Cancer Immunology, Immunotherapy (2019) 68:1391–1400 https://doi.org/10.1007/s00262-019-02364-2

MEETING REPORT

4th Symposium on Advances in Immunology and Immunotherapy, November 29–December 1, 2018, Athens, Greece

Sotirios P. Fortis1 · Athanasios Kotsakis2 · Christophe Le Tourneau3 · Ourania E. Tsitsilonis4 · Vassilis Georgoulias5 · Constantin N. Baxevanis1

Received: 31 March 2019 / Accepted: 2 July 2019 / Published online: 15 July 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019

Keywords Cancer immunology · Immunotherapy · Biomarkers · Immune monitoring · Immunomodulatory mechanisms Abbreviations NTRK Neurotrophic tropomyosin receptor kinase ALK Anaplastic lymphoma kinase PARP Poly (ADP-ribose) ATF Activating transcription factor pTreg Peripheral Treg BCSC Breast cancer stem cells TEX Tumor-derived exosomes CHOP CCAAT-enhancer-binding protein homologous TIGIT T-cell immunoglobulin and ITIM domain protein TIM-3 T-cell immunoglobulin, mucin domain-3 CPI Checkpoint inhibitor TLS Tertiary lympoid structure EMA European agency TME Tumor microenvironment EML4 Microtubule-associated protein-like 4 TP53 Tumor protein 53 EV Extracellular vesicles Treg T regulatory cells FLIP FLICE-inhibitory protein UPR Unfolded protein response GO Gemtuzumab ozogamicin VLP Virus-like particles GPCRs Protein-coupled receptors HNSCC Head and neck squamous cell carcinoma ICIs Immune checkpoint inhibitors Introduction ICIR Immune checkpoint inhibitory receptor irAEs Immune-related adverse events The 4th symposium on advances in cancer immunology and iTreg Induced regulatory T cell immunotherapy was held from November 29 to December 1, miTRAP miRNA trapping by RNA in vitro afnity 2018, in Athens, Greece. The 4th symposium aimed to bring purifcation together many of the leading researchers and clinicians from MM Multiple myeloma Europe and the US to report on most recent advances in can- NSCLC Non-small cell lung cancer cer immunology and immunotherapy and to promote inter- actions between speakers and participants, thus encouraging * Sotirios P. Fortis stimulating discussions and providing network opportunities [email protected] and new collaborations. The main objectives were to gather,

1 share and exchange current knowledge from the feld of Cancer Immunology and Immunotherapy Center, tumor biology and cancer immunology and immunotherapy, Saint Savas Cancer Hospital, 171 Alexandras avenue, 11522 Athens, Greece and to translate and extend this knowledge to the clinic. The main topics included: (1) immune suppression; (2) innate 2 Department of Medical Oncology, University Hospital of Larissa, Larissa, Thessaly, Greece immunity in cancer; (3) monitoring of immunomodulatory mechanisms; (4) combinational treatment; (5) biomarkers 3 INSERM U900 Research Unit, Department of Drug Development and Innovation (D3i), Institut Curie, for immunotherapy; and (6) clinical trials. Paris-Saclay University, Paris, France 4 Overcoming immune suppression Department of Biology, National and Kapodistrian University of Athens, Athens, Greece 5 1st Department of Medical Oncology, Metropolitan General, The tumor microenvironment (TME) contains a suppres- Athens, Greece sor network which promotes tumor growth, invasion and

Vol.:(0123456789)1 3 1392 Cancer Immunology, Immunotherapy (2019) 68:1391–1400 . Detailed investigation of the immunosuppressor by Theresa L. Whiteside (University of Pittsburgh Cancer circuits within the TME will improve our understanding of Institute, Pittsburgh, USA) on the functional role of regula- tumor biology and evolution and at the same time will ena- tory T cells (Treg) in cancer. Although Treg were discovered ble the design of novel therapeutic modalities against can- more than 20 years ago and have been a subject of intense cer. In this session, Vincenzo Bronte (School of interest and investigation ever since, many questions about and Surgery, University of Verona, Verona, Italy) provided their origin, development, functions and clinical signifcance an overview of the role of myeloid-derived suppressor cells in cancer remain elusive. No defnitive marker for human (MDSC) in tumor immune evasion. Low doses of diferent Treg has been identifed; the most frequently used mark- chemotherapeutic agents selectively eliminated the mono- ers HELIOS, neuropilin-1 (NRP-1) and CD15s are not spe- cytic-MDSC (M-MDSC) and enhanced the therapeutic ef- cifc for all Treg. Sakaguchi’s classifcation of Treg based cacy of adoptively transferred, tumor-specifc CD8+ T cells. on expression of CD45RA and FOXP3 identifes fraction II His team found that chemotherapeutics afecting M-MDSC Treg (FOXP3 + CD45RAlow) as the true suppressor cells that all shared the ability to modulate the anti-apoptotic protein accumulate in tumors. Strikingly, there exists a lack of clarity FLICE-inhibitory protein (FLIP) both in vitro and in vivo, in interpretation of Treg responses to checkpoint blockade. and that enforced FLIP expression which partially protected Treg that accumulate at tumor sites and in the circulation of the cells from chemotherapy-induced death. Unexpectedly, patients with cancer are referred to as induced Treg (iTreg). FLIP induction in monocytes regulated a complex transcrip- They difer phenotypically and functionally from peripheral tional program, which included several MDSC-related genes Treg (pTreg) and mediate vigorous suppression of immune such as CD274, CD273, IL-10 and IDO1, promoting their cells utilizing a variety of mechanisms. They may or may transition towards fully immune suppressive monocytic- not be FOXP3+ but co-express numerous immune check- MDSC (M-MDSC). To prove the immunoregulatory power point inhibitory receptors (ICIRs), e.g., cytotoxic CTLA- of this molecular axis, they showed that injection of c-FLIP- 4, PD-1, T-cell Ig and mucin domain-containing protein-3 expressing monocytes was able to control GvHD progres- (TIM3), lymphocyte activation gene 3 (LAG3) and T-cell Ig sion in a setting of xenogeneic transplantation. Patients with and ITIM domains (TIGIT). It was, therefore, expected that pancreatic ductal adenocarcinoma presented an increased the suppressor activity of Treg expressing ICIRs would be frequency of cellular FLICE (FADD-like IL-1β-converting silenced following delivery of checkpoint inhibitory Abs that enzyme) inhibitory protein (c-FLIP) + PD-L1 + CD14 + cells block negative signaling. Unexpectedly, responses of Treg to in the blood and the higher circulating levels of these cell immune checkpoint blockade difered from those of efec- subsets together with increased concentrations of plasma tor T cells: while the blockade released all other immune IL-6 correlated with shorter overall survival. Collectively, cell types from suppression and rejuvenated their anti-tumor these data suggest a critical role of c-FLIP in cancer pro- activities, Treg-mediated suppression, phenotype, frequency motion by regulating the immunosuppressive properties of and stability were not reduced. It appears that Treg are resist- mature myeloid cells. Viktor Umansky (German Cancer ant to ICIR blockade with Abs, and it has been suggested Research Center (DKFZ), Heidelberg, Germany) reported that this resistance may underlie unresponsiveness of cancer on the immunosuppressive network in the melanoma micro- patients to immune checkpoint inhibitors (ICIs). A number environment, where MDSC induced by chronic infamma- of questions and controversies about Treg remain, including tion play a major role. The accumulation and activation of their “transcriptional signature,” the nature of the primary MDSC could be mediated not only by soluble infammatory target of Treg, the role of APC in Treg functions, the redun- factors but also by tumor-derived extracellular vesicles (EV). dancy of Treg suppressive mechanisms and the requirement Recent studies from his laboratory showed that EV isolated for overexpression of multiple ICIRs on Treg. Recent data from Ret mouse melanoma cells induced an upregulation of suggest that Treg are functionally highly diverse regulatory PD-L1 on immature myeloid cells, leading them to acquire cells that are involved in a broad spectrum of immune and the ability to suppress T-cell activation. PD-L1 expression non-immune activities in various tissues and organs. In the and the immunosuppressive ability of EV-generated MDSC tumor microenvironment, Treg are subverted by the tumor, were dependent on the interaction between TLR4 on mye- and their immunosuppressive activities represent a major loid cells and HSP86 on EV followed by NF-κB activation. barrier to successful cancer immune therapies. EV from human melanoma cells or plasma of melanoma patients induced PD-L1 upregulation on normal monocytes The role of innate immunity in cancer leading to their conversion into immunosuppressive cells. Thus, these fndings highlight novel mechanisms of MDSC Chemokines and their specific receptors constitute a generation from normal myeloid cells by melanoma EV, sug- complex network within the TME which depending on gesting the importance of EV targeting for tumor therapy. A the context may promote or suppress tumor growth. diferent mechanism of immunosuppression was described

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Toll-like receptors (TLR) and their agonists play a sig- burden and provide an adjuvant therapy to standard nifcant role in inducing and/or optimizing anti-tumor treatments. immune pathways. Nathan Karin (Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Monitoring of immunomodulatory mechanisms Israel) summarized the role of regulatory chemokines in cancer progression. Chemokines are small (~ 8–14 kDa) Immunomodulatory mechanisms may act in various ways secreted proteins, structurally similar to cytokines. They to establish conditions relevant for the clinical beneft of regulate cell trafcking through interactions with a subset immunotherapies but also other types of cancer therapies. of seven-transmembrane G protein-coupled receptors. In These may include the generation of (1) an immune stimula- oncogenesis, chemokines and their receptors are involved tory TME, (2) robust tumor infltration with lymphocytes, in supporting tumor development and metastatic spread by (3) immunogenic phenotype of cancer cells and (4) func- four major complementary mechanisms: (1) by attracting tionally active anti-tumor efector cells. Theodora Katsila cancer cells to sites of metastatic spread; (2) by support- (Department of Pharmacy, University of Patras, Patras, ing tumor growth through an autocrine loop; (3) through Greece) introduced proteomics and proteogenomics as a mobilization of bone marrow-derived leukocytes from the toolbox toward monitoring immunomodulatory mechanisms. bone marrow to the blood, followed by supporting their Emphasis was put on exosomes (and their cargos) as means colonization at the tumor site and shaping their biological to interpret cell-to-cell communication, also in the context function there; and (4) through chemokines and chemokine of non-classical secretion. The secretome and membrane receptors as immune checkpoints in the regulation of proteome may add an extra information layer to shed light anticancer immunity. Focusing on the last two features. on molecular mechanisms and drug resistance. Coupling Karin’s group, in collaboration with Viktor Umansky from proteomics or proteogenomics to other omics approaches the DKFZ, have recently shown that the chemokine recep- empower data reliability and map inter-individual variabil- tor CCR5 is essential for supporting the mobilization of ity, attempting to defne the so-called ‘actionable genome’. polymorphonuclear MDSC (PMN-MDSC) from the bone Colorectal cancer and glioblastoma multiforme served as marrow to the blood and later their accumulation at the paradigms. Gosse J. Adema (Department of Radiation tumor site to support tumor development and suppress Oncology, RIMLS, Radboud University Medical Center, anti-tumor immunity. They also identifed a chemokine Nijmegen, The Netherlands) considered “in situ cancer vac- that enhances anti-tumor immunity, CXCL10, and devel- cines” following tumor ablation. Tumors can serve as their oped a stabilized form of this chemokine (CXCL10-Ig) own antigenic vaccine “in situ” provided that appropriate for cancer therapy, alone, or in combination with ICIs, tumor ablation approaches are combined with immune acti- and CCR5 blockers. Tina Bagratuni (School of Medicine, vating compounds such as adjuvants or checkpoint mAbs. National and Kapodistrian University of Athens, Athens, Dr. Adema presented data to support the role of saponins as Greece) provided a general overview on TLR. The sys- highly efective adjuvants able to synergize with tumor abla- tematic study of TLRs role and function in tumor cells tion. Subsequent studies revealed that these adjuvants induce can contribute substantially to the development of new an unprecedented level of DC cross-presentation in vitro anti-tumor pharmaceuticals with TLR-dependent mecha- and in vivo. The presence of saponin adjuvants increased nisms of action. To this end, Dr. Bagratuni focused on a cytosolic translocation of antigen, resulting in proteasome- recent study from her group investigating the role of TLR4 dependent cross-presentation in monocytic CD11b­ + DC. in multiple myeloma (MM) cell biology. Their data sup- Strikingly, specifically in this monocytic ­CD11b+ DC port the importance of TLR4-mediated efects in MM and subset, saponins enhanced DC cross-presentation by lipid suggest that TLR4 activation may protect MM cells from body induction. Both pharmaceutical and genetic interfer- apoptosis by suppressing the CCAAT-enhancer-binding ence with lipid body formation inhibited saponin-based protein homologous protein (CHOP)-activating tran- adjuvants (SBA)-induced cross-presentation in these DCs scription factor (ATF) unfolded protein response (UPR) in vitro and in vivo. How lipid bodies afect DC cross-pres- (CHOP-ATF4 UPR) branch. The data from this study entation, type I IFN production and other immune processes deepen our understanding of the function and diversity of are just beginning to be studied in detail. Panayiotis Vergi- TLR4 in MM and the association between innate immune nis (Biomedical Research Foundation of the Academy of signaling and MM cell proliferation. It also ofers new Athens, Athens, Greece) presented data on the role of Treg insights into potential new strategies for MM therapy, as in immune-related adverse events (irAEs) during immuno- it can be assumed that TLR4 targeting could be used as therapy. The advent of ICIs has revolutionized cancer immu- a potential therapeutic strategy that could reduce tumor notherapy. However, despite the enormous success, a signif- cant proportion of patients do not respond, while responses are frequently accompanied by life-threatening (auto)irAEs.

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Therefore, predictive biomarkers of clinical responses are antigen-presenting machinery (APM) components leading urgently needed. Moreover, deciphering new therapeutic to loss or reduced expression of HLA class I molecules. strategies to harness anti-tumor immunity while keeping As a result, tumor cells are not properly recognized by autoimmunity in check remains a daunting task. Achiev- CTL either endogenously induced (e.g., via vaccination or ing these goals has been hampered by the immunosuppres- immune checkpoint blockade) or exogenously transferred in sive nature of the TME. Treg comprise an ideal candidate, the context of adoptive T-cell therapies. Another mechanism since they are physiologically engaged in maintenance of which leads to downregulation of HLA class I molecules is self-tolerance, are the dominant suppressive population in based on the activity of small leucine-rich proteoglycans TME, promote tumor growth, associate with poor progno- (SLRP). When overexpressed, the SLRP biglycan (Bgn) sis and importantly represent a fundamental impediment of leads to upregulation of MHC class I surface expression, success. Towards this, Dr. Verginis while downregulation of Bgn causes reduced MHC class and his group have analyzed the transcriptomic signatures I expression. Interestingly, there is an inverse correlation of CD4 + CD25hiCD127lo Treg from melanoma patients between Bgn and TGF-β expression, since overexpression of that developed or not irAEs after immunotherapy. The fnd- Bgn leads to downregulation of TGF-β receptors as well as ings of this approach demonstrate signifcant alterations in their ligands. Experimental in vivo data showed a signifcant the expression of infammatory genes expressed by Treg in delay in tumor growth by Bgn overexpression, which was patients with irAEs. Decoding Treg signatures in cancer will apparently caused by immune infltrates in those tumors. empower the discovery of personalized predictive biomark- The cancer genome atlas (TCGA) data analyses also con- ers and novel targeted therapy. Dimitris Kletsas (NCSR frmed the role of Bgn in the prognosis of various types “Demokritos”, Athens, Greece) introduced the concept of of cancer. Dr. Seliger also addressed the important role of cellular senescence. He summarized the mechanisms lead- microRNAs (miRNAs) as modulators of the expression of ing to replicative or stress-induced premature senescence, MHC class I APM components as well as of HLA-G, which focusing on the activation of the DNA damage response (i.e., is known to suppress T-cell and NK cell responses and to the activation of the ATM-Chk2-p53-p21WAF1-Rb axis), as a be overexpressed in various types of tumors. The miRNAs common motif in both types of senescence. At the functional identifed by combining miTRAP (miRNA trapping by RNA level, he presented data indicating the putative anticancer in vitro afnity purifcation) with RNA sequencing were role of senescence. The pro-infammatory and catabolic either downregulating or enhancing HLA class I antigens nature of these cells and their role in tissue homeostasis and or HLA-G and thus represent possible therapeutic targets. the development of age-related diseases leads to the sug- SLRPs and miRNAs emerge as new tools, which can be used gestion that the short-term presence of these cells may be by tumor cells to evade immune surveillance, but can be also benefcial in some cases (e.g., in tissue repair), while their therapeutically targeted. chronic presence may be detrimental. In particular, his group has shown that stromal fbroblasts can become senescent as Combinational treatments a result of conventional anticancer treatments (e.g., exposure to ionizing radiation) both in vitro and in vivo. These cells The hostile tumor microenvironment downregulating anti- can enhance the growth of cancer cells through increased tumor immunity and thereby minimizing the clinical efcacy secretion of soluble factors (e.g., matrix metalloproteases) of immunotherapies can now be reversed via application of or insoluble extracellular matrix components (e.g., the pro- diferent immunomodulatory approaches. Ed Lavelle (Adju- teoglycan syndecan 1), which create a permissive environ- vant Research, School of Biochemistry and Immunology ment for tumor growth. The mechanisms leading to these Trinity College, Dublin, Ireland) gave a talk on targeting alterations and especially the participation of TGF-β were strategies to enhance the efectiveness of immunotherapies. considered. Finally, he presented new means for control- He emphasized that while there is great interest in the devel- ling the presence and role of senescent cells in vivo by the opment of cancer vaccines, this is impeded by the lack of use of a new class of compounds which can lead to pref- safe and efective adjuvants that strongly promote cellular erential killing of these cells (senolytics) or to inhibition immunity. To this end, he addressed a number of promising of their infammatory phenotype (senomorphics), and dis- strategies including polymeric nanoparticles. Although the cussed the limitations in their use. Barbara Seliger (Insti- role of particle size in adjuvanticity is contested, there is tute for Medical Immunology, Martin Luther-University now ample information to show that particle size was criti- Halle-Wittenberg, Halle/Saale, Germany) overviewed other cally important in the ability of particulate adjuvants to pro- tumor immune escape mechanisms. Immune escape strate- mote antigen-specifc CD8+ T cells and Th1 responses. In gies developed by the tumors are linked with the biology contrast, a broad range of particle sizes could promote anti- of the tumor itself or occur within the TME. With respect gen-specifc antibody responses. These results indicate than to the former, various types of tumors downregulate their nanoparticles may have signifcant advantages as adjuvants

1 3 Cancer Immunology, Immunotherapy (2019) 68:1391–1400 1395 for cancer vaccines. He then described a second adjuvant efcacy also needs to be taken into consideration. This can system based on chitin-derived polymers with highly dea- be demonstrated using examples of how combinations of cetylated polymers being most efective by promoting acy- anti-PD-1/PD-L1 therapies with certain classes of agents clic GMP-AMP synthase stimulator of interferon genes (chemotherapy, VEGFR-targeted molecules) have led to (cGAS-STING) and nucleotide-binding domain leucine-rich practice-changing progress in specifc indications. Another containing (NLR) pyrin domain-containing 3 (NLRP3) approach will be to examine targeting two promising bio- infammasome-dependent Th1 response. The activation of logical targets with a single, bifunctional (bispecifc) anti- this DNA sensing pathway was proposed to be mediated body. Two clinical-stage examples (anti-PD-L1/TGF-β through induction of mitochondrial reactive oxygen species TRAP, anti-PD-L1/LAG-3) demonstrate how such bifunc- and release of mitochondrial DNA, which is sensed by tional antibodies are anticipated to provide improved clinical cGAS. These adjuvants have unique cellular immunity pro- results with less toxicity compared to combinations of indi- moting properties that are not shared with alum, the most vidual molecules. Enhanced beneft in preclinical models widely used adjuvant, which supports their further investiga- has been shown with respect to specifcity and the physical tion as candidates for inclusion in cancer vaccines. Michael proximity of the target ligands, providing novel insights on I. Koukourakis (Radiobiology and Radiopathology Unit, how synergies may be achieved compared to combinations Democritus University of Thrace, Alexandroupolis, Greece) of separate agents. Rhoda Molife (European Clinical Devel- presented data on the interplay of radiation and immuno- opment, Merck Sharp and Dohme (MSD), London, UK) therapy. The role of host immunity in the efcacy of radio- gave a talk on the novel ICIs and their combinations in geni- therapy against experimental tumors has been recognized tourinary malignancies with a focus on urothelial carcinoma for many decades. Heavily irradiated autologous cancer cells (bladder cancer). Bladder cancer is the ninth most common have been applied as vaccines for the cancer in the world with nearly half a million new cases since 1920. Indeed, irradiated cancer cells function as a vac- recorded per year. Clinically, there are three main disease cine (1) by switching on the IFN type I pathway; (2) by states: non-muscle invasive disease (70%), muscle invasive inducing secretion of cytokines and chemokines, or even (3) disease (20%) and metastatic disease (10%; de novo presen- by restoring HLA class I molecule expression by cancer tation). In 2017, the European Medicines Agency (EMA) cells. DCs are activated and, in turn, activate cytotoxic T approved three checkpoint inhibitors (CPI) as monotherapy cells in lymph nodes. T cells have a direct cytolytic efect on for both untreated and pretreated advanced/metastatic dis- the irradiated local tumor and, also, against metastatic ‘out- ease, thus providing alternate and efective treatments for a of-portal’ deposits (abscopal efects). Handling immune signifcant proportion of patients that are not suitable for checkpoint pathways with novel immunotherapies, in com- frst-line platinum-based chemotherapy, and a more efective bination with radiotherapy-induced tumor vaccination, is therapy for relapsed patients. As such, these agents are suit- expected to become a potent therapeutic tool against locally able partners for combination approaches that may provide advanced and metastatic cancer. Christoph Schultes even more efective treatment options for these patients in (Global Program Lead Oncology, Biopharma | Global R&D, the future. Several of these approaches are being tested in all Merck, Frankfurt, Germany) gave a general overview on disease states (as well as in earlier disease) in phase I to III combinations and bifunctional molecules as the next genera- trials, some of these combine CPI with: (1) chemotherapy; tion of immuno-oncology therapies. He pointed to the fact (2) other CPI; (3) anti-angiogenic agents; (4) poly (ADP- that in attempting to address the broad range of possible ribose) polymerase (PARP) inhibitors and (5) BCG. The immuno-oncology combinations and ascertaining which are results of some of these trials should be reported soon and likely to beneft patients, the industry has adopted a number are eagerly anticipated. Manuel Fernández Bruno (Institute of approaches to ensure that the most promising molecules of Oncology Rosell (IOR), University Hospital Sagrat Cor, are combined in a manner maximizing the beneft/risk ratio Quiron Salud Group, Barcelona, Spain) addressed the for cancer patients. On one hand, this comprises scientifc important topic of combinatorial treatments consisting of and clinical alliances and partnerships, in which assets from ICI and targeted therapies. The identifcation of tumor onco- diferent pipelines can be combined and trials run collabo- genic drivers and the subsequent development of targeted ratively. The selection criteria for molecules in such collabo- therapy represent a recent milestone in the treatment of lung rative ventures may vary signifcantly, though there are gen- cancer. However, ICI that are widely used for the treatment eral trends becoming established in the feld. Fundamentally, of non-small cell lung cancer (NSCLC) have yielded disap- these have to do (1) with the scientifc rationale of specifc pointing results for patients with oncogenic drivers like combinations (2) whether a synergy with anti-PD-1/PD-L1 EGFR mutations. Despite the induction of PD-L1 expression is expected, and (3) with safety aspects of the molecules to due to constitutive oncogenic signaling that has been be combined. Given recent clinical trial setbacks, the impor- reported in NSCLC models harboring microtubule-associ- tance of defining surrogates for clinical monotherapy ated protein-like 4 (EML4), anaplastic lymphoma kinase

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(ALK) rearrangements and EGFR mutations, the combina- failed to demonstrate a survival beneft. Now, for the frst tion of targeted therapies and ICI does not seem to improve time in the history of oncology, drugs were approved by the outcomes, but still have detrimental side efects. Diferent FDA in 2017 in a histology agnostic way, including larotrec- combinations of tumor protein 53 (TP53), EGFR, and serin/ tinib in neurotrophic tropomyosin receptor kinase (NTRK)- threonine kinase 11 mutations, together with PD-L1 expres- translocated cancer patients and pembrolizumab in micros- sion by tumor cells, may represent robust parameters to iden- atellite instability-high cancer patients. They encourage the tify best responders to PD-1 blockade. Elucidating the fac- use of sequencing in patients with recurrent cancer inde- tors responsible for the lack of sensitivity to CPI is a crucial pendent of tumor type, provided they would have access to issue, and novel combination approaches are urgently needed these latter drugs or to clinical trials with drugs matching in this patient population. Other approaches include adoptive molecular alterations that might be identifed. cell therapy in solid tumors, as summarized by Troels Holz Borch (Center for Cancer Immune Therapy-Herlev Hospital, Biomarkers for immunotherapy Herlev, Denmark). He emphasized that across multiple phase II trials, adoptive cell therapy using TIL has consistently Cancer alters various features over the course of its develop- shown response rates of about 40–50% with durable com- ment, generating heterogeneity which contributes to the plete responses in about 15% of treated metastatic melanoma development of resistance to therapies. Therefore, it is of patients. It has been suggested that tumors with a pre-exist- great interest to discover biomarkers to appropriately and ing immune cell infltrate respond well to immunotherapy accurately appreciate the risk of relapse at the time of diag- with ICI, whereas those without do not respond. This would nosis but also to select for patients most likely to respond to implicate that the tumors progressing on CPI should be therapies. Federica Cavallo (Department of Molecular Bio- immunologically ignorant. However, in a recently reported technology and Health Sciences, Molecular Biotechnology clinical trial, it was possible to grow tumor-reactive TIL Center, University of Turin, Turin, Italy) presented data on from 83% of CPI-resistant patients, which is comparable the specifc targeting of cancer stem cells in breast cancer as with previously published fndings. Nevertheless, of 11 a novel therapeutic modality. Breast cancer is still the lead- evaluable patients, only 2 achieved partial responses (18%) ing cause of cancer death in women, with about 30% of the of which only 1 was durable. More data are needed to eluci- afected patients dying because of metastasis and disease date whether the poor clinical outcome of this study was due recurrence. As the normal mammary gland epithelium, to the low number of patients recruited or due to shared breast cancer is hierarchically organized, with a sub-popu- resistance mechanisms with CPI treatment. For other cancer lation of cancer cells with stem cell properties (breast cancer types, the clinical results of TIL therapy has only been stem cells, BCSC) at the top of the hierarchy. BCSC are the reported in small cohorts or case reports. However, it has reservoir for the relapse, metastatic evolution and progres- been shown that growing TIL to a clinically relevant number sion of the disease. Moreover, the transient decrease in the is possible from a wide range of tumors and many trials test- extent of cancer heterogeneity induced by conventional anti- ing their clinical efficacy are ongoing. Christophe Le tumor treatments results in the enrichment of BCSC. There- Tourneau (Department of Drug Development and Innova- fore, targeting BCSC is necessary to obtain tumor eradica- tion (D3i), Institut Curie, Paris-Saclay University, INSERM tion. Her team has recently shown that BCSC overexpress U900 Research Unit, Paris, France) talked about precision xCT, a transmembrane protein that imports extracellular medicine in cancer, based on recent large clinical studies. cystine in exchange for intracellular glutamate, afording Precision medicine has been a reality for a long time in protection against harmful reactive oxygen species generated oncology. Now, the term “precision medicine” has efec- by altered metabolic states and by chemotherapeutic drugs. tively emerged with this advent of high-throughput technolo- Accordingly, the immune targeting of xCT with DNA-, gies, especially sequencing. The use of geneexpression sig- virus-like particles (VLP)-, and bovine herpex virus (BoHV) natures has been shown to be able to help prognosis, 4-based vaccines resulted in reduced tumor growth and especially in early breast cancer. Beyond refning prognosis, metastasis formation in mice and sensitization to chemo- sequencing might have a predictive value and therefore help therapy. Therefore, xCT represents a relevant target for guiding molecularly targeted therapy and immunotherapy. breast cancer treatments. In her second presentation, The- Several types of studies have tried to bring the proof of prin- resa L. Whiteside (University of Pittsburgh Cancer Insti- ciple that sequencing cancer patients might improve their tute, Pittsburgh, USA) reviewed the role of exosomes as outcome, including retrospective studies and prospective biomarkers of tumor progression and response to therapy. clinical trials. While results from retrospective and non- EV are produced by all cells and are present in all body randomized clinical trials were encouraging, no robust con- fuids. They represent a heterogeneous mix of membrane- clusions could be drawn because of methodological issues. bound nanovesicles of various sizes and diferent cellular SHIVA01 has been the only randomized trial to date and it origins. Among them, EV produced by tumor cells, referred

1 3 Cancer Immunology, Immunotherapy (2019) 68:1391–1400 1397 to as tumor-derived exosomes (TEX) have recently emerged signaling leads to rapid integrin activation through afnity as potential noninvasive biomarkers of cancer. TEX are of increase and clustering of integrins on the cell membrane. special interest as liquid biopsies, because their molecular The assay described by Dr. Gouttefangeas is simple (no and genetic profles resemble those of parent tumor cells and sophisticated reagents are needed), is combinable in multi- TEX can be viewed as clinically relevant surrogates of can- parametric fow cytometry, and preserves cell viability. She cer cells. In the tumor microenvironment, TEX carry and presented data to show that the assay is applicable for assess- deliver unique messages from the tumor to normal cells. ing a broad range of virus-, tumor- and vaccine-specifc This TEX-mediated information transfer results in repro- CD8+ T cells. Staining of activated β2-integrins presents the gramming of recipient normal cells to tumor-promoting cells unique advantage of requiring activation times of only sev- which produce their own exosomes and play an active role eral minutes, and could be rapidly implemented in the clini- in tumor progression. In body fuids of cancer patients, TEX cal setting, including for monitoring T-cell-based immuno- represent a fraction of total exosomes, and the ratio of TEX/ therapy. Aristides Eliopoulos (School of Medicine, National non-TEX varies among patients. To study the molecular pro- and Kapodistrian University of Athens, Athens, Greece) fles of TEX, it is necessary to isolate them from body fuids. presented an overview on tumor-associated and nontumor- Isolation and subsetting of TEX are accomplished by immu- associated microbiota. The microbial cosmos that colonizes noafnity capture with antibodies specifc for antigens car- mammals has major physiological roles in the development ried by TEX. Immunocapture ofers an opportunity for iden- and function of the , in host defense against tifcation of proteins, lipids, glycans, nucleic acids and other pathogens and in a plethora of metabolic processes. Imbal- molecular components that TEX carry and use to promote ances in microbiota, termed dysbiosis, have been linked to tumor progression. At the same time, non-TEX can also be various infammatory pathologies such as Clostridium dif- captured, so that the same liquid biopsy can serve as a source cile-associated diarrhea, Crohn’s disease and ulcerative coli- of TEX as well as, e.g., a source of CD3+ exosomes pro- tis, where transplantation therapies with healthy fecal micro- duced by T cells. Once isolated by immunoafnity capture, biota have demonstrated signifcant efcacy. Dysbiosis is molecular profles of TEX and non-TEX can be character- also linked to various types of cancer and experimental evi- ized by Western blot or by on-beads fow cytometry. The dence supports a role for certain microbial species or general same liquid biopsy can inform about a state of the tumor and in tumor growth and metastasis. A fascinating aspect of simultaneously evaluate the competency of immune cells for host–microbial interactions is the infuence of distinct micro- mediating anti-tumor activities. Preliminary data correlating biota clusters on the efcacy of immunotherapy. Specifc protein or miRNA profles of TEX and CD3+ exosomes clusters of ‘tolerogenic’ bacteria correlate with poor from cancer patients’ plasma with disease activity and response to both anti-CTLA-4 and anti-PD1/PD-L1 immu- responses to immune therapies suggest that these exosome notherapy in melanoma patients, whereas ‘immunogenic’ subsets serve as sensitive surrogates of clinical endpoints. bacteria appear to act in concert with ICIs to boost T-cell Anastasios Boutis (Theagenio Cancer Hospital, Thessa- responses and therapeutic efcacy. Modulation of intestinal loniki, Greece) reviewed recent advances in biomarkers for microbial composition through specifc types of diet (includ- immunotherapy beyond simple PD-L1 expression. He ing the Mediterranean diet), fecal microbiota transplantation started his talk with the comment on the landscape of or administration of synthetic stools may serve as adjuvant NSCLC management which has evolved rapidly during the to immunotherapy. Future progress in synthetic biology and last 3 years with the incorporation of ICIs in everyday clini- application of multi‐omic diagnostic approaches is antici- cal practice. However, currently, PD-L1 is the only predic- pated to assist in the identifcation and correction of micro- tive marker in use for immunotherapy, and its use is limited biota defects that compromise therapeutic efcacy. Wolf H. due to intrinsic biological complexity as well as technical Fridman (Laboratory Cancer, Immune Control and Escape, and logistical problems. He also mentioned that tumor muta- Cordeliers Research Centre, Paris, France- University Paris tional burden is the most promising evolving biomarker for Descartes, Paris, France) presented a series of previous and immunotherapy having been tested in several clinical trials most recent data from his laboratory on the immune contex- and consistently shown to correlate with clinical outcome. ture as a prognostic biomarker. In addition to the well-known However, it still lacks validation and until this has been done association between high tumoral infltration of CD8+ T it cannot be routinely implemented in clinical practice. cells and favorable prognosis, he also highlighted the impact Cécile Gouttefangeas (Department of Immunology, Inter- of tertiary lymphoid structures (TLS) as a prognosticator. faculty Institute for Cell Biology, University of Tübingen, TLS are ectopic lymphoid organs found in tumors and their Tübingen, Germany) presented a new method for monitoring microenvironment. They are composed of a T-cell zone con- antigen-specifc T cells which is based on the specifc detec- taining mature dendritic cells which surrounds a B-cell-rich tion of activated integrins using fuorescent multimers of germinal center intricated with follicular dendritic cells. T ICAM1 molecules. During T-cell stimulation, inside-out follicular helper cells are found at the interface of the T-cell

1 3 1398 Cancer Immunology, Immunotherapy (2019) 68:1391–1400 zone and the germinal center. High endothelial venules allow immunotherapy in the feld of mesenchymal tumors. Mes- the entry of T and B lymphocytes into TLS. High density of enchymal tumors comprise numerous tumor entities (> 50 TLS in the tumor core or at its close vicinity was reported to histological subtypes) including benign and malignant sub- be associated with longer patient survival in many , types. Malignant mesenchymal tumors include soft-tissue as illustrated in NSCLC, hepatocellular carcinoma and soft- and bone sarcomas, they represent 1% of adult and 20% of tissue sarcoma. In these cases, the presence of TLS is associ- pediatric cancers and no substantial improvement in overall ated with a strong infltration of memory Th1, CD8+ T survival has been achieved in the last 4 decades. Immune cells and B cells likely with anti-tumor activity. Their clini- infltrates consisting of lymphocytes and macrophages are cal impact is more signifcant than that of CD8+ T cells. In detectable in all of these tumors and the frst immunotherapy contrast, high density of TLS in infammatory regions, at in sarcoma was assessed by William Coley in 1891 by injec- distance of the tumoral nodules bears no positive clinical tion of microorganisms into the tumor mass. Soft-tissue and efcacy, such as in hepatocellular carcinoma. These observa- bone sarcomas are considered as an immune desert com- tions strengthen the fact that not only T cells, but also B pared to a large number of immune privileged cancers and lymphocytes, mature DCs and the overall organization of the this immune desert is mainly explained by a poorly charac- tumor microenvironment are major parameters in the control terized tumor microenvironment. Among the promising of tumor growth and spreading, and have to be considered therapies, targeting immune checkpoints using specifc anti- in designing immunotherapeutic approaches. Rienk bodies would be an alternate, however, the data available on Ofringa (University Hospital Heidelberg and German Can- the expression of such negative regulators are contradictory cer Research Center, Heidelberg, Germany) emphasized the in sarcomas. Cytokine-induced killer cells, oncolytic viruses importance of intratumoral heterogeneity detected via next- and autologous T cells expressing cancer testis antigens are generationsequencing of TCRs. In spite of the fact that pan- potential new therapeutic options as revealed by recent creatic cancer has long been portrayed as a ‘cold’ tumor that results of clinical trials. Even if sarcomas remain an immune cannot be targeted by means of immunotherapy, they found desert, some oasis can be now identifed. Nikolaos Pista- that most primary tumors contain signifcant numbers of maltzian (Oncology Department “MITERA” Hospital in infltrating T cells and that their TCR repertoire is highly Athens, Athens, Greece) talked about immunotherapy in enriched for certain TCR-Vα/β pairs. Moreover, ex vivo bladder (urothelial) cancer. Urothelial cancer is considered expanded T cells isolated from these tumors display HLA- among the most immunogenic cancers, carrying a high restricted activity against autologous tumor cells. They are mutational load and having a prevalence of PD-L1 positivity currently investigating the antigen specifcity of tumor-dom- between 35 and 65% in tumor samples. Apart from intravesi- inant TCRs with a focus on neo-epitopes encoded by the cal administration of BCG, no other form of immunotherapy tumor mutanome. While the outcome of these experiments had made any clinically meaningful impact until now. Dur- is still awaited, they have demonstrated in an orthotopic pan- ing the last 2 years, emerging data from large phase II and creatic cancer mouse model that the dominant TCRs target randomized phase III trials suggest that immunotherapy with neo-epitopes. In parallel, they are isolating TCRs from PD-1 inhibitors like nivolumab and pembrolizumab, and human TCR-locus-transgenic HLA-transgenic mice immu- PD-L1 inhibitors like atezolizumab, have superseded chemo- nized with candidate neo-epitopes, some of which were therapy in terms of response rates and survival. This beneft shown to selectively react against the mutated neo-epitope has been observed in both patient groups: those ineligible and not against its wild-type counterpart. Taken together, for cisplatin administration and those who have been they aim at the development of adjuvant immunotherapeutic exposed to cisplatin, and has been associated with durable strategies for countering the devastating recurrence rate in remissions. All immunotherapy drugs have also exhibited a patients with primary resectable pancreatic cancer. more favorable safety profle compared to chemotherapy. Selecting patients based on PD-L1 expression has produced Immunotherapy and clinical trials conficting results and there is an efort to develop more biomarkers based on immune gene signatures, tumor muta- A deeper understanding of the complex interactions between tion burden and tumor infltrating lymphocytes that will tumor cells and elements of the immune system has paved eventually be used complimentary to each other. There are the way for novel immunotherapy modalities. The feld has also many ongoing trials studying the combination of immu- been revolutionized by therapies based on anti-PD1, anti- notherapy with chemotherapy in the front-line setting, and PD-L1 and/or anti-CTLA-4 antibodies for several types of second-line trials studying anti PD1/PD-L1 agents in com- cancer, with impressive clinical outcomes in melanoma and bination with targeted therapies and other immunotherapies. NSCLC. Dominique Heymann (University of Nantes, Nikolaos Giannakoulas (Internal Medicine and Hematol- Nantes, France and the University of Shefeld, Shefeld, ogy Dept, Faculty of Medicine, University of Thessaly, Lar- UK) presented an overview on the therapeutic advances of issa, Greece) presented current data on the immunotherapy

1 3 Cancer Immunology, Immunotherapy (2019) 68:1391–1400 1399 in blood cancers. Hematological malignancies are a hetero- or carboplatin), 5-FU and cetuximab, (b) platinum, 5-FU and geneous group of disorders mostly of immunological origin. pembrolizumab, or (c) pembrolizumab alone. KEY- A signifcant proportion of patients with hematological NOTE-048 showed survival beneft with pembrolizumab malignancies remain with limited treatment options after monotherapy over the EXTREME regimen in PD-L1 posi- failure of chemotherapy. Allogeneic hematopoietic stem cell tive tumors. Moreover, in all patients, regardless of PD-L1 transplantation and monoclonal antibodies like rituximab expression, pembrolizumab added to platinum and 5-FU have been an essential part of the treatment of hematological resulted in longer OS versus the EXTREME regimen. This malignancies for decades. Newer anti-CD20 antibodies (ofa- important study that will be changing standard treatment tumumab and obinutuzumab) for B-cell malignancies, anti- paradigms awaits fnal analysis and publication. Nivolumab CD38 (daratumumab) and anti-SLAM (elotuzumab) for has been under further investigation in combination with the MM, antibody–drug conjugates like gemtuzumab ozo- CTLA-4 inhibitor ipilimumab in the frst-line therapy set- gamicin (GO) for acute myelogenous leukemia, brentuximab ting. CheckMate 651 is a phase III trial in the frst-line treat- vedotin for Hodgkin’s lymphoma and CD30+ anaplastic ment of recurrent or metastatic HNSCC in which patients T-cell lymphomas, bispecifc antibodies like blinatumomab are randomized to receive either the standard of care for B-cell acute lymphoblastic leukemia have been incorpo- EXTREME regimen or the combination of nivolumab and rated in the treatment of some hematological malignancies. ipilimumab. This study recently completed accrual and Cellular adoptive immunotherapies utilizing T cells geneti- results are pending. Finally, accumulating evidence suggests cally engineered to express chimeric antigen receptors or that the ICIs can work synergistically with radiation. Several T-cell receptors for tumor-associated antigens or neoantigens phase III clinical trials are investigating ICIs combined with have yielded impressive clinical results mostly in hemato- for the treatment of locally advanced logical cancers and in certain solid tumors. Inhibiting HNSCC. Combination approaches and the study of predic- immune checkpoints such as programmed death-1 (PD-1) tive biomarkers are of major interest in future development and its ligand (PD-L1) have yielded remarkable results in of immunotherapy strategies in HNSCC. Monica Neagu some types of solid tumors and recently in some highly (“Victor Babes” National Institute of Pathology, Bucharest, refractory lymphoma subtypes such as relapsed classical Romania) presented current data on immunotherapy for Hodgkin’s lymphoma. Immunotherapy is a pillar of manage- melanoma. With the advent of the new era of immune ther- ment for hematological malignancies and is likely to cure a apy, new rapidly emerging aspects are highlighted from both subset of patients previously considered incurable. Obtain- fundamental research and clinical applications. Actual trends ing maximal beneft with minimal toxicity from all these in melanoma therapy were presented, highlighting the most new immunotherapies remains a challenge. Athanassios recent ones. Therefore, one of the most important points in Argiris (Department of Medical Oncology, Sidney Kimmel melanoma immunotherapy is that, due to the approval of Medical College at Thomas Jeferson University, Philadel- several immune-related therapies, clinical experience is phia, USA) demonstrated the impact of immunotherapy on gained in terms of adverse efects types, their management, head and neck cancer. A major breakthrough in the manage- acquired resistance, modalities to overcome resistance, adju- ment of head and neck squamous cell carcinoma (HNSCC) vant therapies and, prognosis/efcacy biomarkers. Another has been the introduction of ICIs. Currently, pembrolizumab recent trend in this domain is combining newly approved and nivolumab, two agents that target PD-1, are approved in immune-related therapies, looking for the best time frame, both the US and Europe for the treatment of patients with drug(s) concentration and stratifying patients for the combi- recurrent or metastatic HNSCC with disease progression on nation. The limitation of the currently approved drugs has or after platinum-containing therapy (i.e., in second-line triggered the design of new ones like the T-VEC oncolytic therapy and beyond). Pembrolizumab and nivolumab have therapy that was presented. Newly discovered biomarkers or been shown to confer survival advantage over standard ther- combination of biomarkers that can better predict immune- apies (i.e., docetaxel, methotrexate, cetuximab) in two phase related therapy efcacy were also presented. Anna Kou- III clinical trials in platinum refractory HNSCC, KEY- marianou (Fourth Department of Internal Medicine, NOTE-040 and CheckMate 141, respectively. PD-L1 inhibi- Attikon University Hospital, Haidari, Athens, Greece) gave tors, such as durvalumab and avelumab, are being tested in a general overview on immunotherapy and the management clinical trials in HNSCC as well. A further step in develop- of toxicities. Dr. Koumarianou underlined that immuno- ment was the investigation of ICIs either as monotherapy or therapy has recently been added in the therapeutic armamen- in combination regimens in the frst-line treatment of recur- tarium of a large number of cancer clinical trials. Among the rent or metastatic HNSCC (i.e., chemo-naive or platinum- tested immunotherapeutic approaches, immune checkpoint sensitive setting). In the three-arm phase III KEYNOTE-048 blockade has shown remarkable benefit. However, by trial, patients were randomized to receive either (a) the increasing the activity of immune cells, immune checkpoint EXTREME regimen which consists of platinum (cisplatin blockade has been associated with infammatory side efects,

1 3 1400 Cancer Immunology, Immunotherapy (2019) 68:1391–1400 termed irAEs. Novel advances in the technology of mono- and for the selection of patients most likely to respond to clonal antibodies targeting immune checkpoints are expected immune- and targeted therapies, thus opening the way for the to associate with fewer side efects. Although any organ sys- application of precision medicine in cancer immunotherapy. tem can beaficted, most common irAEs involve the mus- Furthermore, the role of exosomes, TLS, microRNAs and culoskeletal, gastrointestinal and endocrine systems, the extracellular matrix molecules as biomarkers and/or novel skin, and the liver. Other systems less often involved are the targets for immunotherapeutic approaches were strongly cardiovascular, pulmonary, hematologic and the central emphasized in this conference. nervous system. Most of the adverse events are reversible and responsive to corticosteroid treatment, except from those affecting the endocrine system that are permanent and Awards require lifelong hormone replacement therapy. Albeit deaths due to immune-related side efects resulting in severe myo- Three poster prizes were awarded to Aikaterini Hatzioan- carditis, pneumonitis, colitis are exceptionally rare, physi- nou (Center of Clinical, Experimental Surgery and Trans- cians have to pay particular attention and provide the sup- lational Research, Biomedical Research Foundation Acad- portive coverage including expert multidisciplinary team emy of Athens, Athens, Greece), Sahitya Saka (Institute when is required. for Molecular Oncology and Experimental Therapeutics, Marien Hospital Herne, Ruhr University Bochum, Medi- cal School, Herne, Germany) and Eleonora Vecchio (Uni- Conclusions and perspectives versity of Catanzaro, Experimental and Clinical Medicine, Catanzaro, Italy). The speakers in this conference addressed many difer- ent aspects of immune regulatory pathways in anti-tumor Acknowledgements We would like to thank all speakers and the attendees for their participation. immunity including the contribution of oncogenes, tumor- suppressor genes and immune genes. The understanding of Author contributions SPF, AK, and CNB wrote the manuscript. CLT, such pathways which may lead either to tumor destruction OET, VG, and CNB edited the manuscript. or tumor escape from immune surveillance clearly provide novel information for the design of improved immunothera- Funding The conference was supported by Bristol-Myers Squibb, peutic modalities. From the presentations and the discus- Novartis, Roche, Roche Diagnostics, Merck Sharp and Dohme Corp (MSD), Amgen, AstraZeneca, Lilly, Aenorasis, Boehringer-Ingelheim, sions which followed the presentations, it also became clear DEMO S.A. Pharmaceutical Industry, Genesis, Merck, Pfzer, Pierre that although immunotherapy has attracted interest as an Fabre, Sanof Genzyme and Specifar. efective cancer treatment, response rates are still low due to immune resistance developed by the tumor cells. Therefore, Compliance with ethical standards to overcome resistance and in this way achieve long-lasting clinical responses, it will be of paramount importance to Conflict of interest The authors declare that they have no confict of better understand (1) the dynamic interactions of immune interest. infltrates within the tumor microenvironment; (2) the role of tumor stroma in regulating intratumoral anti-tumor reactiv- ity; and (3) alterations in tumor phenotype and tumor biol- Publisher’s Note Springer Nature remains neutral with regard to ogy which may generate tumor heterogeneity, and tumor- jurisdictional claims in published maps and institutional afliations. induced suppression. In-depth knowledge of these aspects will lead to the discovery of biomarkers to predict clinical outcomes which will also be valuable for immunomonitoring

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