Ascierto et al. J Transl Med (2017) 15:236 DOI 10.1186/s12967-017-1341-2 Journal of Translational Medicine

MEETING REPORT Open Access Future perspectives in melanoma research “Melanoma Bridge”, Napoli, November 30th–3rd December 2016 Paolo A. Ascierto1,27*, Sanjiv S. Agarwala2, Gennaro Ciliberto3, Sandra Demaria4, Reinhard Dummer5, Connie P. M. Duong6, Soldano Ferrone7, Silvia C. Formenti8, Claus Garbe9, Ruth Halaban10, Samir Khleif11, Jason J. Luke12, Lluis M. Mir13, Willem W. Overwijk14, Michael Postow15,16, Igor Puzanov17, Paul Sondel18,19, Janis M. Taube20, Per Thor Straten21,22, David F. Stroncek23, Jennifer A. Wargo24, Hassane Zarour25 and Magdalena Thurin26

Abstract Major advances have been made in the treatment of cancer with targeted therapy and immunotherapy; several FDA-approved agents with associated improvement of 1-year survival rates became available for stage IV melanoma patients. Before 2010, the 1-year survival were quite low, at 30%; in 2011, the rise to nearly 50% in the setting of treat- ment with Ipilimumab, and rise to 70% with BRAF inhibitor monotherapy in 2013 was observed. Even more impres- sive are 1-year survival rates considering combination strategies with both targeted therapy and immunotherapy, now exceeding 80%. Can we improve response rates even further, and bring these therapies to more patients? In fact, despite these advances, responses are heterogeneous and are not always durable. There is a critical need to better understand who will beneft from therapy, as well as proper timing, sequence and combination of diferent therapeu- tic agents. How can we better understand responses to therapy and optimize treatment regimens? The key to better understanding therapy and to optimizing responses is with insights gained from responses to targeted therapy and immunotherapy through translational research in human samples. Combination therapies including chemotherapy, radiotherapy, targeted therapy, electrochemotherapy with immunotherapy agents such as Immune Checkpoint Blockers are under investigation but there is much room for improvement. Adoptive T cell therapy including tumor infltrating lymphocytes and chimeric antigen receptor modifed T cells therapy is also efcacious in metastatic mela- noma and outcome enhancement seem likely by improved homing capacity of chemokine receptor transduced T cells. Tumor infltrating lymphocytes therapy is also efcacious in metastatic melanoma and outcome enhancement seem likely by improved homing capacity of chemokine receptor transduced T cells. Understanding the mechanisms behind the development of acquired resistance and tests for biomarkers for treatment decisions are also under study and will ofer new opportunities for more efcient combination therapies. Knowledge of immunologic features of the tumor microenvironment associated with response and resistance will improve the identifcation of patients who will derive the most beneft from monotherapy and might reveal additional immunologic determinants that could be targeted in combination with checkpoint blockade. The future of advanced melanoma needs to involve education and trials, biobanks with a focus on primary tumors, bioinformatics and empowerment of patients and clinicians. Keywords: Melanoma, Immunotherapy, Cancer, Checkpoint blockade updates, Combination therapies, Biomarkers

*Correspondence: [email protected]; [email protected] 27 Istituto Nazionale Tumori di Napoli Fondazione “G. Pascale”, Via Mariano Semmola, 80131 Naples, Italy Full list of author information is available at the end of the article

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ascierto et al. J Transl Med (2017) 15:236 Page 2 of 31

Evolving topics in cancer immunotherapy RT induces cancer cell-intrinsic Interferon type I (IFN- Radiotherapy and immunotherapy I) pathway activation and production of IFNβ, which is Te frst evidence that T cells contribute to the local (and required for optimal recruitment to the tumor of BATF3- possibly systemic) response to tumor-targeted radio- dependent dendritic cells. Cancer cell expression of the therapy (RT) was published over 30 years ago [1], but cytoplasmic DNA sensor cGAS and its adaptor STING as only recently it was demonstrated in pre-clinical can- are required for RT-induced IFN-I production. Induc- cer models that the abscopal efect (i.e., tumor regres- ible knockdown of cGAS or STING in the irradiated sion outside of the feld of radiation following RT to one tumor abrogated the induction of anti-tumor CD8 T cells lesion) is immune-mediated [2]. Progress in understand- by 8GyX3 RT and CTLA-4 blockade, and abolished the ing the complex molecular mechanisms that regulate occurrence of abscopal efects. Finally, since cGAS is a T cell activation, migration to tumor site and efector sensor for double-stranded (ds) DNA, we investigated its functions within tumors has led to the identifcation of presence in the cytoplasmic fraction of cancer cells irra- several mechanisms that prevent immune-mediated diated with 8GyX3 or 20 Gy. Tese experiments revealed tumor rejection in most patients. Some of these mecha- that RT induces the accumulation of dsDNA only in can- nisms have been successfully targeted therapeutically by cer cells treated with 8GyX3 but not 20 Gy [10]. In con- using antibodies blocking inhibitory immune checkpoint clusion, the radiation dose and fractionation is a critical receptors such as cytotoxic T-lymphocyte associated determinant of RT synergy with ICBs. Stimulation of protein 4 (CTLA-4) (e.g., ipilimumab) and programmed cancer cell-intrinsic IFNβ production by RT is required death 1 (PD-1) (e.g., nivolumab, pembrolizumab, atezoli- to prime anti-tumor CD8 T cells to poorly immunogenic zumab) [3]. tumors. Tese fndings have important implications for However, most patients do not respond to single agent’s the choice of RT dose and fractionation when used in therapy with immune checkpoint blockers (ICB). In this combination with ICBs. context, RT has been demonstrated to be a good combi- Immunocytokines (ICs) are a class of molecules cre- nation partner for ICBs that increases responses against ated by linking tumor-reactive monoclonal antibodies poorly immunogenic tumors in mice [4], and sometimes (mAbs) to cytokines that are able to activate immune in patients [5, 6]. Proof-of-principle studies have demon- cells. Tumor selective localization is provided by the abil- strated that RT can contribute at three levels to immune- ity of the mAb component to bind to molecules found on mediated tumor rejection: (1) by generating anti-tumor T the tumor cell surface and molecules found selectively in cells; (2) by overcoming T cell exclusion from the tumor; the tumor microenvironment. In this way, the cytokine (3) by enhancing T cell-mediated recognition and kill- component of the immunocytokine is selectively local- ing of cancer cells that survive RT [7]. Tus, RT is under ized to sites of the tumor and can activate immune cells investigation as a modifer of the tumor microenviron- with appropriate receptors for the cytokine. ment shifting so called “cold” tumors, which lack immune It has been previously shown that an intratumoral infltrate (refractory to ICB), to so called “hot” tumors (IT) injection of IC, which consists of an antitumor Ab with lymphocyte infltrate present (responsive to ICB). specifc to disialoganglioside (GD2) linked to interleu- Despite this experimental evidence, the promise of kin (IL)-2, can serve as an in situ vaccine. It enhances RT to convert the irradiated tumor into an in situ vac- local antitumor efects and can generate an adaptive cine and elicit systemic anti-tumor immune responses T cell response directed against distant tumors. Tese capable of mediating abscopal efects (i.e., regression in situ vaccine efects involve T cells as well as NK cells, of metastases outside of the feld of radiation) remains and can result in T cell memory in melanoma and neu- elusive. A number of active clinical trials combining RT roblastoma (NBL) as demonstrated in preclinical stud- with ICBs are ongoing: 18 trials testing radiotherapy with ies. Preclinical studies have shown that tumor-reactive anti-CTLA-4, 51 trials testing radiotherapy with anti- mAbs can mediate in vitro tumor destruction via anti- PD-1/PD-L1, with radiotherapy regimens varying from body-dependent cellular cytotoxicity (ADCC). Based 1.8 GyX28, to 3 GyX10, to stereotactic radiosurgery with on these results clinical approach was developed at the single doses of 20 Gy or more [8]. Children’s Oncology Group (COG) administering this Our group has investigated the impact of RT dose and agent in individuals with smaller burden of cancer (non- fractionation on the ability of RT to synergize with ICBs bulky disease) [11]. Tis approach was tested clinically in and induce abscopal efects. We found that a hypofrac- the minimal disease setting in a pilot COG phase 1 trial tionated regimen of 8GyX3 was efective while a single for children with high risk NBL that were in remission dose of 20 Gy was not [9]. In addition, we recently identi- after autologous hematopoietic stem cell transplantation fed the mechanism underlying this diference in immu- (HSCT) (ASCT) but likely to relapse. To augment ADCC nogenicity of diferent RT regimens. Hypo-fractionated cytokines such as IL-2 to activate natural killer (NK) cells Ascierto et al. J Transl Med (2017) 15:236 Page 3 of 31

and granulocyte–macrophage colony stimulating fac- Tis suggests that in situ vaccination efect can be tor (GM-CSF) to activate neutrophils/macrophages were enhanced by T cell checkpoint blockade, with implica- incorporated. When this same regimen was moved into a tions for clinical evaluation. Preclinical data demonstrate large COG phase 3 trial with immunotherapy the treat- that IT-IC + RT (and anti-CTLA-4) activates innate and ment was statistically superior to the control treatment adaptive immunity, overcomes tumor induced immune for both event-free survival (66% vs. 46% p = 0.01), and suppression, and facilitates use of existing tumors as for overall survival (86% vs. 75% p = 0.02). Tese data in situ vaccines. suggest that other ADCC-mediating mAbs (i.e., rituxi- Te following data support the strategy of increasing mab, herceptin and erbitux) might be considered for tri- the efcacy of the IC vaccine via IT delivery: (1) IT-IC als in which high risk patients likely to relapse receive causes much higher levels of IC in the injected tumor these mAbs in combination with agents known to acti- than IV-IC; (2) Greater IC levels in the tumor enhance vate ADCC (e.g., IL-2 + GM-CSF). NK infltration into the tumor (via FcRs and IL-2Rs), Because the efcacy of anti-GD2 mAb + cytokines in leading to greater ADCC and greater tumor destruction, NBL trial was only 66% and NBL-free survival at 2 years even of larger macroscopic lesions that are unresponsive further enhancement of the clinical potency of ADCC to to IV IC delivery; (3) Some of the IC injected IT circu- obtain even better clinical results was undertaken [11]. lates systemically (via lymphatics and blood vessels), ena- Te ICs were constructed by fusing the human IL-2 gene bling IC delivery to distant sites as efectively as when IC to the chimeric (ch) 14.18 or humanized (hu) 14.18 IgG1 is given IV (possibly with a better PK profle); (4) Te IC- genes to activate IL-2 receptor positive (IL-2R+) efec- facilitated response within the tumor may attract other tor cells with a molecule that bridges them to tumor cells efector cells (T cells and macrophages) to the tumor and then activates them. Indeed it was shown that these site (or to draining lymph nodes), leading to T cell sen- ICs activate GD2-specifc tumor cell binding by IL-2R+ sitization; (5) Te vaccine-like efect resulting in tumor- T cells and NK cells [11]. Ch14.18-IL-2 induces anti-mel- specifc T cell reactivity may impact on distant sites of anoma activity in a SCID-xenograft model and in con- micrometastatic disease (and prevent subsequent growth ventional mice bearing syngeneic tumors expressing GD2 upon experimental tumor re-challenge); and (6) Combin- (B78 melanoma); and anti-NBL activity in conventional ing IC with other treatments that cause localized tumor mice bearing the GD2+ NXS2 NBL [11]. However, when damage (without local immune suppression), should syn- IC (hu14.18-IL-2) was used clinically, anti-tumor activity ergistically augment the antitumor activity of the IC. is accompanied by dose-limiting IL-2-related toxicities suggesting the need to design the reagents and mouse Cancer vaccines in the era of checkpoint blockade models that better simulate the potential activity of IL- A cancer vaccine is a preparation of a tumor antigen 2-based on in vivo immunotherapy in patients [12]. that upon administration stimulates antibody produc- As a single-agent immunotherapeutic approaches can tion or cellular anti-tumor immunity. In fact, it is known have limited efcacy, combining two or more immu- that T cells can mediate remarkable tumor regressions notherapeutic strategies can be synergistic in induc- including complete cure in patients with metastatic can- ing antitumor efects. Tere is a growing enthusiasm cer. Te mutanome, the collective genetic alterations in for testing checkpoint blockade in combination with an individual’s cancer cells, encodes peptides (M-pep- other approaches to augment immune-mediated anti- tides) that can function as unique therapeutic targets as tumor efects. Recently synergistic efect of the combi- neoantigens. nation of anti-CTLA-4 mAb blockage and intratumor M-peptides in individual patients can be identifed by (IT) administration of the IC on smaller tumors (day-7 next-generation sequencing and computational algo- B78, < 50 mm3) was demonstrated although the treat- rithms guided approaches for T cell epitope prediction. ment was less efcacious on larger tumors (day 12, B78 Although there is a correlation between peptide bind- tumors) [13]. Recent studies of mAb/cytokine-based ing afnity for MHC class I and II and immunogenic- immunotherapies for solid tumors have shown IT-IC is ity, other factors also contribute. For example, sufcient more efective for measurable mouse tumors [13, 14] and afnity of the interaction between MHC-bound mutated that IV treatment (mAb or IC) can be efective in mini- peptide and the T-cell receptor (TCR) is essential for the mal residual disease (MRD) setting (COG studies) [15]. recognition of the mutated peptide as ‘foreign’. Te iden- However, combination with immunomodulatory, tifcation of epitopes that drive the immune response in radiation and IT hu14.18-IL-2 administration results in cancer is essential to the understanding and manipula- cure of most large tumors (5-week, 200 mm3, B78) [14]. tion of CD8 T-cell immune responses for clinical beneft. Results show that combining RT and IT-IC in murine Recent studies in mice and humans have suggested that tumor models can eradicate large tumors and metastases. tumor-specifc mutations may have a key role in shaping Ascierto et al. J Transl Med (2017) 15:236 Page 4 of 31

the anti-tumor response; but their identifcation remains synergizes with anti-CTLA-4 therapy and water-based a challenge [16]. To be fully useful in the clinic, it will be peptide vaccination that synergizes with anti-PD-1 ther- necessary to rely on computational predictions, including apy (Hailemichael et al., in press). Given that the adju- structural features of the MHC I-haplotype-specifc and vant choice determines T cell response to cancer vaccine, whole-exome/transcriptome sequencing of a patient’s molecular adjuvants like TLR agonist, CD40 agonists or tumor, which is beginning to be routinely determined. cytokine are investigated. In 2011, Schwartzentruber et al. found that combining IT immunotherapy can be an alternative to inefcient a melanoma vaccine with IL-2 [high-dose IL-2 ± gp100 systemic cancer vaccines. Such therapy could empower peptide in incomplete freund adjuvant (IFA), i.e., water- the immune system to mount T cell responses against in-oil emulsion], improved the response rate and progres- various immunogenic tumor-associated antigens. To sion-free survival in patients with advanced melanoma, mediate systemic tumor regression, intratumoral (IT) versus IL-2 alone [17]. Currently there are 369 open stud- immunotherapy must generate systemic T cell responses ies using cancer vaccines in the USA only, most with lit- that can target distant metastases beyond the initially tle or no evidence of tumor regression. Limited tumor treated tumor mass [21]. regression following treatment with vaccines could also Intratumoral (IT) treatment with 3M-052 (an inject- be due to immunosuppressive tumor microenvironment able, tissue-retained TLR 7/8 agonist) was found to be even in the presence of increased frequency of cancer- a promising approach for the treatment of cancer thus specifc T cells. Tese T cells in the tumor microenvi- establishing a rational strategy for combination therapy ronment are likely those that are not proinfammatory, with IT, tissue-retained TLR7/8 agonist and checkpoint or with poor T cell efector function/wrong phenotype. blockade in metastatic cancer [22]. IT administration of Other tumor phenotypes that account for most of nonre- 3M-052 generated systemic antitumor immunity, and sponders are due to few T cells or poor T cell trafcking sensitized both injected an uninjected wild-type B16.F10 to tumor. Insufcient spontaneous T cell reactivity and/ melanomas to checkpoint blockade therapy with anti- or lacking immune cell infltration to tumor site could be CTLA-4 and anti-PD-L1 antibodies, even when check- one of the limitations of efective anti-tumor response point blockade alone was inefective [22]. [18]. Such tumor-specifc T cell responses could be In conclusion, cancer vaccines can have clinical impact induced through anti-cancer vaccination, but despite by synergizing with checkpoint blockade. To induce bet- great success in animal models, only a few of many cancer ter T cell responses and clinical impact, relevant factors vaccine trials have demonstrated robust clinical beneft. are formulation (linked to possible T cell sequestration), As vaccine adjuvants determine the type and magni- addition of multiple immunomodulators (cytokines, TLR tude of the T cell response after vaccination one possible agonists), combination with checkpoint blockade and explanation for the lack of efcacy of vaccine therapy in use of intratumoral immunotherapy as a vaccine strategy humans is the use of safe, but very weak vaccine adju- need to be considered. vants in clinical trials as opposed to the use of potent, efective vaccine adjuvants in animal models [18]. Vac- Tumor infltrating lymphocytes (TIL) therapy in melanoma cine adjuvants for peptide-based cancer vaccines can Adoptive T cell therapy (ACT) with autologous tumor function as an antigen depot for prolonged release, can infltrating lymphocytes (TILs) is an efective treatment protect antigen from degradation, can increase antigen for patients with metastatic melanoma. TILs that are uptake by antigen presenting cells (APCs) or can induce a expanded in vitro, and reinfused in conjunction with pro-infammatory/pro-immunogenic milieu. IL-2 following a lymphodepletion has been a method Persisting peptide/IFA vaccine depots can induce spe- for treatment of heavily pretreated cancer patients [23]. cifc T cell sequestration, dysfunction and deletion at ATC treatment shows objective responses in about vaccination sites, possibly explaining lack of synergy 50% of patients in clinical trials, with a 20% of complete between gp100 peptide vaccination and ipilimumab in responses. TILs are mainly CD T-cell-based cultures and patients with melanoma [19]. On the contrary, short- better quality of TILs are associated with better clinical lived formulations can overcome these limitations and outcomes including overall survival. Higher T cell infl- result in greater therapeutic efcacy of peptide-based trate in tumors could be accomplished by vaccination, cancer vaccines [20]. target therapy (e.g., BRAF) and the exercise. Recently, Hailemichael et al. demonstrated that IFA- However, TIL therapy infusion products lack strong based vaccination does not synergize with anti-CTLA-4 predictive markers and the limitation of the optimal therapy and that IFA-based vaccination sequesters T responses include inability of T cells to infltrate the cells induced by anti-CTLA-4 therapy. More promising tumor site, immunosuppressive environment, and the results were obtained from virus-based vaccination that quality and quantity of TILs. Eforts are ongoing to Ascierto et al. 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generate more potent TILs or to improve intratumoral In conclusion, exercise, may lead to mobilization and infltration in patients including co-stimulation through tumor infltration of T and NK cells animal models. the 4-1BB/CD137 antibody which increases the CD8+ T Although the mechanism of increase in physical activ- cells frequency. Te use of K562− derived artifcial anti- ity on survival and recurrence in cancer patients are gen presenting cells (APCs), which act as a feeder cells not established as the randomized controlled trials are for T-lymphocytes expansion is also used. Among meth- needed to generate defnite data. However, it can have ods to improve adoptive T-cell therapy, the administra- positive impact on efcacy of immune therapy, includ- tion of checkpoint inhibitory antibodies activating T cells ing ACT such as TILs or other types of ACT (e.g., CARs, or depletion of myeloid derived suppressor cells (MDSC) genetically modifed T cells etc.) as demonstrated in ani- to reduce immunosuppression are proposed. Another mal models. method is re-stimulation of the injected TILs with a tumor vaccine to improve life-span of the antigen specifc System biology session: molecular T cells. Other strategies to counterbalance tumor-driven MicroRNAs and drug resistance to melanoma immune dysfunction are reversing the oxidative stress in Drug resistance is major issue in medical oncology cancer patients by the administration of histamine and because its development limits the long-term efcacy of antioxidants (e.g., vitamin E). current cancer therapies. Understanding the mechanisms Exercise can exert a great anticancer efect [24–26]. behind the development of acquired resistance will ofer Regular exercise delay progression and growth of tumors new opportunities for more efcient combination thera- in numerous experimental models [27, 28]. Furthermore, pies. Some concepts have been demonstrated. therapeutic efect of exercise on cancer is demonstrated Melanoma patients bearing BRAF V600 mutation ben- in cancer patients, for example decreased tumor progres- eft from therapy with BRAF inhibitors [30]. Short term sion in prostate cancer [26]. Multiple studies (n = 88) (6 months) beneft of BRAFi therapy was demonstrated reported on associations between physical activity after [31]. A major issue in the treatment of BRAF mutated diagnosis and prognosis among cancer survivors showing metastatic melanoma is the disease relapse caused by an impact of exercise on cancer survival. Unfortunately, emergence of drug resistance. In most cases BRAFi there are diferences in the models and the type of physi- resistant melanoma bear mutations or molecular aber- cal intervention which make difcult comparisons with rations reactivating the MAPK pathway [32]. Frequent cancer survival. Mechanism(s) for the efect of physical reactivation of MEK in BRAFi resistant tumors led to the activity in cancer remain unknown although dietary and development of BRAFi + MEKi combination therapies. hormonal factors have been postulated (e.g., insulin and Combination therapy with BRAF and MEK inhibitors insulin-like growth factor, infammatory markers). improves survival but is unable to prevent disease relapse Recent fndings demonstrated that voluntary wheel [33]. In tumors resistant to BRAFi + MEKi dual therapy running signifcantly reduces tumor incidence and the same type mutations are found. Terefore, hitting growth in various experimental tumor models [29]. hard two diferent targets in the same pathway doesn’t Exercise, cancer, and immunity are linked as exercise solve the problem and the identifcation of additional decreases tumor incidence and growth by over 60% mechanisms responsible for drug resistance is an unmet across several mouse tumor models. Exercise control of need. tumor growth is mediated through a direct regulation of In a signifcant percent of cases (26%) no new muta- NK cell mobilization and trafcking. Mechanisms involve tions that constitute the basis of resistance could be iden- epinephrine-dependent mobilization of NK cells to the tifed. Other types of molecular changes than mutations circulation and IL-6-dependent redistribution to the as adaptive mechanisms can contribute to drug resistance tumors. In particular, exercise increases NK cell infltra- in melanoma [34, 35]. MicroRNAs that are important tion into tumor site, thereby controlling tumor growth. multifunctional post-transcriptional modulators of gene Epinephrine mobilizes NK cells and blunts the tumor expression afect multitude of cellular pathways. Micro- suppression. In addition, exercise-induced muscle- RNAs also play a key-role in various progression-related derived IL-6 is involved in NK cell redistribution [29]. and invasive properties of human cancers. In addition, For example, subcutaneous B16F10 melanoma model in there is a growing evidences suggesting miRNAs as key female mice demonstrated that 4 weeks of wheel running factors controlling the emergence of drug resistance [36]. prior to tumor cell inoculation reduced tumor growth MicroRNAs (miRNAs) are small noncoding RNAs that by 61% (p < 0.01). Tese fndings indicate that exercise modulate gene expression by mRNA silencing or degra- might deliver a therapeutic efect with increased immune dation, which usually have pleiotropic efects because of cell infltration and generation of an infammatory intra- their ability to target simultaneously multiple mRNAs. tumoral environment [29]. Te frst example of a miRNA-dependent mechanism of Ascierto et al. J Transl Med (2017) 15:236 Page 6 of 31

drug resistance in BRAF mutated melanoma focused on the problem is the use MelArray (Melanoma Targeted a previously poorly characterized miRNA, miR-579-3p. Sequencing) designed by Dr. Michael Krauthammer from Main fndings were: (1) low expression of miR-579-3p is Yale University in collaboration with Dummer Reinhard a negative prognostic factor correlating with poor sur- and Levesque at Dermatology, University of Zurich. Tis vival; (2) expression levels of miR-579-3p decrease dur- array is composed of 190 melanoma mutant genes, TERT ing melanoma progression i.e., from nevi to stage III/ promoter, and 28 introns across eight genes to identify IV melanoma; (3) miR-579-3p acts as oncosuppressor fusion genes such as BRAF, RAF1, ALK, MAP3K8, MET, by targeting the 3′ untranslated region (3′UTR) of two NTRK1, PRKAR1A, and ROS1. Te array has reasonable oncoproteins (BRAF and an E3 ubiquitin protein ligase, sequencing costs; currently about fve time lower than MDM2); (4) moreover miR-579-3p ectopic expression whole exome sequencing. impairs the establishment of drug resistance in human A group of investigators at Yale University tested the melanoma cells; and (5) miR-579-3p is strongly down- MelArray on several tumors and identifed the criti- regulated in matched tumor samples from patients before cal mutations and genomic aberrations. An interesting and after the development of resistance to targeted thera- case was the discovery of the molecular basis of tumor pies [37] and was also identifed in cell lines resistant to heterogeneity in one patient. Te four portions of the BRAF/MEK inhibitors. tumor, all isolated at the same time, displayed ­NRASQ61R, Recently, the assessment of changes in the whole miR- ­IDH1R132C, ­DDX3XP274L, ASPM­ R1763K and TERT 5′Flank NAome profle during the development of drug resist- SNV, but were diferent in CTNNB1 mutations. We iden- ance in vitro in two diferent BRAF-mutated melanoma tifed ­CTNNB1S45Y, ­CTNNB1S45*, and wild-type gene in cell lines was performed by the Nanostring™ platform two other sites. Interestingly, the DDX3X, (DEAD-box (Fattore et al., unpublished). Data revealed a stepwise helicase 3, X-linked) might render the tumor susceptible deregulation of a growing number of miRNAs. Deregu- to RK-33 [38]. However, it is likely that we need to con- lated miRNAs of resistant melanoma cells mostly impact tinue performing WES to fnd out the total number of on pro-infammatory and pro-angiogenetic pathways. mutations and the neoantigens. Besides, conditioned medium from drug resistant Other discoveries are fusion genes in wild type WM266 cells triggers directional cell migration, pro- BRAF and NRAS melanomas, which include PDE8A- motes angiogenesis, and its efect is fully inhibited by RAF1, PDE4DIP-BRAF and NFIA-BRAF. We dem- VEGF antagonists (Fattore et al. unpublished). Per other onstrated that PDE8A-RAF1 is a transforming gene fndings, anti-PD-1 resistant tumors display a transcrip- that activates the MAPK and confers growth factor tional signature (IPRES) resembling that of MAPK-resist- independence in mouse melanocytes. Melanoma cells ant melanomas which is at the basis of cross-resistance. If with PDE8A-RAF1 or PDE4DIP-BRAF were resist- a similar network of miRNAs contributes to cross-resist- ant to vemurafenib but highly sensitive to selumetinib ance between MAPKi and checkpoint inhibitors is still to and SCH772984 (ATP independent MEK1, 2 and ERK be investigated. inhibitors, respectively). Because molecular analyses generate massive amounts Melanoma mutations and ‘precision medicine’ of data, there is a need to partner with computational Te notion of “precision medicine” is to include molec- biology cores. For example, converting the variant call ular markers based on information derived from format (VCF) to actual mutations can be a long pro- genetics, epigenetics, gene expression and proteom- cess. We tested the IBM Watson Genomics supports and ics data to diagnose and treat patients. Tis subject received within minutes the genetic alterations of the is not new as refected by over 900 publications since tumor as well as options for targeted therapy. 2009. Indeed, several institutions already searched for In addition to mutations and genomic aberrations, we a panel of “actionable” mutations (in addition to BRAF need to analyze gene expression that can help in pre- and NRAS), for targeted therapy. Tese include MSK- dicting response to therapy, especially immunotherapy. IMPACT, FoundationOne, and IBM Watson Genomics. RNA sequencing of bulk tumors can provide information Ideally, NextGen Sequencing will be used in the near regarding the composition of TIL subsets in the tumor future to distinguish primary melanomas from benign microenvironment including IFNγ and IFN pathway nevi, to identify tumor heterogeneity, new targets for genes, neoantigen load, and expression of PD-L1 in the therapy, interactions of tumor cells with the microen- tumor tissue and micro-environment. Several programs vironment, the presence of neoantigens, and to moni- were developed to profle leukocyte composition directly tor tumor load, just to name a few. However, the main from RNA sequencing data, and one of them is CIBER- obstacles to achieve these goals are speed of getting the SORT (cell type identifcation by estimating relative sub- results and the high cost of the tests. One way to alleviate sets of known RNA Transcripts) [39]. Ascierto et al. 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In conclusion, a list of ‘omics’ and functional screening become available. For example, the Foundation of Medi- test should include but is not limited to: cine T5 Gene Panel allows Targeted DNA Sequencing of 300 genes across all four classes of genomic alterations 1. Next generation sequencing (NGS): routine analy- to help understand the genomic makeup of a patient’s sis of the molecular changes in the tumor(s) in each tumor. In this assay, next-generation sequencing (NGS) patient; provides therapy targets and biomarkers as is used to analyze cancer specimens for all four classes well as biological input. of genomic alterations (base substitutions, insertions 2. RNAseq: TILs to be profled with increasingly high and deletions, copy number alterations, and rearrange- resolution and accuracy directly from RNA mixtures ments). Te targeted sequencing focuses on annotated of bulk tumor samples. and actionable mutational fndings. In addition, high 3. Non-invasive test, such as CAPP-Seq (cancer per- sequencing depth also overcomes contamination with sonalized profling by deep sequencing of circulating normal tissue and allows for the detection of mutations tumor DNA) [40]. at subclonal level. Genomic profle analysis using T5 4. Proteomics analyses in tissue and blood. Gene Panel demonstrated that the frequency of genetic 5. Functional studies, in vitro and in vivo drug response alterations of CDKN2A and CDKN2B pathway corre- to determine the efects of genomic alterations. lated with short (< 3.5 months) and longer progression free survival (PFS) in melanoma patients. Tis cell cycle Biomarkers for treatment decisions pathway was frequently altered and it is driven primar- Biomarkers have opened the paradigm of ‘precision med- ily by CDKN2A alterations. As far as HGF/MET altera- icine’ by incorporating biomarkers for risk assessment tions are concerned several patients had amplifcation and screening (prognostic); at diagnosis, when markers of genes on the 7q chromosome. Tese patients tended can assist with staging, grading (diagnostic), and therapy to have a shorter PFS than the BRAF-mutant population selection (predictive of response); and to select additional and seven patients exhibited MET (n = 4) and/or HGF therapy or monitor for recurrent disease in clinical man- (n = 6) amplifcations, with co-amplifcation occurring in agement of cancer patients. 3 of 6 patients. PFS was < 3.5 months for 3 of 4 patients Biomarkers such as mutations (NRAS, c-Kit, BRAF) with the MET amplifcation and 5 of 6 patients with the are already critical for proper management of advanced HGF amplifcation (van Herpen et al., submitted). melanoma patients. Based on the recent data of anti- Furthermore, HGF expression may contribute to thera- PD-1 treatment efcacy in PD-L1-positive advanced mel- peutic resistance in BRAF-mutant melanoma. A recent anoma, PD-L1 expression may refect the presence of T study in patients with BRAF-mutant melanoma reported cells secreting IFNγ in the tumor microenvironment [41]. correlation between HGF expression by stromal cells and PD-L1 expression in tumors correlates with the pres- innate resistance to BRAF inhibitor treatment. Response ence of TILs, since IFNγ production by TILs can induce to treatment (BRAFi ± MEKi) was signifcantly lower in expression of PD-L1. Terefore, this category of tumors patients with stromal HGF expression than in patients is likely to respond to TIL targeted therapy unlike tumors without HGF expression (p < 0.05) [42]. Treatment with who do not have T cell infltrate. a BRAF inhibitor increased stromal HGF expression Treatment of systemic metastatic disease (stage IV) in several patients. Besides, in BRAF-mutant cell lines involves new therapeutic strategies. Immunotherapy, treated with a BRAFi and HGF, HGF induced sustained that utilizes antibodies that bind to checkpoint inhibi- activation of ERK and AKT, which was more pronounced tory receptor of T-cells such as CTLA-4 blocking agent under BRAF inhibition than under MEK inhibition. ipilimumab; the anti-PD-1 antibodies, such as nivolumab Tese results suggest that genetic alterations leading to and pembrolizumab have already demonstrated impres- dysregulation of the MAPK pathway (e.g., HGF amplif- sive efcacy [41]. Targeted therapy agents such as selec- cation) could contribute to resistance to BRAFi therapy tive BRAF inhibitors including vemurafenib, encorafenib in patients with BRAF-mutant melanoma [42]. Tese and dabrafenib used alone and/or in combination with data support hypothesis that stromal cells might confer MEK inhibitors such as binimetinib, cobimetinib and innate resistance on cancer cells and cancer drugs. trametinib, have also demonstrated impressive antitumor Oncoprotein-targeted drugs hold enormous promise activity. Terefore, immunotherapy and kinase inhibi- for the future of cancer treatment. However, complete tors have become the backbone of systemic therapy in clinical responses are rare, suggesting that mechanisms melanoma. exist to render a substantial proportion of tumor cells Te feld of genetics has made advances in recent resistant to treatment. Biomarker analysis of tumors fol- years and methods to deliver and share genomic infor- lowing MAPK pathway inhibition of MEK by binimetinib mation for clinical care across diferent tumor types demonstrated that, the tumor genetic landscape was Ascierto et al. J Transl Med (2017) 15:236 Page 8 of 31

concordant with what was reported in the TCGA mela- determine follow-up therapy; these fndings demonstrate noma database. Driven primarily by CDKN2A altera- that, in order to target the large pool of heterogeneous tions, the cell cycle pathway was frequently altered in cells in a patient, the combinational therapy targeting dif- patients with the BRAF mutation. Several patients had ferent pathways is required [46]. amplifcation of genes on the q-arm of chromosome 7. In conclusion, immune checkpoint inhibitors, such as Tree of 7 of these patients had a PFS that was lower than anti-CTLA-4 blockade that activates T cells and enables the median PFS for the BRAF-mutant population, sug- them to destroy tumor cells, are efective cancer treat- gesting that a gene or genes on 7q may promote MEKi ments, but molecular determinants of clinical beneft resistance. Genes of high interest located on 7q include are not defned. Whole-exome sequencing of tumor tis- the HGF/MET pair, as well as BRAF itself (van Herpen sue from melanoma patients treated with ipilimumab or et al., submitted). tremelimumab including mutational profling and HLA Another subtype of melanoma is characterized by analysis identifed the presence of specifc tumor neo- NRAS mutation that occurs in ~ 20% of patients. Despite antigens that might explain the therapeutic beneft. Tis signifcant eforts to develop drug specifc targeting and other published study illustrated the importance of NRAS there are no specifc therapies for NRAS mutated tumor genetics in defning the basis of the clinical beneft melanoma. Despite emergence of immunotherapies as from immunotherapy including CTLA-4 and PD-1 path- efective treatments in melanoma, there is still a need way blockade [47]. to develop therapies for these patients, particularly after failure of NEMO open-label phase 3 study of binimetinib Combination strategy session vs dacarbazine in patients with advanced unresectable/ Overcoming radiation‑induced barriers to an immune metastatic cutaneous NRAS-mutant melanoma who response were previously untreated or had progressed on/after and Radiation therapy is a highly efective local treatment for who showed improvement in PFS [43]. cancer. Over the past 50 years, sporadic events of tumor Pre-clinical data on the BRAF kinase inhibitor vemu- regression in un-irradiated felds, at distant metastatic rafenib showed response rates of more than 50% in sites, known as abscopal efects, have been observed: a patients with metastatic melanoma with the BRAF V600E total of 46 reported cases have been identifed from 1969 mutation. Te next phase 3 randomized clinical trial to 2014 with median radiation dose of 31 Gy, median fol- comparing vemurafenib with dacarbazine in 675 patients low-up of 17.5 months, and median documented time to with previously untreated, metastatic melanoma with notice the abscopal efect was 2 months [48]. the BRAF V600E mutation resulted in improved rates of Te abscopal efect of radiation has more recently been overall and progression-free survival (relative reduction demonstrated to be a result of an antitumor immune of 63% in the risk of death and of 74% in the risk of either response induced by radiation efects in the irradiated death or disease progression, as compared with dacar- tumor. Te rarity of abscopal efects is a consequence of bazine (p < 0.001) [31]. Vemurafenib is a potent inhibi- the fact that at the time of metastasis cancer is associated tor of V600 mutant BRAF but cutaneous side efects are with a profound and tumor-specifc immune-suppressive frequent: peculiar cutaneous profle involving epidermis status. Modern immunotherapy, has ofered the potential and adnexa overlaps with the cutaneous manifestations for a recovery of an immune response and a synergy with of genetic diseases characterized by activating germ line immune activation by radiation. Te combination with mutations of RAS (RASopathy) [44]. immunotherapy has defned a novel role for radiotherapy Te mutational landscape of melanoma was studied by in systemic disease. sequencing the exomes of 147 melanomas: among the Barriers to the potential of radiation to convert a genes, the PPP6C that encodes a serine/threonine phos- tumor into an in situ vaccine have emerged beyond phatase was found exclusively in tumors with mutations the pre-existing immunosuppressive microenviron- in BRAF/NRAS. Tis activating mutation changes Pro29 ment of established tumors. Radiotherapy itself also to serine ­(RAC1P29S) promoting melanocyte proliferation induces some immunosuppressive signals, within and and migration, with possible therapeutic potential [45]. beyond the irradiated feld. Some trials are ongoing to Furthermore, sequencing allows to investigate acquired overcome radiation-induced immunosuppression. For chemotherapeutic resistance of cancer: the sequenced instance, counter-acting RT-induced immune-suppres- exomes of 27 lesions from three metastatic melanoma sion mediated by adenosine, with efect on dendritic patients treated with targeted or non-targeted inhibi- cells (DC) maturation and T cells recruitment was doc- tors showed that BRAF and NRAS co-mutations are not umented in several trials (MEDI9447; NCT02503774; mutually exclusive. However, the sole fnding of dou- NCT01283594). Recent fndings demonstrate in murine ble mutated cells in a resistant tumor is not sufcient to models that CD73-blockade reduces radiation-mediated Ascierto et al. J Transl Med (2017) 15:236 Page 9 of 31

T regulatory cells (Tregs) infltration while promoting trials in combination with radiotherapy as a strategy to CD8+ T cell infltration and combined RT and anti- improve outcomes [50]. CD73 treatment delays tumor progression and prolongs Investigations to defne the optimal dose, fractiona- survival. Tese data suggest that targeted CD73 therapy tion and feld size of radiotherapy when combined with helps radiotherapy by enhancing the adaptive immune immunotherapy are ongoing. When combined RT with response machinery, which may increase the function of concurrent chemotherapy, radiation can cause severe tumor-infltrating T lymphocytes, and subsequently lead treatment-related lymphopenia (TRL) (< 500 cells/mm3) to improved survival in cancer patients. that is associated with reduced survival. Severe TRL was Another approach regards overcoming TGFβ activa- observed in more than 40% of glioma patients 2 months tion by radiation-induced ROS, a mechanism that hin- after initiating chemo-radiation, an efect that was inde- ders priming of anti-tumor T cells. Vanpouille-Box et al. pendently associated with shorter survival from tumor demonstrated that antibody-mediated TGFβ neutraliza- progression [51]. Yovino et al. modeled these efects in tion during radiation therapy efectively generates CD8+ an attempt to predict the consequences of radiation- T cell responses to multiple endogenous tumor antigens related variables, such as the number of fractions, the in poorly immunogenic mouse carcinomas. Generated T feld size and the dose rate on circulating lymphocytes cells were efective at mediating regression of irradiated [52]. Te model proposed is based on the assumption tumors and non-irradiated lung metastases or synchro- that a single radiation fraction delivers 0.5 Gy to 5% of nous tumors (abscopal efect). Gene signatures associ- circulating cells. Consequently, 99% of circulating blood ated with IFNγ and immune-mediated rejection were would receive ≥ 0.5 Gy after 30 daily fractions. Reduc- detected in tumors treated with radiation therapy and ing the number of fractions and the feld size signifcantly TGFβ blockade in combination but not as single agents. decreases the exposure of circulating mononuclear cells However, up-regulation of programmed death ligand-1 during treatment [52]. Confrming this model, a ret- and -2 (PD-L1 and 2) in neoplastic and myeloid cells and rospective study in locally advanced pancreatic cancer PD-1 on intratumoral T cells limited the persistence of demonstrated signifcantly less severe radiation-induced tumor rejection, resulting in rapid recurrence. Addition lymphopenia from stereotactic body radiation than of anti-PD-1 antibodies enhanced the immune response standard radiotherapy, that utilizes larger felds and more and extended survival of the animals treated with radia- fractions of radiation [53]. Novel approaches are needed tion and TGFβ blockade. Tus, TGFβ is a fundamental to limit radiation to circulating lymphocytes given the regulator of radiation therapy ability to generate an in situ association of lymphopenia with poorer survival in tumor vaccine and its systemic efects are enhanced by patients. PD-1 blockade. Te combination of local radiation ther- In conclusion, radiotherapy induces both pro-immu- apy with TGFβ neutralization and PD-1 blockade ofers nogenic and immunosuppressive efects. RT reduces a novel individualized strategy for vaccinating patients circulating lymphocytes rendering blood an “organ at against their tumors [49]. risk”, especially for combination with immunotherapy. Tumor-infltrating myeloid cells (TIM), including Multiple immunotherapy strategies may be required to CD11b (Integrin subunit alpha, ITGAM)þF4/80 (EMR1) circumvent both pre-existing and RT-induced immune- b protein positive tumor associated macrophages, and suppression with a strategy that combines hypo-fraction- CD11bþGr-1 (LY6G)þ myeloid-derived suppressor cells ated, short courses of RT to small targets likely to be key (MDSC) respond to cancer-related stresses and promote to the success of RT + immunotherapy. tumor angiogenesis, tissue remodeling, and immunosup- pression. While the role of myeloid derived suppressor Where are we really with clinical trials of combination cells is complex, radiation increases both macrophages immunotherapy? and MDSC. Enhanced macrophage migration induced by Tere are two ways to think about combination conditioned media from irradiated tumor cells was com- immunotherapy: pletely blocked by a selective inhibitor of CSF1R. Mecha- nistic investigations revealed the recruitment of the DNA 1. Indirect methods: eradicate the tumor with immu- damage-induced kinase ABL1 into the nucleus where it nogenic stimulus for example including radiotherapy, bound the CSF1 gene promoter resulting in CSF1 gene chemotherapy, oncolytic viral therapy or targeted transcription. When added to radiotherapy, a selective therapy. inhibitor of CSF1R suppressed tumor growth more efec- 2. Direct methods include activating T cells through tively than irradiation alone. Te CSF1/CSF1R signaling agonistic or antagonistic antibodies through co-stim- to recruit TIMs likely limits the efcacy of radiotherapy. ulatory targets or turning of inhibitory receptors on Tus, CSF1 inhibitors should be evaluated in clinical T cells, respectively. Ascierto et al. J Transl Med (2017) 15:236 Page 10 of 31

Tere are several targets with emerging data in mela- 2-year OS rate was 64% with the combination compared noma, and this report selects a few for discussion. with 54% for ipilimumab alone (HR, 0.74; 95% CI 0.43– OX40 is a co-stimulatory receptor that can potentiate 1.26). Te median OS at 2 years in patients randomized T cell receptor signaling on the surface of T lymphocytes. to either the combination or monotherapy has not been In an OX40 phase I Trial, a mouse mAb that agonizes reached [60]. human OX40 signaling in patients with advanced cancer However, demonstrating overall survival benefts in showed an acceptable toxicity profle and regression of at randomized trials will be increasingly difcult due to the least one metastatic lesion in 12/30 patients [54]. Maxi- limited number of patients with melanoma available for mum tolerated dose was not reached (some fevers/chills, trials and higher landmark overall survival metrics. New rash, fatigue, arthralgias); tumor regressions without for- ways to test combinations are required. Should we add mal RECIST partial responses; two patients with mela- combinations to PD-1 non-responders or start combi- noma had mixed responses [54]. Phase I investigations of nations for “biomarker unfavorable” patients? Multiple OX40 agonists are ongoing in multiple solid tumors and combinations are partnering with PD-1, but should we in combination with PD-1 and PD-L1/2 partial responses reconsider ipilimumab combinations in the PD-1 refrac- (RCC and UCC) were reported in 2016 [55]. tory setting? Other co-stimulatory agonist drugs have been clinically Neoadjuvant Trials are also being conducted. Teir evaluated in early phase trials with some early activity advantages are quick interpretation of drug efect, and such as urelumab and utomilumab targeting activating they have pre-and post-treatment tissue available for receptor CD137/4-1BB. Ongoing studies in combination pharmacodynamic analyses. Whether efects on macro- with PD-1: utomilumab plus pembrolizumab have also metastatic disease in the neoadjuvant setting resemble been conducted with results indicating that this combi- efcacy against micrometastases in the neoadjuvant set- nation is well tolerated with some responses being seen ting remain unknown. Tere is also the question of tox- in solid tumors [56]. icity of systemic therapy in patients otherwise cured by Another strategy involves blocking an intratumoral surgery alone. enzyme “checkpoint” called indoleamine 2,3 dioxygenase In conclusion, combinations are now testing multiple T (IDO) a tryptophan-catabolizing enzyme that induces cell regulators, intratumoral “checkpoints”, and standard immune tolerance by T-cell suppression. IDO depletes anticancer agents. Reconsidering clinical trial endpoints tryptophan and produces toxic kynurenine. Epacadostat other than overall survival will be needed. Neoadjuvant is an oral, potent, selective inhibitor of IDO1 and pre- studies may ofer unique opportunities. liminary results from ongoing study of epacadostat with pembrolizumab showed promising clinical activity and Sequencing and combinations of checkpoint inhibitors acceptable safety profle. A dose expansion (epac 50, 100, with targeted agents in melanoma and 300 mg BID + pembrolizumab 200 mg IV Q3 W) Immunotherapy has been a highly promising approach was then implemented (n = 22), showing a ORR in 19 of melanoma treatment. Targeting BRAF mutation in untreated patients equal to 58%, and median PFS has not patients harboring mutated gene has been shown to be been reached [57]. Epacadostat is now in phase 3 study also highly efcacious. Dual BRAF and MEK inhibition with pembrolizumab. is associated with high response rates and median PFS Combinations have been studied based on preclini- (mPFS) of 9–12 months and superior survival compared cal rationale and clinical evidence for efcacy as single to single-agent BRAF inhibitors (BRAFi) [61–63]. agents. Early phase combination dose-fnding studies Comparison of systemic therapy for advanced (unre- and then ultimately randomized studies with an overall sectable Stage III or Stage IV) melanoma reported in survival endpoint have been then implemented. Ipili- Table 1 shows similar efcacy in the median patient mumab and GM-CSF combination has better overall response, PFS and survival in selected group of patients. survival than ipilimumab alone (HR 0.64 for OS) in a Tus, combining targeted therapy with immunother- phase 2 study [58]. In a 2-year assessment of the phase apy may lead to enhanced anti-tumor response and result 2 CheckMate-069 trial, the 2-year OS rate with the com- in durable responses and prolonged survival (Fig. 1). bination of nivolumab + ipilimumab was 69% compared Te critical issue for designing efective combination with 53% for ipilimumab alone (many patients crossed of anti-BRAF and immunotherapy relies on determina- over to nivolumab), for patients with BRAF wild-type tion of optimal selection of targeted and immune ther- melanoma [59]. Te median OS among patients was not apy agents and identifcation of biomarkers predictive of been reached with the combination regimen and was treatment response that will lead to rational combinato- 24.8 months with ipilimumab monotherapy (HR, 0.58, rial “regimens”. Retrospective analysis of overall survival 95% CI 0.31–1.08). In the overall study population, the (OS) revealed improvement in patients treated with Ascierto et al. 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Table 1 Comparison of systemic therapy for advanced (unresectable stage III or stage IV) melanoma Treatment RR (%) PFS (med), months OS (med/2-years)

Single-agent BRAFi 50 6–8 18.7 months/~ 40% Combo BRAFi and MEKi 65–70 9–12 15 months/~ 50% Ipilimumab 10 2–3 12 months/~ 30% (20% 5 year survival) Anti-PD-1 mAb (nivolumab or pembrolizumab) 25–45 ~ 6 18–24 months/~ 50% Combo ipilimumab and nivolumab ~ 60 11–12 Unk./~ 64%

Fig. 1 Melanoma survival curves depending on the type of therapy (modifed from [150])

ipilimumab compared with those treated with BRAF genetic ways of reactivating the MAPK pathway have inhibitors frst [64]. Another retrospective analysis from been identifed. Besides, mechanisms of intrinsic resist- four major academic centers suggests that either BRAFi ance are not less diverse; one of the major drivers of or anti-PD-1 may be efective regardless of treatment intrinsic resistance seen in AXL-high at baseline [67]. sequence in patients with BRAFV600-mutant melanoma, Reduction in circulating tumor DNA (ctDNA) BRAF but clinical outcomes to front-line therapy are superior level is associated with tumor regression by RECIST: [65]. maximum ctDNA BRAFV600 level reduction occurs in Te data suggests that BRAFi as a salvage strategy may Cycle 2 and 3 time points in vemurafenib treated patients not be highly active in the subgroup that fails anti-PD-1 and in at least Cycle 4 in patients treated with the com- and front-line BRAF/MEK inhibitor therapy should be bination of dabrafenib and trametinib. Average, a detect- considered. Patients who benefted from BRAF-directed able increase in BRAFV600E level was seen ~ 50 days therapy for ≥ 6 months had a 34% overall response in patients treated with BRAF directed therapy prior to rate (ORR) to subsequent anti-PD-1 (11 of 32 patients radiographic PD [68]. responded). Patients who benefted for < 6 months subse- Recommendations from the “SWITCH 2.0” Trial for quently had a 15% ORR to anti-PD-1 (4 of 26; p = 0.04). optimal strategies for treatment of melanoma patients Shared “phenotype “to predict response for BRAFi and include: anti-PD-1 therapy may exist and studies of molecular characterization of “responders vs. resistant phenotype” 1. BRAFV600 positive tumor melanoma patients, front- are on the way. If successful, the identifed profle may line treatment. serve as a selection marker for prospective studies. It is 2. Group 1 treated with BRAFi/MEKi until RECIST suggested that there is a diference in the molecular pro- 1.1 progressive disease (PD) and then switched to fle of tumors responding to CTLA-4 vs PD-1. In the ideal ipilimumab/nivolumab, possibility to switch again to scenario, molecular profles identifying targeted therapy alternative BRAFi/MEKi. responders should not overlap with immune therapy 3. Group 2 treated with BRAFi/MEKi until BRAF level responders to provide a beneft of combination therapy. frst increase (or set at four cycles) and switched Resistance is a clinical problem and genetic mecha- prior to RECIST 1.1 to ipilimumab/nivolumab with nisms of acquired resistance are diverse [66]. Multiple Ascierto et al. J Transl Med (2017) 15:236 Page 12 of 31

possibility to switch back to BRAFi/MEKi again if PD dose interruption and/or reduction. Treatment-related (alternative type of inhibitors). serious AEs including grade 3 blood creatinine phos- 4. Primary endpoint: PFS to ipilimumab/nivolumab phokinase levels increased (cobi-related), grade 4 sepsis between Groups 1 and 2, ORR, PFS from switch 1 to (cobi-and/or vem-related), grade 3 diarrhea and ALT/ the end of second round of BRAF/MEK (where the AST levels increased (atezo- and/or cobi- and/or vem- strategy used). related); all 3 patients continued on study treatments 5. Compare molecular profles at diagnosis, after each after interruption [73]. line of therapy and at the time of ultimate resistance In conclusion, atezolizumab + cobimetinib demon- development (will difer based on intrinsic patient strated encouraging antitumor activity. However, it is characteristics and types of therapy). not known whether the efect is additive or synergistic. Overall, ORR was 45% and mPFS was 12.0 months (ate- Building rational combinatorial “regimens” is the zolizumab monotherapy in cutaneous melanoma: ORR, important factor. BRAFi efects on tumor microenvi- 33%; mPFS, 5.5 months). Te clinical beneft of atezoli- ronment predict optimal combination with anti-PD-1/ zumab + cobimetinib was seen regardless of BRAF sta- PD-L1 inhibition [69]. Preclinical data predicted synergy tus. BRAF mutant: ORR was 40%; mPFS, 11.9 months; between MAPK targeting and PD-1/PDL1 inhibition [70, BRAF wild type: ORR was 50%; mPFS, 15.7 months. 71]. Atezolizumab + cobimetinib had a manageable safety A phase Ib dose-escalation and -expansion study profle in metastatic melanoma, similar to that observed (NCT01988896) study combining atezolizumab and with atezolizumab alone or cobimetinib + vemurafenib cobimetinib in metastatic melanoma suggested higher combination. Te combination atezolizumab + cobi- ORR/disease control rate [durable response rate (DCR), metinib + vemurafenib had a manageable safety profle in overall response rate (ORR) + stable disease] and longer patients with BRAF V600 mutant metastatic melanoma, PFS with the combination. Updated safety and efcacy but liver toxicity seems to be higher. Te triple combina- data (Oct 12, 2016) showed that RECIST v1.1-confrmed tion demonstrated promising antitumor activity, uncon- ORR was 45.0% in patients with non-ocular melanoma frmed response rate was 83% (95% CI 64.2, 94.2). (median duration of response was not reached); DCR Te question remains whether to start the treatment was 75.0%; mPFS was 12.0 months (95% CI 2.8–not eval- with the combination of diferent agents including tar- uable). ORR was similar for patients with BRAF-mutant geted agents, immunotherapy with checkpoints inhibi- and wild-type melanoma; adverse events (AEs) were tors, novel approaches) or add/switch on progression. experienced in all patients and related grade (G) 3–4 in Other challenges in the optimal use of BRAF/MEK and 54.5% were most common; related serious AEs in 13.6% immunotherapy are: [72]. A phase 3 trial evaluating atezolizumab + cobi- metinib vs anti-PD-1 therapy in patients with BRAF wild- 1. Incorporating novel agents-both targeted (AXLi) and type advanced melanoma is planned. immunotherapy (TLR9, T-VEC). Targeted therapy with MEK inhibitor cobimetinib 2. Postponing large phase 3 trials until we have identi- (cobi) + BRAF inhibitor vemurafenib in BRAFV600- fed better markers for patients’ selection. mutant melanoma can result in anti-cancer immune acti- 3. Use of the data from early phase trials. vation and rapid clinical response. Inhibition of PD-L1 4. Need for model systems-surrogate markers (in vivo using atezolizumab can lead to anti-cancer immune imaging, liquid biopsies). activity and durable responses. Combining these agents may enhance antitumor immune activity and potentially Innovative combination strategies: oncolytic and systemic improve both rate of clinical response and durability therapy [69]. In this study 13/14 patients (93%) showed responses Oncolytic therapy is a therapeutic modality of direct (RECIST v1.1), including 1 CR and 12 PRs and 11/13 injection at the tumor site agents that may induce a pts continue in response. One patients with PR had a local and systemic efect that is immunologically medi- 100% reduction in target lesions. Responses were uncon- ated and produce regression. Te feld of oncolytic frmed, and median DOR and PFS were not evaluable virotherapy encompasses the use of viruses with natu- due to limited follow-up at the time of data cut (Feb 15, ral or engineered tumor-selective replication to infect 2016). Functional biomarkers data of T-cell activation are and kill tumor cells. Te main intralesional (IL) agents ongoing. currently in phase 3 trials are viral based (Talimogene Triple combination treatment was generally well tol- laherparepvec, TVEC, HF-10, CAVATAK) and non-viral erated as no unexpected AEs including grade 5 AEs based (PV-10, IL-12). Te mechanism of action is cell occurred. All AEs were manageable and reversed with lysis (viral replication, chemical and mechanical ablation) Ascierto et al. J Transl Med (2017) 15:236 Page 13 of 31

and indirect “bystander response” by induction of innate 1–2 injections. An open-label, randomized controlled immune response and adaptive immune response. trial of single-agent intralesional PV-10 versus systemic For an anticancer immune response to lead to efective chemotherapy or intralesional oncolytic viral therapy is killing of cancer cells, a series of stepwise events must be currently ongoing to assess treatment of locally advanced initiated and allowed to proceed and expand iteratively. cutaneous melanoma in patients who have failed or are We refer to these steps as the cancer-immunity cycle not otherwise candidates for targeted therapy and have which manages the delicate balance between the recogni- failed or are note candidates for at least one immune tion of nonself and the prevention of autoimmunity [74]. checkpoint inhibitor. An international multicenter, open- Intralesional oncolytic therapy in soft tissue and skin label, sequential phase study of intralesional PV-10 in metastases has shown good safety profle and a durable combination with pembrolizumab in stage IV metastatic response rate. Local–regional control of tumor growth is melanoma patients with at least one injectable cutane- clinically important, whilst the systemic therapy may not ous or subcutaneous lesion (NCT02557321) is ongoing. always be possible or appropriate. Newer IL agents that In the phase 1b portion of the study, all participants will have the ability to trigger a systemic immune efect can receive the combination of IL PV-10 and pembrolizumab be clinically useful for combination treatment. Melanoma (i.e., PV-10 + standard of care). In the subsequent phase intra-lymphatic metastasis occurs in 3–10% of primary 2 portion of the study participants will be randomized 1:1 melanoma patients and it manifests clinically as local/ to receive either the combination of IL PV-10 and pem- in-transit recurrences. High risk groups for this pres- brolizumab or pembrolizumab alone (i.e., PV-10 + stand- entation are characterized by thick, ulcerated, positive ard of care vs. standard of care). sentinel lymph nodes (SLN), lower extremity and greater Te Intratumoral DNA-encoded IL-12 Electroporation than 50% risk of distant disease and death. Intralesional (IT-pIL12-EP) is a DNA plasmid encoding interleukin-12 approaches may be applicable in melanoma treatment of (IL-12), a potent pro-infammatory cytokine. It is deliv- these population of patients. ered directly to tumor in vivo by electroporation. Trans- Current clinical trials with IL agents include monother- fection of the plasmid stimulates local immune response apy with PV-10 (phase III trial is ongoing, IL-12 admin- and subsequently, systemic efect. Tirty patients with istered by electroporation (EP), oncolytic Picornavirus, stage IIIB-IV melanoma received up to four cycles of and Coxsackievirus A21 (CAVATAK™). Combination tri- IL-12 EP into superfcial cutaneous, subcutaneous, and als with TVEC, PV-10, HF10-oncolytic HSV1 and Reovi- nodal lesions on days 1, 5 and 8 of each 12-week cycle. rus (HF-10) are also ongoing. Te treatment induced objective tumor responses in a PV-10 is an investigational new drug containing a pro- signifcant proportion of patients (31%) and treatment prietary injectable formulation of Rose Bengal disodium was well tolerated [76]. (10% RB). It is a small molecule fuorescein derivative CAVATAK, is a bioselected oncolytic strain of Cox- lysing primary tumors by entering lysosomes, activat- sackievirus A21 (CVA21) is another oncolytic immuno- ing tumor-infltrating lymphocytes at the local site and therapy agent. Following intratumoral injection, CVA21 regression of distant tumors. Necrotic tumor cells have preferentially infects ICAM-1 expressing tumor cells, been shown to facilitate antigen presentation and the resulting in viral replication, cell lysis, and a systemic secondary tumors are rejected in immuno-competent anti-tumor immune response. Te phase II CALM animals. Responses are tumor specifc and no immune study investigated the efcacy and safety of IT CVA21 in response was reported in immuno-compromised ani- patients with advanced melanoma. Te primary endpoint mals. Adoptive transfer of spleen cells can convey of the study was achieved in 38.6% evaluable patients immunity because T cell subsets have increased expres- with durable responses observed in both injected and sion of IFNγ. A phase 2 study of intralesional PV-10 in uninjected melanoma metastases, suggesting the genera- refractory metastatic melanoma assessing efcacy and tion of systemic host anti-tumor responses [77]. safety of PV-10 in 80 patients with refractory cutane- Talimogene laherparepvec (T-VEC) is an intralesional ous or subcutaneous metastatic melanoma showed oncolytic virus based on a modifed herpes simplex encouraging results. Te best overall response rate for virus type-1. It selectively targets tumor cells, causing targeted lesion was 51%, and the complete response rate regression in injected lesions and inducing immuno- was 26%; median time to response was 1.9 months, and logic responses that mediate regression at uninjected/ median duration of response was 4.0 months, with 8% distant sites. In a randomized phase 3 trial, T-VEC of patients having no evidence of disease after 52 weeks met its primary endpoint of improving the durable [75]. Regression of bystander lesions strongly correlated response rate vs granulocyte–macrophage colony-stim- with response in target lesions and responses occur early ulating factor (GM-CSF) in patients with unresectable as 56% of lesions achieved complete response (CR) after melanoma [78]. Responses were observed in injected Ascierto et al. J Transl Med (2017) 15:236 Page 14 of 31

and uninjected regional and visceral lesions. Explora- A phase 1b on subjects treated with MK-3475 (pem- tory analyses suggested survival diferences in favor of brolizumab) until CR disease progression per irRC, T-VEC in patients with untreated or stage IIIB/IIIC/ or intolerance of study treatment, up to a maxi- IVM1a disease. T-VEC therapy was generally well tol- mum of 24 months of study treatment is ongoing erated, the most common adverse events being fu-like (NCT02263508). In phase 3, subjects will be treated symptoms [79]. with T-VEC plus pembrolizumab (arm 1) or placebo plus OPTiM was a randomized, phase 3 trial of T-VEC or pembrolizumab (arm 2) until 24 months from the date of GM-CSF in patients with unresected melanoma with the frst dose of pembrolizumab or end of treatment due regional or distant metastases. Te primary endpoint to disappearance of injectable lesions, complete response, was durable response rate (DRR): partial or complete disease progression per irRC-RECIST or intolerance of response (CR) continuously for ≥ 6 months starting study treatment. within 12 months. Objective response rate with T-VEC HF10 is not genetically engineered but is a nonselec- was 26% (95% CI 21, 32%) with 11% CR, and with GM- tive clone from the non-neuroinvasive HSV-1 strain CSF was 6% (95% CI 2, 10%) with 1% CR. DRR for T-VEC HF. Apart from loss of UL56 gene, sequencing of HF10 was 16% (95% CI 12, 21%) and 2% for GM-CSF (95% CI revealed an overall 99.1% similarity to HSV-1 strain 17, 0, 5%), p < 0.0001. DRR by stage (T-VEC, GM-CSF) was with mutations in genes involved in regulation of syncy- IIIB/C (33, 0%), M1a (16, 2%), M1b (3, 4%), and M1c (8, tia formation including UL1, UL20, UL22, UL24, UL27, 3%). Interim OS showed a trend in favor of T-VEC; HR and UL53. HSV strains can spread from cell-to-cell via 0.79 (95% CI 0.61, 1.02) which did not reach statistical infection across the junctions between the membranes signifcance. Most common adverse events (AEs) with of adjacent cells (wild-type strains (syn+) or by fusion of T-VEC were fatigue, chills, and pyrexia. Serious adverse the infected cell with adjacent uninfected cells leading to events (AEs) occurred in 26% of T-VEC and 13% of GM- the formation of multinucleated polykaryocytes or syn- CSF pts. No ≥ grade 3 AE occurred in ≥ 3% of pts in cytia [syncytial mutant strains (syn)]. Interestingly, HF10 either arm [80]. induces syncytia formation in vitro, and did not result in Preliminary data suggest higher CR and OR rates than the cytopathic efect observed in hrR3 infected cells [85]. either agent alone and earlier responses after ipilimumab It is characterized by greater replication ability, result- initiation during T-VEC + ipilimumab than with ipili- ing in lower efective dose and no toxicity resulting from mumab alone [81]. A phase 1b demonstrated that of 17 inserted exogenous gene (ex. GM-CSF). Attenuation of pts with investigator assessed response, ORR was 41% neurovirulence to be attributable to the lack of the UL56 (24% CR, 18% PR); 35% had stable disease (SD). Median gene (lack of UL56 gene decreases HSV-1 pathogenicity time to response was 2.9 months. Activated CD8 T cells without afecting viral replication ability). In addition to numbers signifcantly increased from baseline 1.8× after local oncolytic tumor destruction, systemic anti-tumor T-VEC alone and 2.9× during T-VEC + ipilimumab immune response observed. treatment; Gr 3/4 AEs occurred in 32% as determined by Preliminary data of the antitumor activity of fow cytometry [82]. HF10 + ipilimumab combination in an ongoing phase ORR was higher for T-VEC in combination with ipili- 2 trial in melanoma on 43 patients enrolled and treated mumab vs ipilimumab alone [83]. Confrmed ORR was at abstract data cut-of 01 Feb16 show that majority of 35.7% (T + I) and 17.5% (I); unconfrmed ORR was 50% HF10-related AEs are ≤ G2, similarly to HF10 monother- (T + I) and 27.5%. AEs were comparable between arms apy and consistent with other oncolytic viruses. No DLTs except for increased fatigue, chills, and pyrexia in the or ≥ G4 AEs were reported. G3 AEs were experienced by T + I arm [83]. 11.6% of pts. Of 37 efcacy evaluable patients, BORR by Te combination of T-VEC and pembrolizumab dem- irRC at 24 weeks is 37.8% (13.5% CR and 24.3% PR) and onstrated improved clinical beneft in a phase 1b/2 study disease stability rate is 56.8% (18.9% SD) [86]. in unresectable stage IIIB-IV melanoma with all patients Pro and con arguments of the role for IL monotherapy having started on T-VEC + pembrolizumab ≥ 6 months are reported in Table 2. prior [84]. Per immune related criteria (irRC), in 21 pts, In conclusion, soft tissue and cutaneous metastases are confrmed/not yet confrmed objective response rate a major clinical problem in melanoma. Oncolytic intral- (ORR) was 48%/57%; CR rate was 14%/24%. Median time esional approaches may have value as local direct efect, to response was 17 wks. Circulating CD8+ T cells includ- systemic immune efect and low toxicity. Several agents ing those expressing defned immune modulatory recep- in development appear promising such as TVEC that was tors (e.g., Tim3, BTLA) became elevated during therapy approved by US and EU regulators. Combination thera- with T-VEC initially but decreased after pembrolizumab pies are likely to be the future and may be the best way to began on d 36 [84]. integrate them into clinical practice. Ascierto et al. J Transl Med (2017) 15:236 Page 15 of 31

Table 2 Potential role of intralesional monotherapy Yes No

Not all patients are candidate for systemic therapy (co-morbidities, toxicity) Systemic therapies in 2015 are safe and efective After progression on other therapies Melanoma is a systemic disease Alternative to surgery? Surgery is an instant CR Neoadjuvant potential Not yet proven

System biology session: immunology eliminates the tumor response induced by the agonist Rational design of combination immunotherapy antibody [89]. We also found, that blocking PD-1/PD-L1 Efective immunotherapy is a balance between induction pathway while activating OX40 drives the CD8 T cells of immune response (NK, B cell, CD4 cells, CTL) and into apoptotic cell death [89]. Accordingly, it is crucial inhibition of suppression (Tregs, MDSC, TAM, IDO). to know that combining two immune modularity agents Biologically rational design of immunotherapeutic com- is not a straightforward approach and such combination bination is crucial for the success of such approach. may not induce a desirable response if not treated in the Te PD-1 receptor is a negative regulator of T-cell proper sequence. In a combination approach, any of the efector mechanisms that limits immune responses agents could lead to changes within the tumor microen- against cancer: lambrolizumab was tested in patients vironment that produce a specifc immune efect which with advanced melanoma and showed a high rate of sus- may infuence the outcome of the second immunomodu- tained tumor regression, with mainly grade 1 or 2 toxic lator in a potentially positive or a negative manner. efects [87]. In patients with melanoma, ipilimumab pro- Naturally occurring T regulatory cells (Tregs) express longs overall survival, and nivolumab produced durable the transcription factor Foxp3, which is a master regula- tumor regression in a phase 1 trial. Based on their dis- tor of Tregs development and function. Te Foxp3 gene tinct immunologic mechanisms of action and supportive was frst identifed as the defective gene in the mouse preclinical data, a phase 1 trial of nivolumab combined strain Scurfy. Scurfy is an X-linked recessive mutant that with ipilimumab in patients with advanced melanoma is lethal in hemizygous males within a month after birth. showed a rapid and deep tumor regression in a substan- Hypoxia, or low oxygen tension, is a major regulator of tial proportion of patients; a total of 16 out of 53 patients tumor development and aggressiveness. Accordingly, had tumor reduction of 80% or more at 12 weeks, includ- strategies to decrease Tregs are important to be included ing 5 with a complete response [88]. Accordingly, pre- within the combination repertoire to further enhance liminary data of early clinical trials of such combination the outcome of therapeutic approaches. Cyclophospha- proved better than the efect of each of the single agents. mide used in low metronomic doses have been shown to Other immunomodulatory agents that may come into decrease Tregs in the microenvironment and enhance the play in the spectrum of combination therapy include anti-tumor immune efect [90]. Based on that, we found immune agonists antibodies of activating receptors such that combining antigen-vaccine with anti-PD-1 and a as anti-OX40 and anti-GITR. Anti-OX40 enhances T single dose of low-dose cyclophosphamide can improve cell responses and is associated with increased T cell immune outcome [91]. Utilizing Listeria as a platform expansion/proliferation, efector function, T cell sur- for cancer vaccine to generate antigen-specifc anti- vival, T cell memory development as well as enhancing tumor immune response, we found that Listeria exhibit vaccine therapeutic efect. Accordingly, the combina- a bystander immune response leading to the decrease tion of anti-OX40 and anti-PD-1 is a rational strategy in in the ratio of Tregs to CD4 T-cells [92]. It has been immune therapy combination development. Few clini- reported that anti-glucocorticoid-induced tumor necro- cal trials are currently being conducted combining these sis factor receptor (GITR) agonist antibodies leads to two agents. Interestingly, we found that the sequence of tumor regression only in the context of Tregs depletions such combination is crucial for its success or failure. In [93]. Based on that and since Listeria-based immunother- addition, combining OX40 agonist antibody enhances apy is able to decrease the ratio of Tregs to CD4 T-cells, specifc immune response in pre-primed animals with we hypothesized that combining Listeria-based vaccine tumor specifc antigens. Tis enhancement leads to bet- with GITR-agonist antibody would exhibit synergis- ter tumor response that is immune-dependent. However, tic anti-tumor immune response. Indeed, listeria-based when anti-PD-1 is combined concomitantly with ago- (Lm) immunotherapy combined with agonist anti-GITR nist anti-OX40, surprisingly, we found that it completely antibody provide a potent synergistic treatment strat- abrogates the immune response of OX40 and hence egy that simultaneously targets both the efector and Ascierto et al. J Transl Med (2017) 15:236 Page 16 of 31

suppressor arms of the immune system, leading to sig- of Batf3+ DC recruitment, failed T cell priming and nifcantly improved anti-tumor efcacy [94]. response to checkpoint blockade [98]. Genetic land- In summary, unlike most of chemotherapeutic scape of the T cell-infamed tumor microenvironment approaches to cancer, combination immunotherapy is across Te Cancer Genome Atlas (TCGA) solid tumors not a random approach of drug coupling, but rather it was investigated in order to describe the spectrum of should be based on biologically based rational approach the T cell-infamed tumor phenotype across histologies to therapy that is intended to redesign the immune com- [99]. Studies of mutational burden compared with the T position of the tumor microenvironment. cell-infamed tumor microenvironment showed no cor- relation between gene expression and mutational bur- Bioinformatics approaches to investigate mechanisms den in any cancer type [99]. Antigen is not rate-limiting driving the non‑T cell‑infamed tumor microenvironment in non-T cell-infamed tumors that lack of spontaneous Tumors with substantial susceptibility to anti-PD-1 anti- immune infltration in solid tumors is unlikely due to lack body are numerous (melanoma, non-small cell lung can- of antigens. Rather a clear linear association with antigen cer, renal cell carcinoma—clear cell carcinoma etc.) and presenting machinery and Batf3 lineage DCs are sug- registrational trials are on-going in many cancer histolo- gested. As an example of this approach, gene expression gies (gastroesophageal cancer, hepatocellular carcinoma, in testicular germ cell tumor (GCT) revealed a T-cell- mesothelioma etc.) [95]. infamed tumor microenvironment in 47% of testicular Te T cell-infamed tumor microenvironment is char- GCTs, including seminoma (83%) and nonseminoma acterized by expression of immune-inhibitory pathways (17%) tumor subtypes [100]. Expression of alpha-fetopro- and predicts patient outcomes to immunotherapy [96, tein (AFP) RNA correlated with lack of the T-cell signa- 97]. Te molecular mechanisms that explain the T cell- ture, with increasing AFP RNA inversely correlating with infamed versus non-infamed tumor microenvironments the infamed signature and expression of IFNγ-associated could include though not necessarily be limited to: (1) genes. Tese data suggest that GCTs can respond to somatic diferences at the level of tumor cells charac- anti-PD-1 and that gene expression profling supports terized by distinct oncogene pathways activated in dif- investigation of immunotherapy for treatment of GCTs ferent patients and mutational landscape and antigenic [100]. Te presence of intratumoral CD8+ T cells has repertoire; (2) germline genetic diferences at the level been associated with clinical beneft to immunotherapy of the host (patient) characterized by polymorphisms in and patients can be categorized based on the presence immune regulatory genes; (3) environmental diferences or absence of a T cell-infamed tumor microenviron- characterized by commensal microbiota. Data for each ment. Molecular mechanisms underpinning the absence of these is now available for melanoma and investiga- of a T cell response are beginning to be understood with tions are on-going to spread these concepts across tumor identifcation of the WNT/β-catenin pathway playing a types. major role in melanoma including activating mutations Regarding cancer cell intrinsic properties and specif- CTNNB1 and Inactivating mutations in negative regula- cally distinct oncogene pathways, some of T cell exclu- tors of Wnt pathway (Axin1, Axin2, APC1, APC2) [101]. sion have been elucidated. Only a subset of patients Pathway activation without mutation includes overex- responds to blockade of immune-inhibitory receptors pression of Wnt ligands, Fzd receptors, β-catenin [101]. treatments and the therapeutic beneft is preferentially Other tumor-oncogenic pathways correlating with T-cell achieved in patients with a pre-existing T-cell response exclusion in the non-T-cell-infamed tumor microen- against their tumor, as evidenced by a baseline CD8+ vironment and the resistance to immunotherapies are T-cell infltration within the tumor microenvironment. also under investigation. Upregulation of genes encod- It is critical to understand molecular mechanisms of a ing immune checkpoint proteins PD-L1, IDO, FOXP3, spontaneous anti-tumor T-cell response in melanoma- TIM3, and LAG3 was associated with T-cell-infamed cell-intrinsic oncogenic pathway that contributes to a tumors, suggesting potential for sensitivity to checkpoint lack of T-cell infltration in melanoma [98]. Te corre- blockade. Conversely, β-catenin, PPAR-γ, and FGFR3 lation between activation of the WNT/β-catenin sign- pathways were activated in non-T-cell-infamed tumors aling pathway and absence of a T-cell gene expression [102]. Furthermore, PTEN loss in melanoma associates signature was the frst tumor-intrinsic molecular sign- with non-T cell-infamed tumor microenvironment and aling pathway identifed to mediate immune exclusion. resistance to anti-PD-1 [103]. Activated β-catenin signaling results in T-cell exclu- Te next hurdle in cancer immunotherapy is over- sion and resistance to anti-PD-L1/anti-CTLA-4 mono- coming the non-T cell-infamed tumor microenviron- clonal antibody therapy [98]. On a mechanistic level, ment. Terefore, a number of clinical trials are ongoing β-catenin represses CCL4 chemokine, leading to lack on diferent targets, promoting innate immunity/type I Ascierto et al. J Transl Med (2017) 15:236 Page 17 of 31

IFNs like intratumoral STING agonists, inducing tertiary homeostatic equilibrium between Intestinal Epithelial lymphoid structures and modulate stroma (anti-CD40). Cells (IEC) and intraepithelial lymphocyte, leading to Examples are phase 1 study of stereotactic body radio- the apoptotic demise of IEC in the presence of micro- therapy followed by pembrolizumab in advanced solid bial products. Compensation of mice with B. fragilis + B. tumor, phase 1 study of SEA-CD40 in advanced malig- cepacia was able to protect against subclinical toxicity. nancies, phase 1b/2 study of BBI608 in combination with Furthermore, we saw an increase in IFNγ and a decrease immune checkpoint inhibitors in advanced solid tumors in IL-10 production in B. fragilis/Bacteroides thetaio- and phase II dual cohort study of gut microbiota modula- taomicron-specifc memory CD4+ T cell responses in tion with pembrolizumab in melanoma. metastatic melanoma patients post-CTLA-4 blockade. In conclusion, anti-PD-1 or PD-L1 immunotherapy is Feces from metastatic melanoma patients were analysed becoming standard therapy in many tumor types. T cell and grouped into three clusters (A, B and C) based on gene signatures correlate well with available biomarkers genus composition. Germ free (GF) mice transplanted (including gene expression, mutational load, PD-L1, TCR with feces from Cluster C patients had a signifcantly clonality) and clinical beneft. Future immunotherapy greater response to CTLA-4 blockade compared to mice drug development should be stratifed toward engaging which received Cluster B feces and were found to facili- either the infamed or non-infamed tumor microenvi- tate the outgrowth of benefcial B. fragilis. Te efcacy ronment (infamed like IDO, Tregs, MDSCs and non- of anti-CTLA-4 therapy in Cluster B transplanted mice infamed like RT, STING, CD40) and molecular pathways could be improved by compensation mice with certain leading to immune exclusion (e.g., β-catenin/PTEN). bacteria. In conclusion, gut microbiota impacts ther- apy-induced antitumor immunosurveillance and that The gut microbiome and cancer therapeutics the therapeutic coverage of immune checkpoint block- Te gut microbiota is composed mostly of bacteria, but ade could be broadened when a favorable microbiota is also fungi, archaea and viruses. Evolutionary develop- present. ment of the host immune system has been closely asso- ciated with the microbiota in a symbiotic relationship Next target for immune checkpoint blockade (eubiosis). Tis diverse microbiome is associated with Tere is ample evidence that high-level spontaneous and many diseases including asthma, allergy and infamma- vaccine-induced tumor antigen-specifc T cells may exist tory bowel disease, and is involved in numerous func- in patients with advanced and progressive melanoma. tions including, digestion of complex carbohydrates, Tis paradoxical coexistence of T cell immune responses direct competition for limited nutrients and occupation with melanoma progression has led us to investigate the of ecological niches that may otherwise be colonized by multiple immunoregulatory pathways driving T-cell dys- pathogenic microorganisms [104]. Furthermore, the gut function in the tumor micro environment (TME). Te microbiota can afect distant organs [105]. Dissecting upregulation of inhibitory receptors by T cells chronically the relationship between the microbiota, pathogens and activated by tumor cells in the TME represents a major the host may provide novel insights into disease patho- mechanism of tumor-induced T cell dysfunction. Target- genesis, as well as novel avenues for the prevention and ing inhibitory pathways with blocking antibodies have treatment of intestinal and systemic disorders including transformed the standard of care for patients with mela- cancer. noma and other solid tumors. Anti-PD-1 antibodies are During cancer treatment, there is a complex interplay a potent therapy for melanoma, which provide clinical between the gut microbiota and anticancer therapies. benefts to 30–40% of patients with advanced melanoma. Tese therapies can exert cytotoxic efects on intestinal Beyond PD-1, group at the University of Pittsburgh bacteria, leading to dysbiosis. However, the gut micro- has worked on identifying additional inhibitory path- biota can in turn infuence both the therapeutic activity ways that may cooperate with PD-1 to dampen T cell and the side efects of anticancer agents, via pharma- responses to melanoma. Tere are numerous inhibitory codynamics and immunological mechanisms [106]. receptors expressed by T cells in the TME that bind to Te antitumor efcacy of immune checkpoint inhibi- their respective ligands expressed by antigen-presenting tors including anti-CTLA-4 has been demonstrated to cells and tumor cells [108]. require gut microbiota, in particular, gram-negative bac- Te rationale for optimal combinatorial immune check- teria [107]. Burkholderia cepacia acts in conjunction with point blockades is based on the identifcation of inhibi- Bacteroides fragilis to restore sensitivity to anti-CTLA-4. tory or activating receptors expressed by a signifcant Adoptive T cell transfer of T cells primed with B. fragi- number of tumor antigen-specifc CD8+ T cells. Te evi- lis ameliorates the antitumor efects of CTLA-4 block- dence exists of additive/synergistic efects on tumor-anti- ade in germ free mice. Anti-CTLA-4 compromises the gen specifc CD8+ T cell expansion and function upon Ascierto et al. J Transl Med (2017) 15:236 Page 18 of 31

dual blockade targeting non-redundant inhibitory path- in PSA [expressed as slope log (PSA) or PSA doubling way. Mouse tumor models support the in vivo efcacy of time (PSADT)], validated tumor growth measures, and the dual blockade with a nontoxic and safe profle. Based tumor growth rate in men with Stage D0 prostate can- on this hypothesis, the group has focused on the novel cer. HLA-A*0201 positive men were randomized to T cell inhibitory receptor with immunoglobulin (Ig) and receive epitope-enhanced (29-37-9V) and wild-type immunoreceptor tyrosine-based inhibition motif (ITIM) (27–35) TARP peptides administered as a Montanide/ domains called TIGIT. TIGIT is expressed by T cells GM-CSF peptide emulsion or as an autologous peptide- and NK cells. It competes with the costimulatory mol- pulsed dendritic cell vaccine, every 3 weeks for a total of ecules CD226/DNMA-1 for binding to the same ligands fve vaccinations with an optional 6th dose of vaccine at CD155/PVR and CD112 expressed by antigen-presenting 36 weeks based on immune response or PSADT criteria cells and tumor cells [109]. with a booster dose of vaccine for all patients at 48 and TIGIT was shown to be co-expressed with PD-1 by 96 weeks [111]. Per protocol, Peripheral Blood Mononu- tumor antigen-specifc CD8+ T cells in melanoma and clear Cells (PBMCs) were collected by apheresis, PBMCs TIGIT ligands are highly expressed in metastatic mela- were enriched for monocytes by counter-fow elutria- noma [110]. Dual TIGIT/PD-1 blockade increases the tion (20–80%) and aliquots of the monocytes were cryo- expansion and function of human tumor antigen (TA)- preserved. Subsequently, thawed monocytes culture for specifc CD8+ T cells. In addition, others have also 3 days with IL-4 + GM-CSF and then culture for 1 day observed that dual PD-1/TIGIT blockade promotes with LPS + IFN-1. On the last day of culture the DCs tumor regression in multiple mouse tumor models [110]. were pulsed with HLA-A2-restricted TARP peptides, TIGIT is also upregulated by human Tregs in the harvested and infused. 72% (n = 41) of patients reach- TME, and TIGIT high CD4+ TILs are highly immuno- ing 24 weeks and 74% reaching 48 weeks had a decreased suppressive. Targeting TIGIT with Fc-engineered mAbs slope log (PSA) compared to their pre-vaccination base- may prove efcacious to deplete Tregs in patients with line (p = 0.0012 and p = 0.0004 for comparison of overall advanced melanoma. changes in slope log (PSA), respectively). TARP vaccina- In conclusion, TA-specifc CD8+ T cells in the periph- tion also decreased by 50% in median tumor growth rate ery and at the tumor sites co-express a number of inhibi- (g): pre-vaccine g = 0.0042/day, post-vaccine g = 0.0021/ tory receptors in addition to PD-1, including TIGIT. day (p = 0.003). 80% of subjects exhibited new vaccine- TIGIT ligands are highly expressed in the TME of mela- induced TARP-specifc IFNγ ELISPOT responses but noma and many other solid tumors. Dual PD-1/TIGIT these responses did not correlate with decreases in blockade augments the expansion and function of human slope log (PSA). Tus, vaccination with TARP peptides TA-specifc CD8+ T cells in vitro and promotes tumor resulted in signifcant slowing in PSA velocity and reduc- regression in multiple mouse tumor models. Tese data tion in tumor growth rate in most of patients with PSA served as the rationale for ongoing clinical trials with biochemical recurrence [111]. dual PD-1/TIGIT blockade in patients with advanced Tis study suggests TRAP DC vaccines could be cancers, including melanoma. improved by understanding lot-to-lot variability and better understanding of the mechanisms of action and Advancing dendritic cell cancer immunotherapy biomarkers associated or predictive of clinical response. In stage D0 prostate cancer, PSA recurrence without evi- TARP DCs are autologous therapies and a unique prod- dence of measurable disease is a clinical problem with uct is manufactured for each vaccine, consequently, limited treatment options. Immune therapy could delay patient factors can contribute to product variability. the initiation of androgen deprivation therapy. In fact, a Sources of variability of cellular therapies are starting pilot study of vaccination with epitope-enhanced T cell materials (patient biology in terms of genetic, gender, receptor alternate reading frame protein (TARP) and race and age), patient disease (type, stage and treatment); TARP peptide-pulsed dendritic cells for the treatment manufacture related (starting material, separation, of stage D0 prostate cancer has recently been completed response to stimuli and metabolism). [111]. TARP is a novel 58 amino acid protein, expressed A very recent study a subset of stage D0 prostate on 85–95% of prostate cancer specimens, which origi- cancer patients treated with TRAP DC vaccines aimed nates from prostate epithelial cells, not infltrating T to identify DC markers correlating with clinical and cells. It is expressed on normal prostate epithelium and immunologic response to the vaccination regimen. over-expressed in prostate cancer. TARP expression is TRAP DCs from 18 vaccinated patients were exten- associated with conventional markers of unfavorable sively characterized [112]. Peptide-pulsed DC prepa- and more aggressive tumor behavior. Te study inves- rations were analyzed by gene expression profling, tigated TARP peptide vaccination’s impact on the rise cell surface marker expression and cytokine release Ascierto et al. J Transl Med (2017) 15:236 Page 19 of 31

secretion, and correlated with clinical and immuno- administration of non- or low-permeant cytotoxic drugs, logic responses. Characteristics of the fnal DC prod- such as bleomycin. Cell membrane permeabilization per- uct were: > 95% for all products (CD80, CD83, CD86, mits the drug to enter the target cells and eventually to CD123, CD11c, CD38, CD54, and HLA-DR); variable trigger cell death through multiple DNA breaks, which expression (CD14: range 14–90%, CCR7: range 5–90%, are lethal for dividing cells. viability: range 37–91%, DC yield range 6–48%). Com- Electroporation and electroporation-based therapies parison of TARP DCs associated with PSA clinical are promising approaches for the treatment of cancer. response and non-response showed in responders a Te following medical applications of electroporation trend toward higher levels of CCR7 and a trend toward have already been developed and brought to clinics: lower levels of CD14. Hierarchical clustering and prin- cipal component analysis of the gene expression data 1. Electroporation to transfer drugs and small mol- revealed that DCs clustered by patient with no separa- ecules is the combination of EP and cytotoxic drugs tion of responders and non-responders. A class com- that do not freely cross the plasma membrane. ECT parison of clinical responders and non-responders was the frst application that reached the clinical found 55 diferentially expressed genes (false discovery stage. ECT works very efciently and without major rate = 65%). Te weighted gene coexpression network side efects. It selectively kills the tumor cells and analysis (WGCNA) method was used for further analy- spares the normal non-dividing cells in the volume sis of the gene expression data. WGCNA is a systems exposed to electric pulses (EPs) (usually eight short biology method, describing correlation patterns across pulses of 100-μs duration). However, ECT remains microarray samples, it is not biased and it fnds clus- a local treatment with no obvious efects on distant ters or modules of highly correlated genes. Tis analysis metastases. found that DCs showing lower expression of a tolero- Two anti-cancer molecules bleomycin and cisplatin genic gene signature induced strong antigen-specifc have met these prerequisites and are currently used immune response and slowing in PSA velocity, a surro- in the clinical practice of ECT. Tese drugs, once gate for clinical response. Tese DCs were also charac- internalized into cells via the local delivery of EPs, terized by lower surface expression of CD14, secretion generate DNA lesions, either both single-strand and of IL10 and MCP-1 (CCL2), and greater secretion of double-strand DNA breaks (if bleomycin is used) MDC (CCL22). When combined, these four factors dis- or adducts and intra-strand and inter-strand DNA criminate DCs inducing strong immunologic response. bonds (if cisplatin is used), ultimately leading to cell In conclusion, tolerogenic TARP DCs are associated death. with a poor immunological and clinical response and After cell electroporation, large amounts of the cyto- candidate DC potency markers include CD14, IL-10, toxic molecules enter cells by difusion, regardless of CCL2 and CCL22. However, lot-to-lot variability is a the cell type. Electroporation thus turns bleomycin critical issue for DC-based immunotherapies. Tere is and cisplatin into very efcient drugs in all tumor no best method to identify cell therapy biomarkers and types, as verifed in preclinical and clinical studies. both hypothesis and discovery driven approaches are A wide range of tumors has been treated by ECT, efective. Product variation can be used to better under- mainly using bleomycin. Tese now include primary stand mechanism of action and identify potency markers. tumors (basal cell carcinoma) and metastases of Work is ongoing to improve the consistency of DC-based head and neck carcinomas, Kaposi’s sarcoma, breast immunotherapies. adenocarcinoma, and melanoma. Clinical trials are nowadays addressing the treatment of deep-seated Biomarkers session tumors (primary pancreatic carcinoma and bone, The promising alliance of electrochemotherapy liver, and brain metastases). and immunotherapy In addition, ECT demonstrates potent anti-vascular Electroporation (EP) consists in the delivery of a lim- efects. A transient vasoconstriction is observed ited number of short and intense electric pulses which following EPs delivery alone, and moreover, the are defned by an intensity E and a duration t. Above a endothelial cells forming tumor blood vessels are certain threshold of the E and/or t parameters, cell mem- also sensitive to ECT (as any proliferative cell in the brane defects appear and result in cell permeabilization. treated region). Consequently, these phenomena After a given lag time, cell membrane integrity is restored result in tumor starvation (lack of oxygen and growth leading to cell survival. factors) and thus contribute to cancer cell death. Electrochemotherapy (ECT) consists in the deliv- Nowadays, ECT is used in routine in about 140 Euro- ery of short and intense electric pulses following the pean cancer centers. Ascierto et al. J Transl Med (2017) 15:236 Page 20 of 31

Technology was developed in the Cliniporator EU 3. Irreversible electroporation (IRE) consists in the use project. Ten, the EU-funded ESOPE clinical study of excessive electroporation to cause cell death. Dif- demonstrated an objective response rate of 85% in ferent approaches can lead to this outcome including ECT treated tumor nodules, regardless of the tumor the use of very long or very intense electric pulses. histology and drug used or route of its administra- Tis local ablative treatment is not selective against tion [113]. It also established the standard operating the tumor cells, killing also the normal cells in the procedures for ECT use in the clinic i.e., a series of volume of tissue exposed to the EP [114]. eight electric pulses (EPs) of 100 μs and appropriate 4. Tere is also an interest in the combination of anti- feld amplitude must be delivered using either inva- tumor ECT with immunotherapy for long-term and sive (EPs of 1000 V/cm) or non-invasive electrodes systemic anti-tumor responses. Intratumoral recruit- (EPs of 1300 V/cm), depending on the depth and ment of dendritic cells (DCs) expressing CD80/ on the size of the nodules to treat. Recently, in addi- CD86 maturation markers, circulating monocytes tion to treating cutaneous and subcutaneous tumors and splenic T lymphocytes was observed after ECT the treatment of deep-seated tumors was applied in treatment in animal model. Tese studies high- clinical trials. Clinical trials are also ongoing for bone light immune system activation after the treatment. metastases, liver metastases, brain tumors, pancreas, Recently, the mechanisms underlying this immune colorectal cancers (endoscopic electrodes), as dem- activation were identifed. ECT induces an immuno- onstrated in number of published reports. genic cancer cell death (ICD) through the liberation 2. Electroporation to transfer nucleic acids inside the of ATP and HMGB1 and the translocation of cal- cells, the electroporation-based gene transfer, consti- reticulin to the cell surface. Immunogenic cell death tutes a subcategory of gene therapy. Electroporation is responsible for the generation of tumor-specifc T mediated gene therapy, namely, electrogenetherapy cells [113, 114], that are able to kill non-ECT-sensi- (EGT), is an approach rapidly expanding in cancer tive cancer cells within the primary tumor. Interest- and non-cancer therapeutic domains. Gene electro- ingly, cancer stem cells, thought to be responsible for transfer can be achieved by frst permeabilizing the cancer recurrence and metastasis, seem sensitive to cell membrane thanks to short and intense electric both extracellular ATP and T cell recognition. Tese pulse deliveries and second by driving electrophoreti- tumor-specifc T cells can also target metastatic nod- cally the DNA toward the electroporated membrane ules, although there is a lack of direct evidence in the thanks to one or several long and low-voltage elec- absence of a complementary immune stimulation. tric pulses. It is expected that a protein of interest is Immunotherapy agents (e.g., cytokines, therapeutic produced and epitope presentation occurs on MHC antibodies, immune checkpoint blockers, and genes) molecules. mount immune responses that are synergistic with DNA vaccination consists of the administration the one triggered by ECT. Overall, this ECT-driven of a DNA encoding a cancer antigen of interest. immune activation might be responsible for control- Encoded antigen will be responsible for the genera- ling local relapses and metastatic spread. tion of a pool of specifc B and T cells, from which Antigen presenting cells (APCs), mostly DCs, are of some will remain as memory cells for long-term great importance for the outcome of vaccination as protection. Tumor-specifc CD8+ T cell generation, they ensure efective T cell priming and maintenance. associated with the secretion of T1 cytokines (e.g., Terefore, EPs appear to play a pivotal role in anti- tumor necrosis factor alpha, TNFα and IFNγ) was cancer DNA vaccination, not only by enhancing the demonstrated. Gene electrotransfer is one of the transgene expression but also by recruiting APCs in most efcient non-viral techniques and has proven the electroporated tissues: in vivo electroporation its efciency in many tissues, should they be superf- can also serve as an adjuvant for DNA vaccination cial (e.g., skin) or internal (e.g., liver and muscle). Te [115]. ECT is more efcient in immunocompetent main limit to the use of gene electrotransfer is related mice as compared with immunodefcient animals to its application to not easily accessible organs. [114, 116]. It has been applied in humans as mono- Te two most advanced EGT strategies are related therapy and in combination approaches [117–119]. to immunotherapy, namely, DNA vaccination and Recently Mozzillo et al. reported that the combina- cytokine-based anti-cancer therapies. Intratumoral tion of ipilimumab and ECT may be benefcial for the administration of an IL-12-encoding plasmid in con- treatment of metastatic melanoma [118]. Te study junction with EPs demonstrated local and systemic reports that the volume of distant non-ECT-treated anti-tumor efects and improved cure rates of tumors tumors decreased or was stabilized in 9 patients out in animal models as well as in patients. of 15, possibly through ipilimumab-induced Tregs Ascierto et al. J Transl Med (2017) 15:236 Page 21 of 31

depletion. Local ECT treatment of cutaneous lesions by the heterodimeric transporter associated with antigen of melanoma was followed by ipilimumab admin- processing (TAP) complex into the lumen of the endo- istration resulting in the complete regression of all plasmic reticulum for loading of beta2 microglobulin the cutaneous and visceral metastases for at least (β2m)-associated HLA class I α and β chain dimers with 1 year. Interestingly, vitiligo-like lesions developed the help of the chaperone molecules ERp57, calnexin, cal- exclusively around the sites of previous ECT, suggest- reticulin and tapasin. Te resulting trimers then travel to ing that a prior ECT-driven immune activation was the plasma membrane and tumor antigen derived pep- enhanced by ipilimumab [120]. Electrochemotherapy tides are presented to cognate T cells. in combination with ipilimumab or pembrolizumab Te role played by HLA class I APM in the response or nivolumab was also studied [119]. Combination to therapy with immune checkpoint inhibitors has rekin- of ECT with immunotherapy is likely to transform dled interest in the characterization of the defects in the ECT from a local to systemic therapy [121]. ECT HLA class I APM component expression and/or function has numerous advantages such as: no side efects; in malignant cells, since these defects may represent one is not immunosuppressive; stimulates the immune of the mechanisms underlying the resistance to immune system; rarely causes bleeding even after needles checkpoint inhibitor-based therapy. Extensive review insertion; possess anti-hemorrhagic efects; high of the literature describing HLA class I APM compo- specifcity against tumor cells; treatment planning nent expression in many types of solid tumors showed procedures are available; standardized equipment that these defects are present in all types of solid tumors is available; has reasonable cost; is efcient and fast; tested with a frequency of at least 40%; in some cancer and requires minimal post treatment care. In conclu- types the frequency of HLA class I APM defects can be sion, ETC-based strategies represent highly promis- as high as 75% [122]. Tese defects have functional rel- ing approaches to induce anticancer systemic efects evance, since they cause resistance of cancer cells to cog- and beneft for patients. nate T cells’ recognition and lysis [123] and have clinical relevance in at least some cancer types [122]. In most HLA class I antigen processing machinery defects cases HLA class I APM component downregulation or in malignant cells in the era of immunotherapy loss is associated with poor clinical course of the disease. of malignant diseases with immune check point inhibitors Tis association has been suggested to refect the escape Te results of a growing number of clinical trials in large of malignant cells from immune surveillance. Defective populations of patients with many types of malignan- HLA class I-tumor antigen derived peptide processing, cies have convincingly shown that immunotherapy with HLA synthesis and complex assembly can cause abnor- checkpoint inhibitors as monotherapy or in combina- malities in expression of HLA class I APM components tion with other agents including immunomodulators and/or function. As a result, the malignant cell recogni- can induce long lasting clinical responses in patients tion by cognate T cells is defective. Furthermore, in a lim- with malignant diseases. However, these therapeu- ited number of patients (mostly melanoma) who respond tic efects occur only in a small number of the treated to T cell-based immunotherapy, disease recurrence was patients with a given type of cancer. Furthermore, the associated with HLA class I APM component down reg- frequency of responses is markedly diferent among the ulation/loss [124]. However, in a few cancer types high various types of cancer. Several lines of evidence includ- expression of HLA class I has been found to be associ- ing the increased lymphocyte infltration in tumors, the ated with poor prognosis [122]. Tis unexpected associa- correlation between mutation load in tumors and clini- tion has been suggested to refect more important role cal outcome in patients treated with immune checkpoint of NK cells than that of T cells in the control of tumor inhibitors are consistent with the possibility that clinical growth in these malignancies. High HLA class I expres- responses refect the recognition and elimination of can- sion by tumor cells can inhibit the anti-tumor activity of cer cells by cognate T cells unleashed by immune check NK cells due to interaction of killer inhibitory receptors point inhibitors. Te interactions between tumor cells (KIR) on NK cells with HLA class I that is a KIR-ligands. and cognate T cells are mediated by HLA class I-tumor Diferent types of HLA class I defects have been identi- antigen derived peptide complexes. Teir synthesis and fed in malignant cells: expression require a fully functional HLA class I antigen processing machinery (APM). Specifcally, proteasome 1. Total HLA class I antigen loss to selective loss of the isoforms and their active subunits degrade down mostly, HLA class I allospecifcities encoded in the genome although not exclusively, endogenous proteins to pep- of a tumor bearing patient. Te latter include loss of tides with the correct length and sequence for HLA class the gene products of one or two HLA class I loci or I binding. Proteasomal degradation products are shufed of the HLA class I allospecifcities encoded by the Ascierto et al. J Transl Med (2017) 15:236 Page 22 of 31

genes present in the major histocompatibility com- a limited number of studies has correlated the expres- plex region of one of the parental chromosomes 6. sion of HLA class I APM components in tumors with 2. Down-regulation represents the most frequent type response to immune checkpoint inhibitor therapy or has of abnormality in APM components, as complete analyzed the expression of HLA class I APM compo- lack of expression has been described only for a few nents in tumors from patients who have not responded APM components such as TAP1 and tapasin, and for to this type of therapy or have acquired resistance to it. each of them in a few cases. Since expression of the However, studies begin to analyze the role of interactions HLA class complex components is determined by the between HLA class I antigens and checkpoint molecules co-dominance of the two HLA genes (one of pater- in the clinical course of the disease. High HLA class I nal and the other one of maternal origin) as well as antigen expression in intrahepatic cholangiocarcinoma β2m, multiple factors may contribute to the level of and in esophageal cancer has been found to be associated the HLA I complex expression. with good prognosis only when PD-L1 is not detected 3. Structural mutations of the genes encoding HLA on tumor cells [127, 128]. One might argue that when class I APM components have been found at most PD-L1 binds to PD-1 on cognate T cells and inhibits in 5% of the cases. Structural abnormalities in β2m their anti-tumor activity, HLA class I antigen expression which caused lack of HLA class I antigen expression may not play a major role in the interactions of tumor by tumor cells have been reported to be associated cells with cognate T cells. In contrast, when PD-L1 is with resistance to immune checkpoint inhibitors. not expressed on tumor cells and cognate T cells are able A patient with advanced melanoma who acquired to attack tumor cells, HLA class I antigen expression is resistance to the anti-PD-1 antibody, pembroli- necessary to recognize and kill malignant cells. Te lat- zumab, harbored a homozygous β2m truncating ter scenario is likely to mimic what happens in the clini- mutation [125]. In addition, β2m alterations have also cal setting when the PD-1/PD-L1 axis is disrupted with been described in two brain metastases from patients inhibitors. Tese fndings imply that selection of patients with mismatch repair defcient colorectal cancer who to be treated with immune checkpoint inhibitors should were resistant to immune check point-based therapy consider the expression of HLA class I APM components [126]. by the tumor cells. When this machinery is not fully 4. Epigenetic mechanisms appear to be the most fre- functional, HLA class I independent immunotherapeutic quent cause of defects in HLA class I APM compo- strategies appear to be the strategies of choice. nent expression [122]. Tey include histone acety- A word of caution about the absolute requirement of lation defects and methylation of the promoters of a fully functional HLA class I APM in tumor cells for the genes encoding HLA class I APM components. response to anti-PD-1 therapy. About 70% of patients Strategies to counteract the epigenetic mechanisms with Hodgkin disease carry β2m-inactivating mutations have been developed. Tey include the use of his- in their tumor cells, which cause lack of HLA class I tone acetylation inhibitors and demethylating agents. expression [129]. Nevertheless, these patients show dra- Tese approaches have been shown to restore HLA matic response rates to anti-PD-1 therapy [130]. Whether class I APM component expression and/or function this surprising result refects the targeting of HLA class II in vitro, in animal model systems and/or in clinical antigen bearing tumor cells by CD4 cells remains to be settings. Tese results emphasize the need to char- determined. acterize the expression and function of HLA class I APM in tumors from patients who are resistant or Prognostic and predictive markers for patients treated acquire resistance to immune checkpoint inhibitor with CTLA‑4 and PD‑1 inhibitors therapy. Te resulting information may contribute Recently it was demonstrated that patients who dis- to the successful design of therapies which combine continue nivolumab in combination with ipilimumab immune checkpoint inhibitors with strategies which due to drug toxicity derive an OS beneft similar to that restore the expression and/or function of HLA class observed in the overall population at 18-month follow-up I APM components. Tis combinatorial therapy is [131]. Diferent diagnostic tests have been approved to expected to restore patients’ sensitivity to immune determine PD-L1 expression in tumor, with diferent cut- checkpoint inhibitor therapy at least in some cases of positivity. Response by PD-L1 expression level (5%) in and to enhance the efcacy of this type of therapy in Checkmate067 showed that patients with PD-L1 positive others. tumors treated with nivolumab in combination with ipili- mumab had higher ORR [132]. Despite the potential role of HLA class I APM in the A recent study evaluated the expression of PD-L1 in response to therapy with checkpoint inhibitors, only immunotherapy-naïve metastatic melanoma patients and Ascierto et al. J Transl Med (2017) 15:236 Page 23 of 31

demonstrated that PD-L1 expression was frequently dis- p < 0.05). Early increase of ALC and delayed increase cordant between primary tumors and metastases as well of CD4+ T cells and early increase of ALC and delayed as between intrapatient metastases [133]. A positive uni- increase of CD4+ and CD8+ T cells were also signifcant variate association between PD-L1 expression in locore- [134]. gional metastases and melanoma-specifc survival was LDH, routine blood count parameters, and clinical observed, but not for primary melanoma. In locoregional endpoints OS and best overall response following pem- lymph node metastasis, PD-L1+/TIL+ patients had the brolizumab treatment were investigated in 616 patients best outcome, and PD-L1+/TIL− patients had poor out- [137]. Te biomarkers studied for pembrolizumab, by come [133]. multivariate model with Cox regression analysis for Studies of survival rate and PD-L1 expression in the overall survival, were the pattern of visceral metastases; CheckMate 066 trial demonstrated that, patients treated serum levels of LDH; relative lymphocyte count; and rel- with nivolumab had improved overall survival regard- ative eosinophil count. Biomarkers showed independent less of PD-L1 status, as compared with dacarbazine- positive correlation (all p < 0.001), that was subsequently treated patients however the median overall survival confrmed in the validation cohort (n = 257; all p < 0.01). was not reached in either PD-L1 subgroup treated with Te probability to survive 12 months after the frst dose nivolumab. In the dacarbazine group, the median overall was 15% in patients with none out of four favorable fac- survival was slightly longer in the subgroup with positive tors, in contrast to 84% for patients with all four favora- PD-L1 as compared with the subgroup with negative or ble factors present. Because of the large diferences in indeterminate PD-L1 status. outcome according to the number of favorable factors, it Te assessment of biomarkers associated with clinical remains unclear whether the combination model is pre- outcome following ipilimumab treatment in advanced dictive of pembrolizumab treatment beneft [137]. melanoma patients showed that low Serum Lactate Table 3 summarizes the prognostic and predictive Dehydrogenase (LDH), absolute monocyte counts markers for checkpoint inhibitors. (AMC), and MDSCs as well as high absolute eosinophil Te presence of circulating T cells responding to counts (AEC), Tregs, and relative lymphocyte counts Melan-A or NY-ESO-1 had strong independent prognos- (RLC) is associated with favorable outcome following tic impact on survival in advanced melanoma, suggesting ipilimumab [134]. Patients (43.5%) presenting with the that these could be targets for immunotherapy [138]. In best biomarker signature (a baseline signature of low cohort A patients (84 patients with follow-up after analy- LDH, AMC, and MDSCs as well as high AEC, Tregs, and sis), the presence of T cells responding to peptides from RLC) had a 30% response rate and median survival of NY-ESO-1, Melan-A, or MAGE-3 and the M category 16 months. In contrast, patients with the worst biomark- were signifcantly associated with survival. NY-ESO-1 ers (27.5%) had only a 3% response rate and median sur- and Melan-A (hazard ratios, 0.29 and 0.18, respectively) vival of 4 months [135]. remained independent prognostic factors in Cox regres- Te impact of γδ T-cells on OS and of the frst dose of sion analysis and were superior to the M category in ipilimumab in melanoma patients was assessed in 109 predicting outcome [138]. Median survival of patients pos- melanoma patients 109 healthy controls [136]. Patients sessing T cells responding to NY-ESO-1, Melan-A, or both with higher frequencies of Vδ1+ cells (≥ 30%) had was 21 months, compared with 6 months for all others. poorer OS (p = 0.043). In contrast, higher frequencies of Melan-A responses were found in 42 and 47% of patients Vδ2+ cells (≥ 39%) were associated with longer survival in cohort A and B (24 months survival after frst occur- (p = 0.031) independent of the M category or lactate rence of distant metastases; cohort B), respectively. In dehydrogenase level. Besides, frequencies of both Vδ1+ Table 3 Biomarkers for checkpoint inhibitors: prognostic and Vδ2+ cells demonstrated to be candidate biomarkers for outcome in melanoma patients following ipilimumab (in italics) and predictive details [136]. Ipilimumab Pembrolizumab Changes in blood counts and frequency of circulating immune cell populations analyzed by fow cytometry Lactate dehydrogenase Stage III-IVB/IVC were investigated in 82 patients to compare baseline val- Relative lymphocyte counts (RLC) Lactate dehydrogenase (LDH) ues with diferent time-points after starting ipilimumab. Absolute eosinophil counts (AEC) Relative lymphocyte counts (RLC) Endpoints were OS and best clinical responses [134]. Absolute monocyte counts (AMC) Relative eosinophil counts (REC) ALC increases at 2–8 weeks (p 0.003) and CD4 Tregs: unknown = + MDSC: unknown and CD8+ T cells 8–14 weeks (p = 0.001 and p = 0.02) after the frst dose of ipilimumab were correlated with γδ T-cells: uncertain Increase CD4 CD8 T cell: uncertain improved survival and with clinical responses (all + + Ascierto et al. J Transl Med (2017) 15:236 Page 24 of 31

contrast, the proportion was only 22% for NY-ESO-1 and to patients with melanoma without testing for PD-L1 23% for Melan-A in those who died within 6 months [138]. expression. Tis is in contrast with non-small cell lung Identifying peptides recognized by individual T cells carcinoma, where approvals for the agent itself were is important for understanding and treating immune- often accompanied by approval for the Companion or related responses. A peptide–Major Histocompatibility Complementary diagnostic. Complex (MHC) multimers labeled with individual DNA Te publicly available data on PD-L1 IHC testing in barcodes to screen > 1000 peptide specifcities in a sin- patients with metastatic melanoma as of October 2015, gle sample that detect low-frequency CD8 T cells specifc shows that practitioners tested approximately 37% of for virus- or cancer-restricted antigens was investigated their patients for PD-L1 expression vs. testing 98% of [139]. Several neoepitope-specifc T cells in tumor-infl- patients for BRAF mutations (Source: BrandImpact/ trating lymphocytes were identifed. Barcode-labeled BrandImpact Dx). When melanoma patients were tested, pMHC multimers enable the combination of functional only 20% of surgical pathology labs were running CDxs, T-cell analysis with large-scale epitope recognition profl- with more labs either running a laboratory developed ing to characterize T-cell recognition in various diseases, tests (LDT) for PD-L1 IHC or sending the test out to be including in clinical patients samples [139]. performed by a Reference Laboratory. Since the PD-L1 In conclusion, prognostic and predictive markers for IHC Complementary Dx for melanoma was approved by patients treated with CTLA-4 and PD-1 inhibitors are: FDA after this survey was conducted, it is possible that the proportion of oncologists ordering PD-L1 testing as 1. Clinical outcome of frst shot which may be the most well as surgical pathology labs performing the test will relevant biomarker. increase in subsequent surveys. 2. Established prognostic markers like tumor-stage and Tere are numerous commercially available PD-L1 LDH which are valid in checkpoint inhibitor therapy. immunohistochemistry (IHC) assays (Table 4). It is 3. Lymphocyte and eosinophil counts which may have impractical for most surgical pathology laboratories to predictive value. host four diferent IHC assays for PD-L1, thus there is 4. Preexisting functional antitumor T cell responses great interest in understanding the comparative perfor- which should be better analyzed. mance of these assays. To that aim, the Blueprint PD-L1 IHC Assay Com- Update on PD‑L1 and related markers parison Project was initiated. It is an industrial-academic FDA approvals for immune checkpoint blockade agents collaborative partnership to provide information on the have been numerous both in melanoma and other analytical performance of these four PD-L1 IHC assays tumors. A number of these approvals have been accom- [140]. Phase 1 was conducted by staining n = 40 NSCLC panied by immunohistochemical (IHC) assays for PD-L1 with each of the four PD-L1 IHC assays (using clones in the form of complementary or companion diagnostics 22C3, 28-8, SP142, and SP263). Both the percent of tumor (CDxs). cells and immune cells staining for PD-L1 were assessed Complementary diagnostic for melanoma was by three pathologists, and their scores were averaged. approved approximately 5 years after anti-CTLA-4 was Tree of the four assays (22C3, 28-8, and SP263) were frst approved, and as such, practitioners were well- closely aligned on tumor cell staining whereas the fourth accustomed to administering checkpoint blocking agents (SP142) showed consistently fewer tumor cells stained.

Table 4 Commercially available PD-L1 IHC assays BMS Merck Roche Astra Zeneca mAb clone 28-8 22C3 SP142 SP263 Automated Yes Yes Yes Yes Diagnostic partner Dako Dako Ventana Ventana Machine Link 48 Link 48 Benchmark ULTRA Benchmark ULTRA Scoring Tumor cells (membrane) Tumor cells (membrane) Tumor cells/or immune (membrane) Tumor cells (membrane) Positive cutof 5% (also studied 1% and 10% 1% for trial enrollment TC3 (> 50%) or IC3 (> 10%) 25% ≥thresholds ≥ ≥ Other≥ analysis at > 50% TC2/3 or IC2/3 (> 5%) ≥ TC 1/2/3 or IC 1/2/3 (> 1%) TC0 and IC0 (0%)

The scoring systems noted below often depend on the line of therapy and the tumor type being tested. An increasing number of assays from diferent companies are reaching the market Ascierto et al. J Transl Med (2017) 15:236 Page 25 of 31

All the assays demonstrated immune cell staining, but prognostic algorithms. While genomic studies and gene with greater variability than with tumor cell staining. signatures contribute global assessments of the tumor A second study tested 90 archival NSCLCs stained by microenvironment, emerging data reinforces the con- diferent PD-L1 IHC assays and scored by 13 patholo- tribution of spatially-resolved approaches to protein gists. Te assays tested in this second study included: (1) expression. Future biomarker panels for patients with 28-8 assay on Dako Link 48; (2) 22c3 assay on Dako Link melanoma will likely include multiplex IHC/immuno- 48; (3) SP142 assay on Ventana Benchmark (LDT mim- fuorescent studies as well as genomic studies and gene icking the IUO test); and (4) E1L3N antibody on Leica signatures. Assay development will thus require the inte- Bond (LDT). Similar to the Blueprint study, the assay gration and prioritization of these diferent modalities. using the SP142 antibody was an outlier detecting sig- nifcantly less tumor cell PD-L1 expression. It was also Understanding responses to cancer therapy: lessons an outlier with respect to detecting immune cell PD-L1 learned from mouse and man expression [141]. Te Cancer Genome Atlas (TCGA) program per- To determine whether the performance characteris- formed a systematic multi-platform characterization of tics of the SP142 assays were due to the SP142 antibody 333 cutaneous melanomas at the DNA, RNA, and pro- itself or to the assay conditions, we conducted a study tein levels to create a catalog of somatic alterations and where we held all of the other assay conditions (antigen describe their potential biological and clinical signif- retrieval, bufers, amplifcation systems) essentially con- cance. TCGA represents the largest integrative analysis stant and changed only the antibody. In this study, we of cutaneous melanoma; it establishes a framework for assessed archival melanoma cases, and compared the melanoma genomic classifcation (BRAF, RAS, NF1, and 5H1, SP142, SP263, 22C3, and 28-8 antibody clones. We Triple-WT), it identifes additional subtypes that may found that all antibodies could demonstrate similar stain- beneft from MAPK-and RTK-targeted therapies and ing properties regarding labeling PD-L1 display by both multi-dimensional analyses identify immune signatures tumor cells and immune cells, if all other assay conditions associated with improved survival [144]. were constant. Tese fndings indicate that it is likely the Terapeutic targeting of oncogenic BRAF mutations assay conditions (beyond the primary antibody itself) results in rapid regression of melanoma tumors in most that contribute to the diferences observed between the patients. Clinical trials showed that treatment with BRAF SP142 assay and the other studied assays [142]. inhibitors resulted in a survival beneft over then stand- In addition to PD-L1, there is a number of other can- ard of care therapy with dacarbazine substantiating the didate biomarkers which have been nominated, includ- FDA approval of the frst of these agents (vemurafenib) in ing PD-1, CD8, and mutational load. PD-L2 has not been 2011. However, most of patients progressing on therapy well-studied yet, due to the lack of well-validated com- within 6 months that is the limitations of this therapy. mercially available antibodies. We next used the TCGA Insight into resistance mechanisms has led to advances in dataset to assess how these diferent markers related to therapy, including the use of combined BRAF and MEK PD-L1 expression. We found that PD-L1, PD-L2, PD-1, inhibition, which was shown to improve progression free cytolytic activity (CYT), and mutational load are all posi- survival in patients; even with the combination, most of tive prognostic features in patients with melanoma. Nota- patients progress within a year. Tere is a critical need to bly, PD-L1, PD-L2, PD-1 and CYT are all closely linked to identify pre-treatment biomarkers of response and resist- each other and to T1/IFNγ expression in patients with ance, as well as early on-treatment markers of resistance melanoma, while mutational load is not. When principal which are potentially actionable. component analysis was conducted on these fve vari- Substantial research eforts internationally focused on ables, PD-L1, PD-L2, PD-1 and CYT all formed the frst response and resistance to targeted therapy for mela- principal component, with each contributing near equally, noma in biopsies of patients treated with BRAF inhibi- while mutational load was orthogonal and formed the tors. Te team of researchers at the MD Anderson CC second principle component. We then assessed the rela- performed genomic analysis in pre-treatment and pro- tive contribution of these factors to survival using condi- gressing lesions, with immune profling in pre-treatment, tional inference modeling, which revealed that PD-1/CYT on-treatment and progressing lesions. Trough such expression (i.e., an infamed tumor microenvironment) as studies, multiple genomic mechanisms of resistance have the most impactful feature, followed by mutational den- been identifed and published. sity in tumors that were less infamed [143]. Although oncogenic mutations contribute to tumor Additional studies are underway to perform similar escape via multiple mechanisms, including through analyses as they relate to predicting response or resist- immune evasion mutations can make tumors more ance to checkpoint blockade, as well as further refning immunogenic. Terefore, in addition to studying Ascierto et al. J Transl Med (2017) 15:236 Page 26 of 31

genomic mechanisms of resistance, immune profling in tumor biopsies including mutational load at baseline was also performed in tumors over the course of therapy with evidence that patients with a higher mutational load to gain insights into anti-tumor immunity was researched have improved responses to checkpoint inhibitor therapy out. Specifcally, T cell infltrate and markers of cytotox- [47] though this is not perfectly predictive and better bio- icity, the expression of immunosuppressive cytokines and markers are needed. In addition, immune markers are VEGF as well as PD-1 and PD-L1 were assessed. Trough being studied including CD8 T cell density and distribu- these studies, immune mechanisms of response and tion as well as IFNγ response gene signatures [96, 97]. resistance to targeted therapy were also identifed. Treat- Cohort of patients with metastatic melanoma who ment with BRAF-targeted therapy increased melanoma initially received Ipilimumab and then went onto PD-1 antigens expression (e.g., MART) and CD8+ T cells blockade therapy, and isolated tissue samples from mul- infltrate resulting in more favorable TME. Furthermore, tiple time points on therapy and embarked on a deep decreased the level of immunosuppressive cytokines and molecular and immune profling of these tumors was VEGF, within 2 weeks of starting therapy providing sup- carried out [146]. In this study, immune signatures in port for potential synergy of BRAF-targeted therapy and pre-treatment tumor biopsies assessed by a 12-marker immunotherapy [69]. However along with these favorable immunohistochemistry panel largely failed to predict changes, an increase in expression of the immunomodu- response to PD-1 blockade, but signatures in on-treat- latory molecule PD-L1 early during treatment was noted, ment biopsies were highly predictive [146]. Gene expres- suggesting a possible immune mechanism of resistance to sion profling using Custom 795 gene NanoString set was therapy. Interestingly, these favorable immune efects are used to see if responses to immune checkpoint blockade not likely to be solely related to increased melanoma anti- can be predicted via gene expression profling. Gene gens but to overall changes in the tumor microenviron- expression profles at pre-treatment time point largely ment. Hypothesis that combining targeted therapy and failed to predict response to immune checkpoint block- immune checkpoint blockade would enhance responses ade. However, signatures in on-treatment PD-1 samples to therapy was tested in mouse models. A synergy was were strongly predictive [146]. found in these studies, with delayed tumor outgrowth Tese data suggest that clinically useful biomarkers for and prolonged survival when mice were treated with response to immune checkpoint blockade exist, but spe- BRAF targeted therapy and PD-1 blockade, compared cifc time point to assess them might be critical. Te study to either therapy alone [70]. Tere was a modest CD8 needs to be validated in additional cohorts, the emphasis, T cell infltrate in the setting of BRAF inhibitor mono- should rather be on assessing adaptive immune responses therapy, but when PD-1 blockade was added to a back- in early on-treatment samples for response to checkpoint bone of a targeted therapy a dramatic increase in CD8 T therapy (and should be incorporating this into clinical cell infltrate in the tumors was observed [70]. Based on trials) instead on pre-treatment biomarkers. Importantly, this observation, multiple trials are underway combining we are also bringing immune checkpoint inhibitor treat- these strategies for melanoma as well as other cancer. ment to patients with earlier stage disease, and have an In addition, these treatments are also being assessed ongoing phase 2 trial to test the hypothesis that treat- in patients with earlier stage disease. Upfront surgery is ment with neoadjuvant (+ adjuvant) immune checkpoint the standard treatment for patients with clinical stage inhibitors will be associated with a high pathological III melanoma. However, most patients treated with complete response (path CR) rate and immune infltrate upfront surgery will relapse and die (~ 70%). A phase 2 (NCT02519322). In this trial, patients with stage IIIB or clinical trial to test the hypothesis that treatment with stage IIIC melanoma are randomized to treatment with neoadjuvant and adjuvant BRAF/MEK inhibitors would PD-1 monotherapy versus combined CTLA-4 and PD-1 improve RFS in patients with bulky stage III melanoma blockade. Preliminary fndings are available [147] though and a BRAF mutation (vs upfront surgery and standard of mature data will not be available for some time. care adjuvant therapy) is currently underway, and trans- In addition to the infuence of the tumor and micro- lational assessments to identify molecular and immune environment in modulating responses to therapy in determinants of response are ongoing [145]. melanoma and other cancers, there is a growing role of Furthermore, mechanisms of responses to immune environmental factors that may contribute to therapeu- checkpoint blockade targeting CTLA-4 and PD-1 tic response and resistance. Pioneering work by Gajew- through translational research in human samples are also ski and Zitvogel demonstrated that bacteria in the gut studied. Not all patients respond to immune checkpoint could modulate therapeutic responses to immune check- inhibitor therapy particularly as monotherapy. Tremen- point blockade in pre-clinical models [107, 148]. We dous eforts are underway to identify predictive biomark- recently studied this in a cohort of patients with meta- ers of response to immune checkpoint blockade based static melanoma, and found that diferential bacterial Ascierto et al. J Transl Med (2017) 15:236 Page 27 of 31

3 4 signatures existed in responders versus non-responders PA, USA. IRCCS “Regina Elena” National Cancer Institute, Rome, Italy. Radia- tion Oncology and Pathology, Weill Cornell Medical College, New York City, to immune checkpoint blockade (specifcally anti-PD-1 NY, USA. 5 Department of Dermatology, University of Zurich Hospital, Zurich, therapy), and reported these fndings at the annual meet- Switzerland. 6 INSERM (National Institute of Health and Medical Research), 7 ing of the American Society of Clinical Oncology in Institut Gustave Roussy, Villejuif, France. Massachusetts General Hospital, Boston, MA, USA. 8 Department of Radiation Oncology, Weill Cornell Medi- 2017. Additional studies are currently underway to better cal College, New York City, NY, USA. 9 Division of Dermatologic Oncology, understand the mechanism behind this, and to develop Department of Dermatology, Eberhard Karls University, Tübingen, Germany. 10 strategies to enhance therapeutic responses via modula- Department of Dermatology, Yale University School of Medicine, New Haven, CT, USA. 11 Georgia Cancer Center, Augusta University, Augusta, tion of the microbiome. GA, USA. 12 Department of Hematology/Oncology, University of Chicago Another innovative technique is the use of Raman Comprehensive Cancer Center, Chicago, IL, USA. 13 CNRS (National Center spectroscopy, a noninvasive and label-free optical tech- for Scientifc Research, France), University Paris-Saclay, Gustave Roussy, Villejuif, France. 14 Division of Cancer Medicine, Department of Melanoma Medical nique that provides detailed information about the Oncology‑Research, University of Texas MD Anderson Cancer Center, Houston, molecular composition of a sample. It can potentially TX, USA. 15 Department of Medicine, Memorial Sloan Kettering Cancer Center, 16 predict skin toxicity due to tyrosine kinase inhibitors New York City, NY, USA. Weill Cornell Medical College, New York, NY, USA. 17 Department of Medicine, Roswell Park Cancer Institute, Bufalo, NY, USA. treatment. Raman spectra of skin of patients undergoing 18 Pediatrics, Human Oncology and Genetics, University of Wisconsin, Madison, treatment with MEK, EGFR, or BRAF inhibitors, which WI, USA. 19 UW Carbone Cancer Center, Madison, WI, USA. 20 Johns Hopkins 21 are known to induce severe skin toxicity were collected University School of Medicine, Baltimore, MD, USA. Center for Cancer Immune Therapy (CCIT), Department of Hematology, University Hospital (three patients were included for each inhibitor) [149]. Herlev, Herlev, Denmark. 22 Department of Immunology and Microbiology, Te algorithm, based on partial least squares-discrimi- University of Copenhagen, Herlev, Denmark. 23 Clinical Center, National 24 nant analysis (PLS-DA) and cross-validation by boot- Institutes of Health, Bethesda, MD, USA. Department of Surgical Oncol- ogy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. strapping, can identify patients with risk for cutaneous 25 Medicine, Immunology and Dermatology Institute, University of Pittsburgh adverse events. For MEK and EGFR inhibitors, discrimi- School of Medicine, Pittsburgh, PA, USA. 26 Cancer Diagnosis Program, Division 27 native power was more than 90% in the viable epidermis of Cancer Treatment and Diagnosis, NCI, NIH, Rockville, MD, USA. Istituto Nazionale Tumori di Napoli Fondazione “G. Pascale”, Via Mariano Semmola, skin layer; whereas for BRAF inhibitors, discriminative 80131 Naples, Italy. power was 71% [149]. Raman spectroscopy can detect skin toxicity induced by TKI treatment on locations not Acknowledgements This research was funded in part through the NIH/NCI Cancer Center Support afected from a dermatological and histological point- Grant P30 CA008748. A special thanks to 3P Solution of Napoli for their sup- of-view. It has better discriminative power in the case of port and cooperation in organizing the meeting and especially to Lucia Politi. MEK and EGFR inhibitors which are known to be associ- Competing interests ated with inhibition of MAPK pathway on healthy tissue PAA has/had a consultant/advisory role for BMS, Roche-Genentech, MSD, contrary to BRAF inhibitors. Tere is good correlation Array, Novartis, Amgen, Merck Serono, Pierre Fabre, Incyte. He also received (> 80%) of Raman signature of the skin and drug concen- research funds from BMS, Roche-Genentech, and Array. He is also Section Editor for Combination Strategies for Journal of Translational Medicine. SD has tration into the blood in the case of EGFR inhibitors and received honorarium from Eisai Inc., Lytix Biopharma, Nanobiotix, and EMD VE skin layer. Serono for advisory services. RD has intermittent, project focused consulting and/or advisory relationships with Novartis, Merck Sharp & Dhome (MSD), Bristol-Myers Squibb (BMS), Roche, Amgen, Takeda, Pierre Fabre outside the Abbreviations submitted work. CG reports personal fees from Amgen, grants and personal ALC: absolute lymphocyte counts; ATP: adenosine triphosphate; AFP: fees from BMS, personal fees from MSD, grants and personal fees from alpha-fetoprotein; BRAFi: BRAF inhibitors; COG’s: Children’s Oncology Group; Novartis, personal fees from LEO, personal fees from Philogen, grants and per- CR: complete response; CYT: cytolytic activity; DCs: dendritic cells; IDO: sonal fees from Roche. JL reports consultancy for Actym, Amgen, Array, Astra- indolamine 2,3 dehydrogenase; DRR: durable response rate; GCT: germ cell Zeneca, BeneVir, Bristol-Myers Squibb, Castle, CheckMate, EMD Serono, Gilead, tumor; HLA: human leukocyte antigen; HIF: hypoxia-inducible factor; ICB: Novartis, Merck, 7Hills, and Clinical Trial Support to Institution for AbbVie, immune checkpoint blockers; ICD: immunogenic cell death; IHC: immunohis- Boston Biomedical, Bristol-Myers Squibb, Celldex, Corvus, Delcath, Five Prime, tochemistry; IFN-I: interferon type I; IEC: intestinal epithelial cells; LDH: lactate Genentech, Immunocore, Incyte, MedImmune, Macrogenics, Novartis, Phar- dehydrogenase; MHC: major histocompatibility complex; miRNAs: microRNAs; macyclics, Merck, Tesaro. LM has a consultant/advisory role for IGEA. MP has/ mAb: monoclonal antibody; NK: natural killer cells; NGS: next generation had a consultant/advisory role for BMS, Merck, Novartis, and Array BioPharma. sequencing; ORR: objective response rate; PBMCs: peripheral blood mono- He has also received honoraria from BMS and Merck and a research grant from nuclear cells; RT: radiotherapy; T-VEC: talimogene laherparepvec; TIL: tumor BMS. IP has/has consultant role for Amgen and Roche. DFS is a Section Editor infltrating lymphocytes; TME: tumor micro environment; WGCNA: weighted for Cell, Tissue and Gene Therapy for the Journal of Translational Medicine. HZ gene coexpression network analysis. reports research contracts (Bristol-Myers Squibb, Merck and Tesaro). SA, GC, CD, RH, WWO, PTS, MT have no competing interests. Authors’ contributions PAA and MT prepared the manuscript collaboratively with input of SA, GC, SD, Availability of data and materials RD, CD, SF, SCF, CG, RH, SK, JL, LM, WWO, MP, IP, PS, JMT, PTS, DFS, JAW, HZ. All Not applicable. authors read and approved the fnal manuscript. Consent for publication Author details Not applicable. 1 Unit of Melanoma, Cancer Immunotherapy and Innovative Therapy, IRCCS Istituto Nazionale Tumori “Fondazione G. Pascale”, Naples, Italy. 2 Oncology & Ethical approval and consent to participate Hematology, St. Luke’s University Hospital and Temple University, Bethlehem, Not applicable. Ascierto et al. J Transl Med (2017) 15:236 Page 28 of 31

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