest that the degree of T cell infiltration in the tumour MDSCs and secrete TGF-β, which creates a reactive is an important predictor of response of patients to immunosuppressive stroma that lacks an inflammatory cancer immunotherapy (Teng et al. 2015). Furthermore, milieu required for T cell infiltration and activation the phenotype of T cells and their localisation within (Hegde et al. 2016). In these types of cancers, checkpoint the tumour microenvironment are likely to impact the inhibitor immunotherapy responses are uncommon results (Teng et al. 2015, Chen & Mellman 2017). In an (Tothill et al. 2008, Calon et al. 2015, Ryner et al. attempt to categorise cancers according to their immune 2015). Non-inflamed tumours are also characterised by phenotype, three broad types of cancer were suggested cytokines and cell types that are associated with immune to exist: Inflamed tumours, immune excluded and suppression and tolerance, such as regulatory T cells, immunologically ignorant, also referred to as immune MDSCs and M2 macrophages (Chen & Mellman 2017). deserts (Hegde et al. 2016). Inflamed tumours with pre- M1 and M2 macrophages promote Th1 and Th2 responses, existing immunity are characterised by the presence of respectively, where M1 macrophages initially fight to tumour infiltrating lymphocytes (TILs) with high density sterilise a wound in the case of an infection followed by of functional IFN-γ-secreting CD8+ T cells and CD4+ T cells. M2 macrophages, which secrete growth factors to promote These T cells often express inhibitory receptors such as growth, proliferation, wound healing and the induction PD-1 and CTLA-4 and other markers indicative of an of anti-inflammatory regulatory T cells (Cao et al. 2010, exhausted phenotype. The inflamed phenotype correlates Mills 2012). Cancers with insignificant levels of specific in some cancers with genetic instability and occurrence antigen are analogous to sterile wounds. The presence of of tumour-specific mutations in peptides that can be M2 macrophages promotes tumour growth while tumour- recognised by T cells, so-called neoantigens (Schumacher secreted factors such as TGF-β and prostaglandins inhibit & Schreiber 2015, Hegde et al. 2016). Inflamed tumours the presence of M1 macrophages and maintain wound are also characterised by the increased expression of healing M2 macrophages at tumour sites, shielding the proinflammatory cytokines including interleukin-1β tumour from T cells (Mills 2012). Other non-inflamed (IL-1), IL-2, IL-12, IL-23 and TNF-α (Chen & Mellman tumours show minimal infiltration of immune cells or 2017). Current checkpoint inhibitor therapies act by expression of immune-related genes possibly because they reinforcement of pre-existing T cell anti-tumour responses are non-immunogenic or represent early immune escape and are therefore most effective in inflamed tumours. In mutants. Tumours with immune desert phenotype are Endocrine-Related Cancer Endocrine-Related the presence of an active T cell tumour response, IFN-γ rare responders to checkpoint inhibitor therapies. produced by T cells and other immune cells upregulate Tumours with higher mutation and neoantigen the expression of PD-L1, which counters T cell receptor burden correlate with durable clinical benefit to and CD28-mediated activation signals of T cells (Hui et al. checkpoint inhibitor therapies. In addition, neoantigen 2017, Kamphorst et al. 2017). In clinical studies of PD-1 signatures for predicting therapy responses are also or PD-L1 antagonists, high T cell density and the adaptive beginning to emerge (Snyder et al. 2014, Rizvi et al. 2015, increase of PD-L1 expression on tumour and immune Van Allen et al. 2015). In agreement with these findings, cells are often used as features of an active anti-tumour patients with DNA repair pathway deficiencies, another T cell response (Chen & Mellman 2017). Other predictive determinant of mutational burden, were also associated biomarkers that correlate with active cellular immunity with better response (Le et al. 2015, Rizvi et al. 2015). include the expression of granzymes in T cells and chemokines such as C-X-C motif ligand 9 (CXCL9) and Role of tumour mutational load in cancer immunity CXCL10 (Chen et al. 2012). Although a response to PD-L1 inhibitor therapy is most common in inflamed tumours, A number of recent publications found correlations it is not assured, suggesting that immune cell infiltration between the mutational load uniquely present in cancer is necessary but may not be sufficient for an effective cells (neoantigens) and T cell infiltration of tumours anti-cancer response (Chen & Mellman 2017). Myeloid- (Brown et al. 2015, Li et al. 2016, Senbabaoglu et al. derived suppressor cells (MDSCs) are premature myeloid 2016). High-throughput epitope screening of cancer progenitor cells produced that suppress T cell function. mutanomes revealed that only a small fraction of The expansion of MDSCs is further promoted by tumour- mutated peptides in cancer cells produced T cell reactivity derived cytokines (Delitto et al. 2016). Some inflamed (Lu et al. 2014, Yadav et al. 2014, Linnemann et al. 2015,

http://erc.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/ERC-17-0146 Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 06:28:41PM via free access Thematic Review S Gasser et al. Tumour microenvironment 24:12 T285 and therapeutic implications

Schumacher & Schreiber 2015). The parameters that are derived from gene products that are expendable define immunogenic peptides are not well understood, for tumor progression, they are likely subject to loss by but may include MHC affinity and hydrophobicity in key downregulation, mutation or gene deletion (Lu et al. residues (Chowell et al. 2015, Chen & Mellman 2017). 2014). Examining the intra-tumour heterogeneity (ITH) A number of overexpressed antigens in various cancers in lung cancer and melanoma showed that tumours with that are lowly expressed in normal cells have also been both a high clonal (present in all tumour cells) neoantigen targeted for immunotherapeutic vaccines. These included burden and low neoantigen ITH had significantly longer antigenic peptides from genes such as renal tumour progression-free survival after checkpoint inhibitor antigen 1 (RAGE-1) for renal carcinoma, preferentially therapy (McGranahan et al. 2016). Conversely, tumours expressed antigen in melanoma (PRAME) for melanoma with high subclonal (present in a subset) neoantigen and other cancers, erb-b2 receptor tyrosine kinase 2 fraction were poor responders (McGranahan et al. (ERBB2) for epithelial cancers and Wilms’ tumour 1 2016). The enrichment of subclonal neoantigens may (WT-1) for leukaemia (Vigneron 2015). Vaccines using be due to the immune shaping of the tumour during its translation-optimised E7 protein of HPV16 expressed in natural development or may be induced by cytotoxic cervical cancer induce significant E7-specific cytotoxic T chemotherapy exposure (McGranahan et al. 2016). It cell response in vivo (Liu et al. 2002). Most neoantigens might be necessary therefore to target multiple tumour arising from passenger mutations are not related to the antigens in cancer immunotherapies at the same time to tumourigenesis process and are therefore likely to be target subclonal neoantigens and to prevent outgrowth unique to every patient. The development of algorithms of less immunogenic tumour variants (Mittal et al. 2014). to predict candidate neoantigens and immunogenicity The use of peptides vaccines and CAR-T cell of peptides with higher precision are the topic of intense technology for the treatment of endocrine cancers might current research efforts in academia and the private sector be challenging as several independent whole-exome (Weber et al. 2009, Zhang et al. 2015). Such algorithms sequencing projects of pancreatic cancers (PC) tend to will be invaluable for the design of peptide vaccines show fewer mutations than lung cancer and melanoma, and chimeric antigen receptor (CAR) T cell technology, with one base pair per megabase for PC compared to where chimeras are generated by combining side-chain approximately 10 base pairs per megabase for lung variable fragments with the ζ subunit of CD3, CD28 cancer and melanoma (Alexandrov et al. 2013). Thus, the or CD137 to artificially link tumour surface antigen lower rate of somatic mutations may contribute to the Endocrine-Related Cancer Endocrine-Related specificity with activation signal transmission in T cells poor immunogenicity of PCs and render these cancers upon antigen binding (Delitto et al. 2016). Vaccines have resistant to checkpoint immunotherapy (Delitto et al. had poor track record, as they largely focus on antigens 2016). Recent genomic profiling found that although not specific to tumours (non-neoantigens). It is possible the predicted number of neoantigens in pancreatic that peripheral tolerance contributed to the low efficacy cancer samples is low, nearly all pancreatic ductal of non-neoantigen-based vaccines. The recent use of adenocarcinoma (PDAC) yielded potential targetable adoptive cell transfer with melanoma-associated antigen neoantigen candidates (Bailey et al. 2016). It is important (MAGE-A)-specific CAR-T cells resulted in significant to note that the identification of neoantigens may change clinical responses in some patients but has also been the potential for immunotherapy in pancreatic cancers, accompanied by fatal on-target/off tumour adverse but must be accompanied by strategies to overcome events (Zajac et al. 2017). The shift of focus on targeting the immunosuppressive tumour microenvironment to neoantigens may circumvent issues of tolerance and tissue unmask these neoantigen-specific responses Bailey( et al. specificity to improve efficacy; however, the challenge 2016, Yarchoan et al. 2017). of heterogeneous antigen expression in tumours and difficulties in conducting randomised trials remain due to T cell exhaustion and response to cancer immunotherapy the personalised nature of neoantigens (Zajac et al. 2017). Similarly, most CAR-T cells have focused on antigens that Many TILs present in the tumour microenvironment are not tumour specific (CD19/CD20) Johnson( & June display an exhausted phenotype (Chen & Mellman 2017). CD19 is expressed on the majority of malignant B 2017). These cells are characterised by the expression cells, but is also expressed by normal B cells and follicular of immune checkpoint inhibitors such as PD-1, TIM3 dendritic cells (Wang et al. 2012). As most neoantigens and LAG3 (Chen & Mellman 2017). Recent evidence

has shown that the response of PD-1/PD-L1-based Activation of stimulator of IFN genes (STING) immunotherapy depends on the presence and expansion can induce anti-tumour effects via type I interferons of T cells with a less exhausted phenotype (Im et al. 2016). in various cancers including PCs in animal models Encouragingly, such T cell populations might be enriched (Gasser et al. 2016). Administration of STING agonists in patients via different means. Inhibition of MEK and resulted in marked increase of CD8+ T cell infiltration PD-1 can impede the rate of T cell exhaustion, or in the and upregulation of PD-L1 (Fu et al. 2015). Consequently, case of PD-L1 inhibitors, reverse the exhaustion of T cells the combined use of STING agonists and PD-1 blockade expressing intermediate levels of PD-1, but not T cells enhanced anti-tumour immunity (Demaria et al. 2015) expressing high levels of PD-1 (Chen & Mellman 2017). and led to regression of tumours that were resistant to The enrichment of non-exhausted TILs in endometrial PD-1 therapy (Fu et al. 2015). cancers may hold great promise for these and other poorly PCs were found to frequently exhibit an immune immunogenic cancers. The combined use of MEK and infiltrate exclusion profile characterised by elevated levels PD-1/L1 inhibitors has yielded synergistic and sustained of TGF-β (Delitto et al. 2016). Moreover, it has recently pancreatic tumour regression in vivo (Ebert et al. 2016). been shown that the exclusion of T cells from PCs was However, combined blockage of PD-L1 and CTLA4 leads attributed to local immunosupression mediated by the to a more favourable treatment outcome when compared production of CXCL12 by fibroblast activation protein to MEK inhibitors. A possible reason is that inhibition (FAP) expressing carcinoma-associated fibroblasts. of CTLA4 enhances priming and proliferation of naïve Depletion of FAP+ stromal cells restored the effects of T cells, whereas MEK inhibitors block T cell priming PD-L1 and CTLA4 inhibitors. Blocking of the CXCL12 (Buchbinder & Desai 2016). Hence, these data support receptor not only restored T cell infiltration, but also acted the conclusion that the continuous replenishment of synergistically with PD-L1 inhibition in the clearance TILs by naïve T cell priming is necessary to efficiently of tumour cells (Feig et al. 2013). Production of heme reject tumours (Chen & Mellman 2017). It is tempting oxygenase-1 by FAP+CD45+ M2 macrophages can further to speculate that careful sequential use of CTLA4 and drive immunosuppression in PCs (Arnold et al. 2014). MEK inhibitors may further increase the response rate The immune excluded phenotype of PCs is partially in endometrial cancers by allowing efficient priming of regulated by macrophages residing outside of the tumour tumour-specific T cells and preventing the exhaustion microenvironment. Depletion of these macrophages of such T cells. The recent discovery that epigenetic restored T cell immunotherapy efficacy (Beatty et al. 2015). Endocrine-Related Cancer Endocrine-Related changes contribute to the exhaustion of T cells may Other major contributors of the immunosuppressive yield new combination partners for PD-1/L1 inhibitors tumour microenvironment in PCs include suppressive (Chang et al. 2014). myeloid cells and regulatory T cells (Tregs) (Morse et al. 2009, Delitto et al. 2016). The administration of the attenuated Toxoplasma gondii vaccine strain CPS in Pancreatic cancer microenvironment Panc02 cancer models activated proinflammatory The median overall survival of advanced pancreatic cancer myeloid cell populations and increased the expression of patients have remained poor under current standard of care costimulatory molecules, the secretion of IL-12, and the therapies with tumours frequently developing resistance recruitment of T cells into the tumour microenvironment (Lim et al. 2015, Irigoyen et al. 2017). Unfortunately, (Sanders et al. 2015). A modified ISOCOM vaccine was immune checkpoint inhibitors have shown little effects shown to be ineffective for advanced cancers due to Treg- in pancreatic cancer patients (Brahmer et al. 2012, mediated immune suppression (Nicholaou et al. 2009). The Delitto et al. 2016). However, combination of high-dose response to ISOCOM tumour vaccines in an orthotopic radiotherapy with anti-PD-L1 significantly improved model of PC was improved by the use of low-dose CpG tumour responses in pancreatic ductal adenocarcinoma TLR9 agonist as adjuvant, which resulted in tumour (PDAC) cell allografts (Azad et al. 2017). Radiotherapy regression by breaking Treg-mediated immunosupression induced a large population of tumour cells susceptible (Jacobs et al. 2011). Such findings are yet to be validated to cytotoxic killing, and the early administration of for patients with advanced cancers in clinical studies. anti-PD-L1 prevented the growth of immunosuppressive T cells were also shown to induce dormancy or pro cells and increased recruitment and activation of T cells mote multistage carcinogenesis depending on cytokine (Azad et al. 2017). signals present in the tumour microenvironment.

In a Simian virus 40, large T antigen-induced pancreatic (Ward 2014). Similar to papillary thyroid cancer (PTC), tumour mouse model, IFN-γ-producing CD4+ T cells follicular epithelial dysplasia lesions in chronic lymphocytic arrested tumour growth by preventing the activation thyroiditis (CLT) showed strong diffuse staining for HBME-

of αvβ3 integrin, tumour-angiogenesis and multistage 1, cytokeratin 19, galectin-3 and cyclin-D1 compared to carcinogenesis via IFN-γ and TNFR1 signalling. However, the normal thyroid (Chui et al. 2013). Functional TLR3 in the absence of combined IFN-γ and TNFR1 signalling, is overexpressed in both Hashimoto’s thyroiditis (HT) the same tumour-protective CD4+ T cells strongly and PTC, which induces the production of immune cell enhanced angiogenesis and multistage carcinogenesis attracting cytokines and chemokines. In accordance, cell (Muller-Hermelink et al. 2008). This permanent infiltration in PTC resembles the immune cell infiltrates

growth arrest in G1/G0 required the activation of in HT (Harii et al. 2005, McCall et al. 2007). Although p16INK4a, STAT1, IFN-γ and TNFR1 signalling, which the molecular mechanisms that contribute to thyroid was essential for sustained Rb hypophosphorylation autoimmunity and cancer remain to be clarified, these (Braumuller et al. 2013). observations suggest a link between inflammation and Although IFN-γ generally exhibits anti-cancer activity, thyroid cancer. it can also induce the expression of PD-L1 and CD80 Inflammation in thyroid cancer depends on the in PCs. The binding of these two ligands to inhibitory exposure to radiation and iodine intake, and more receptors correlated with higher tumour infiltration importantly, the presence of concurrent chronic of Tregs, poorer anti-cancer responses and shortened inflammatory conditions and the balance of pro-tumour postoperative survival (Loos et al. 2008). and anti-tumour activity elicited by immune cells that MDSCs are important mediators of immune infiltrate the thyroid cancer microenvironment Ward( suppressive tumour microenvironments (Gabrilovich & 2014). Although many patients respond well to current Nagaraj 2009). In PDAC patients, the accumulation of therapy, 10–30% of thyroid cancer patients eventually neutrophil-like CD13high MDSCs was found to correlate develop recurrent disease and tumour metastasis (Ward with shorter survival (Zhang et al. 2017). The suppression 2014). Early-stage lymph node metastasis is a characteristic of anti-cancer T cell responses by CD13high MDSCs was of thyroid cancer. These clinically undetectable micro- mediated via expression of arginase-1 (Zhang et al. 2017), metastasis to cervical lymph nodes are present in 45% of which depletes l-arginine leading to downregulation of PTC patients at diagnosis and are difficult to distinguish the TCR chain required for TCR signalling and T cell from inflammatory lymph nodes, hindering the curability Endocrine-Related Cancer Endocrine-Related ζ growth arrest (Munder 2009). MDSC expansion was found of the disease (Li et al. 2014). TLR3 is also implicated in to be further driven by tumour-derived cytokines such as PTC recurrence and metastasis, which provides further VEGF, G-CSF, GM-CSF, IFN-γ, TGF-β, IL-1β, IL-6, IL-10 and evidence for an inflammatory component influencing CCL12 in PCs (Delitto et al. 2016). disease progression (McCall et al. 2007, Li et al. 2014). Gemcitabine-based combination therapies remain Ligands responsible for activating TLR3 have not been the first line of treatment for PCs (Lim et al. 2015, Irigoyen elucidated, but possibly include nucleotides from viruses et al. 2017). Interestingly, gemcitabine doses that have no or immunological cell death. direct effects on cell growth or cytotoxicity can induce The compromisation of the innate immune system γδ T cell-mediated lysis of PC cells by upregulating the in PTC is suggested by the decrease in cytotoxic NK cell expression of ligands for the activating immune receptor infiltration with disease progression from thyroid nodular NKG2D (Miyashita et al. 2017). In contrast, high doses of goitre to PTC (Gogali et al. 2012). Different subsets of NK gemcitabine stimulate phosphorylation of Erk1/2, which cells can be found in the tumour microenvironment of reduces the expression of NKG2D ligands (Miyashita et al. PTC (Poli et al. 2009). Cytotoxic function of NK cells is 2017). Hence, low-dose gemcitabine may be useful in enriched among CD56dim cells, while CD56bright NK cells context of cancer immunotherapy of PC patients. predominately have immune regulatory functions. High expression level of CD16 on CD56dim NK cells also makes them efficient mediators of antibody-dependent cellular Thyroid cancer microenvironment cytotoxicity (Poli et al. 2009). The cytotoxic CD16+CD56dim In contrast to PC, thyroid cancers are highly inflammatory NK cell population predominates in healthy thyroid tumours (Ward 2014). Multiple similarities exist between tissue, thyroid nodular goitre and PTC. CD56bright NK thyroid cancers and thyroid autoimmunity, which may cells predominately infiltrate early-stage PTC tissue, but represent a risk factor for thyroid cancer development this population is subsequently replaced by the CD56dim

cytotoxic subpopulation of NK cells in advanced stages of NCI-H295R ACC cells to LPS, but also reduced basal and PTC (Gogali et al. 2013). These findings suggest a specific growth factor-induced ACC cell viability (Kanczkowski et al. enrichment of CD56bright NK cells at earlier stages of PTC 2010). Hence, treatment of ACC patients with intratumoural development. However, the mechanisms that mediate the TLR4 agonist may create a more inflammatory tumour enrichment or the activity of either NK cell subset in PTC microenvironment that better responds to checkpoint will require further research in the future (Gogali et al. inhibitors or other cancer immunotherapies. 2013). Tumour-associated macrophages (TAMs) in poorly Pituitary carcinoma differentiated thyroid carcinoma are associated with capsular invasion and poor survival (Ryder et al. 2008). It has In pituitary adenomas, constitutively produced recently been shown that CXCL16/CXCR signalling drives intratumoural IL-6 contributes to the excessive hormone macrophage polarisation in the PTC microenvironment, secretion in endocrine-active tumours via autocrine and resulting in cells with a M-2-like phenotype characterised paracrine stimulation of the IL-6 receptor (Thiele et al. by colony stimulating factor-1 receptor (CSF-1R) and C-C 2003). This results in tumour expansion by concomitantly motif chemokine ligand and receptor 2 (CCL2/CCR2) stimulating tumour cell proliferation and tumour overexpression, which enhanced the PTC cell migration neovascularisation (Renner et al. 2009). In nude mice potential and reinforced angiogenesis (Ryder et al. 2013). studies, it was found that cytokines binding to the shared In anaplastic thyroid cancer, expression of CD163, a gp130 subunit of the IL-6 receptor contributed to tumour marker for M2 macrophages, correlated with CXCR4 progression (Graciarena et al. 2004, Renner et al. 2009). expression and more aggressive clinical characteristics The authors have not identified which cytokines were (Kim et al. 2016). The potentially CXCR4-mediated responsible for the effect. If future studies identify IL-6 as recruitment of macrophages could be countered by an important contributing factor in these mice, this model CXCR4 inhibitors or by ablation of macrophages from may serve as a good model for human pituitary adenomas. the tumour microenvironment via CSF-1R depleting It is not clear what signals trigger IL-6 production in human antibodies (Ries et al. 2014, Escamilla et al. 2015). Such pituitary adenomas, but an in vitro study showed that TLR4 compounds are currently in early clinical development by agonists can induce IL-6 expression in human pituitary several companies. adenoma cell lines (Tichomirowa et al. 2005). However, LPS also directly suppressed cell growth of the TLR4-positive Endocrine-Related Cancer Endocrine-Related pituitary carcinoma cell line AtT20 (Tichomirowa et al. Other endocrine tumours 2005). It is unclear whether TLR signals are triggered in pituitary adenoma and which effect of TLR4 agonists Adrenalcortical tumour would dominate in the tumour. Moreover, not all pituitary Adrenalcortical carcinoma (ACC) is a rare malignancy adenomas express TLR4 and only a subset of cells expresses with variable but generally dismal prognosis TLR4 in TLR4-positive tumours (Tichomirowa et al. 2005). (Bilimoria et al. 2008). Mitotane is the only FDA-approved A better understanding of these cellular processes may cytotoxic treatment, which has minimal effects on 5-year allow the targeting of gp130-dependent pathways for the survival rates (Aufforth & Nilubol 2014). Immunotherapy treatment of pituitary carcinomas. approaches have not been successful due to the secretion of immunosuppressive glucocorticoids, the lack of known Current therapeutic approaches in ACC-tumour-specific antigens and insufficient knowledge endocrine cancer of the ACC immunosuppressive microenvironment (Papewalis et al. 2006). In addition, adrenal masses are Chemotherapy treatment in the past was generally often clinically unapparent and with few reliable markers, believed to be immunosuppressive (Delitto et al. 2016). histological differentiation of benign and malignant However, the combination of certain cytotoxic therapies lesions remains difficult Mansmann( et al. 2004). In the with immunotherapy may assist in synergistic anti- normal adrenal, TLR4 expression was detected throughout tumour immunity (Zitvogel et al. 2011). Cell death the adrenal cortex, whereas the TLR4 coreceptor CD14 induced by chemotherapy and radiation may provide expression was found only in the inner cortical zones. proinflammatory ‘danger’ signals in the otherwise No staining was observed in epithelial cells of ACCs. tolerogenic milieu of the tumour microenvironment The restoration of TLR4 and CD14 not only sensitised (Delitto et al. 2016). The subsequent immunologic response

will depend on the type of cell death, the magnitude of with other cytotoxic agents such as capecitabine or cellular stress and the pattern of regulatory cytokines at erlotinib (Lim et al. 2015, Irigoyen et al. 2017). Gemcitabine play (Lake & Robinson 2005). Release of tumour antigens was found to have positive effects on the ability of by dying cells and damaged tissue may also boost existing intratumoural DCs to present antigens to tumour-specific tumour-specific immune responses or potentially induce T cells (McDonnell et al. 2015). Importantly, gemcitabine novel T cell responses against cancer. More importantly, primed the host for a strong anti-tumour response after current chemotherapy and immunotherapy combinations a second insult with a virus expressing the same antigen have started to harness the immunomodulation effects (Nowak et al. 2003). Together, these findings supported of chemotherapy (Lake & Robinson 2005), resulting in the rationale behind the combination of gemcitabine improved antigen presentation and cytotoxic activity with immunotherapy, especially when administered in the immunosuppressing tumour microenvironment. before a tumour vaccine or CD40 agonists (Table 1). A search in http://www.clinicaltrials.gov for clinical CD40 agonists are powerful activators of APCs and trials of endocrine cancers using immunomodulatory promote T cell and other cytotoxic immune responses agents has yielded a list of mostly on-going, clinical (Vonderheide & Glennie 2013). trials (Table 1). A partial list of pancreatic cancer A number of other clinical trials aim to enhance immunotherapies has been selected for discussion here, the anticancer immune response by providing as additional pancreatic cancer-specific clinical studies proinflammatory cues such as TLR agonists, TNF or IL-12. have been extensively reviewed recently by Delitto and These agents primarily act on innate immune cells, which coworkers (Delitto et al. 2016). are important for the development of adaptive T cell responses against cancers.

Immune checkpoint inhibitors Vaccines CTLA4 and PD-1 are two inhibitory receptors on T cells that counter priming or activation of T cells (Buchbinder Another approach to enhance cancer-specific T cells & Desai 2016). After activation of naïve T cells, CTLA4 responses in endocrine cancers is the use of cancer is expressed on the cell surface and competes with the vaccines. Cancer vaccines consist of peptide or protein costimulatory receptor CD28 for binding of its B7 ligands antigens present in tumour cells and adjuvants that on antigen-presenting cells (APCs) and tumour cells stimulate APCs. Protein antigens are generally less prone Endocrine-Related Cancer Endocrine-Related (Sansom 2000). PD-1 expression levels increase with to degradation and require a weaker stimulant to be continuous activation of T cells. Its ligands are expressed immunogenic (Azmi et al. 2014). A number of different on APCs, immune cells, tumour-associated stromal cells peptide/protein and adjuvant combinations are currently and tumour cells. Recent reports have shown that PD-1 explored in clinical trials for endocrine cancers. suppresses intracellular signalling pathways initiated by CD28, but PD-1 may also counter T cell receptor Telomerase peptide vaccines Telomeres caps signals (Hui et al. 2017). PD-1/PD-L1 antagonists were shorten as cells divide, which eventually drives cells into found to restore the effector functions of T cells in the senescence or cell death (Muraki et al. 2012). Cancer cells periphery that have been turned off following extended frequently activate the expression of telomerase reverse antigen exposure, and are characterized by an exhausted transcriptase (hTERT) to maintain telomere ends of DNA phenotype described earlier in this review. Monoclonal (Maida & Masutomi 2015). The ability of hTERT peptides antibody-based antagonists against PD-1 (Nivolumab and to induce cancer cell-specific cytotoxic T cell activity was Pembrolizumab), PD-L1 (MEDI4726 and Atezolizumab) explored in the phase III TeloVac study. The MHC class and CTLA4 (Ipilimumab and tremelimumab) have II telomerase vaccine GV1001 was given with a GM-CSF already shown success in the treatment of other cancers, adjuvant and gemcitabine/capecitabine chemotherapy particularly in advanced melanoma and lung cancer (Table 1). The sequential arm of this study was based (Lipson et al. 2015, Postow et al. 2015). Due to the limited on the idea that a brief course of chemotherapy before effectiveness of monotherapies in endocrine cancers as vaccination may stimulate an endogenous anti-cancer discussed earlier, combination strategies are currently immune response, which would be further boosted by explored for the treatment of endocrine cancers (Table 1). the vaccine. However, no difference in survival was Therapies based on gemcitabine remain the first line observed in the chemotherapy group compared to the of treatment for pancreatic cancer, often in combination sequential chemotherapy/vaccine group (Middleton et al.

Table 1 Immunotherapies under evaluation in clinical trials for endocrine cancers.

Immune therapy agent Phase; status Mechanism Endocrine cancers treated Other agents NCT number Modulators of innate and adaptive immunity Prembrolizumab II; recruiting Anti-PD1 Thyroid, adrenocortical NCT02721732 Prembrolizumab II; recruiting Anti-PD1 Thyroid, NCT02628067 neuroendocrine Prembrolizumab II; not yet recruiting Anti-PD1 Thyroid, NCT03012620 neuroendocrine Prembrolizumab I; recruiting Anti-PD1 Thyroid, pancreatic Enoblituzumab NCT02475213 (MGA271) Prembrolizumab II; not yet recruiting Anti-PD1 Thyroid Lenvatinib NCT02973997 Prembrolizumab II; not yet recruiting Anti-PD1 Thyroid NCT03072160 Prembrolizumab II; recruiting Anti-PD1 Thyroid NCT02688608 Prembrolizumab II; recruiting Anti-PD1 Adrenocortical NCT02673333 Prembrolizumab I/II; not yet recruiting Anti-PD1 Gastroenteropancreatic Somatuline Depot NCT03043664 Neuroendocrine Prembrolizumab II; recruiting Anti-PD1 Neuroendocrine NCT02939651 MEDI4736 II; not yet recruiting Anti-PDL1 Neuroendocrine Tremelimumab NCT03095274 (anti-CTLA4) MEDI4736 I; recruiting Anti-PDL1 Pancreatic Tremelimumab NCT02639026 (anti-CTLA4), radiation Ipilimumab I; recruiting Anti-CTLA4 Thyroid, pancreatic Enoblituzumab NCT02381314 (MGA271) Nivolumab II; recruiting Anti-PD1 Adrenocortical NCT02720484 Nivolumab II; recruiting Anti-PD1 Pancreatic Ipilimumab (anti- NCT02866383 CTLA4), radiation Nivolumab II; recruiting Anti-PD1 Adrenal cortex, adrenal NCT02834013 Ipilimumab Anti-CTLA4 gland pheochromocytoma, pancreatic, pancreatic neuroendocrine, parathyroid gland, thyroid, pituitary gland Endocrine-Related Cancer Endocrine-Related Nivolumab II; not yet recruiting Anti-PD1 Neuroendocrine NCT02923934 Ipilimumab Anti-CTLA4 Ibrutinib II; recruiting Btk inhibitor Pancreatic, carcinoid NCT02575300 RO7009789 I; recruiting CD40 agonist Pancreatic Nab-paclitaxel, NCT02588443 gemcitabine VTX-2337 I; active, not recruiting TLR8 agonist Pancreatic Cyclophosphamide, NCT02650635 pegfilgrastim CYT-6091 I, completed Recombinant TNF Adrenocortical, NCT00436410 pancreatic Recombinant I, completed Recombinant Thyroid, pancreatic, ABI-007, carboplatin, NCT00004074 interleukin-12 interleukin-12 parathyroid, trastuzumab adenocortical MGD009 I, recruiting Antibody for Thyroid, pancreatic NCT02628535 B7-H3 and CD3 Vaccines GV1001 III; completed Telomerase Pancreatic GM-CSF, gemcitabine, NCT00425360 peptide vaccine capecitabine GV1001 III; recruiting Telomerase Pancreatic Gemcitabine, NCT02854072 peptide vaccine capecitabine TG01 Mutant Ras GM-CSF, gemcitabine NCT02261714 peptide vaccine CEA RNA-pulsed DC I, completed DC cell based Pancreatic NCT00004604 cancer vaccine vaccine

(Continued)

Table 1 Continued.

Immune therapy agent Phase; status Mechanism Endocrine cancers treated Other agents NCT number Ras peptide cancer II, completed Mutant RAS Aldesleukin (IL-2), NCT00019331 vaccine peptide vaccine sargramostim (GM-CSF), detoxPC TRICOM-CEA(6D) I, completed DC cells loaded Pancreatic NCT00027534 with TRICOM- CEA(6D) antigen GI-6207 II, recruiting Recombinant Thyroid NCT01856920 yeast-based vaccine express mutant full length human CEA PSMA/PRAME I, completed DNA vector Panreatic, thyroid, NCT00423254 (MKC1106-PP) pPRA-PSM with neuroendocrine synthetic peptides E-PRA and E-PSM Recombinant I, completed Recombinant Pancreatic, thyroid With or without NCT00028496 fowlpox-CEA(6D)/ fowlpox virus Sargramostim TRICOM vaccine vector encoding (GM-CSF) CEA/TRICOM

2014). Several reasons may have a contributed to the angiogenesis through integrin regulation (Conway et al. failure of this study including the enrolment of late- 2006). The MKC1106-PP vaccine is directly administered stage patients, limited efficacy of chemotherapy due to into lymph nodes, which may allow for more efficient stromal characteristics of pancreatic cancer, effects of the priming of T cells by directly targeting APCs present in chemotherapy on the immune response and choice of the lymph nodes (Smith et al. 2011). A phase I trial was peptide or adjuvant (Emens & Middleton 2015). able to induce PRAME- and PSMA-specific T cells in the blood in 15 out of 24 patients (Weber et al. 2011). In 7 Carcinoembryonic antigen vaccines The TRICOM- patients, the disease was controlled for at least 6 months Endocrine-Related Cancer Endocrine-Related CEA(6D) vaccine encodes the carcinoembryonic antigen (Weber et al. 2011). (CEA) and a TRIad of COstimulatory Molecules (B7-1, ICAM-1 and LFA-3) (TRICOM) (Palena et al. 2004). The vaccine is either administered via a recombinant fowlpox Conclusion and future perspectives virus vector or directly loaded onto DCs to enhance CEA The recent success of immunotherapy for cancer has presentation on APCs. CEA-TRICOM vaccines are generally generated enormous enthusiasm with much effort safe and can generate significant CEA-specific immune focusing on checkpoint inhibitors and cancer vaccines. responses (Marshall et al. 2005). When administered with Although some patients have benefited from favourable IFN-α, CEA-TRICOM can led to significantly increased responses, immunotherapies have proved ineffective overall survival of patients compared to vaccine alone for many cancer patients including endocrine cancer (Marshall et al. 2005, Duggan et al. 2016). patients. A better understanding of how to manipulate the tumour microenvironment to elicit an effective Melanoma and prostate peptide vaccines The cancer-directed immune response will be essential. Many MKC1106-PP vaccine consists of a DNA vector, pPRA-PSM, challenges remain, such as how to select and deliver encoding segments of two tumor antigens, PRAME tumor antigens and what immune suppression or evasion and PSMA (Weber et al. 2011). Both PRAME and PSMA mechanism to target in different cancer patients. Only are expressed in a range of tumours (Silver et al. 1997, a fraction of candidate neoantigens selected by current Conway et al. 2006, Lerut et al. 2015). The former facilitates neoantigen prediction algorithms trigger an anti-tumour tumour progression by inhibiting cellular differentiation response (Mardis 2017). A more detailed understanding through repression of retinoic acid receptor signalling in of antigen processing, trafficking and MHC loading cancer cells (Oehler et al. 2009), and the latter is expressed will be necessary for improving prediction algorithms in the neovasculature of solid tumours mediating (Mardis 2017). The newly established tumour neoantigen

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Received in final form 25 July 2017 Accepted 27 July 2017 Accepted Preprint published online 28 July 2017