Battle of the γδ T cell subsets in the gut

© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).In a study in Science, Reis et al. describe a temporal segregation of γδ T cell activities in colorectal cancer (CRC). Initially tumor surveillance is orchestrated by interferon-γ (IFN-γ)-producing and cytotoxic γδ T cell subsets, but once the tumor thrives, it becomes infiltrated by interleukin (IL)-17+ γδ T cell subsets that promote its growth.The authors acknowledge funding from Fundação para a Ciência e Tecnologia (Ministério da Ciência, Tecnologia e Ensino Superior, Portugal): PTDC/MED- ONC/6829/2020 (to B.S-S.) and 2021.01953. CEECIND contract (to S.M.).info:eu-repo/semantics/publishedVersio

Therapeutic avenues for γδ T cells in cancer

© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.γδ T cells are regarded as promising effector lymphocytes for next-generation cancer immunotherapies. In spite of being relatively rare in human peripheral blood, γδ T cells are more abundant in epithelial tissues where many tumors develop, and have been shown to actively participate in anticancer immunity as cytotoxic cells or as "type 1" immune orchestrators. A major asset of γδ T cells for tackling advanced cancers is their independence from antigen presentation via the major histocompatibility complex, which clearly sets them apart from conventional αβ T cells. Here we discuss the main therapeutic strategies based on human γδ T cells. These include antibody-based bispecific engagers and adoptive cell therapies, either focused on the Vδ1+ or Vδ2+ γδ T-cell subsets, which can be expanded selectively and differentiated or engineered to maximize their antitumor functions. We review the preclinical data that supports each of the therapeutic strategies under development; and summarize the clinical trials being pursued towards establishing γδ T cell-based treatments for solid and hematological malignancies.This study was funded by Fundação para a Ciência e Tecnologia of the Portuguese Ministério da Ciência, Tecnologia e Ensino Superior (PTDC/MED-ONC/6829/2020 to BS-S and 2021.01953.CEECIND to SM).info:eu-repo/semantics/publishedVersio

Dissecting the role of microRNAs in effector versus regulatory CD4+ T cell differentiation during (auto)immune responses in vivo

Introduction: MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. They have been implicated in the regulation of the differentiation and function of CD4+ T cell subsets, key players in host defense against pathogens, but also responsible for immune-mediated diseases depending on the correct vs incorrect balance, respectively, between pro-inflammatory effector CD4+T cells, including the IFN-γ-producers T helper 1 (Th)1 and the IL-17-producers Th17 cells, and anti-inflammatory regulatory T cells (Treg). While individual miRNAs were found to regulate the differentiation of specific CD4+ T cell populations, an approach based on in vivo responses is still missing and is key to understanding how miRNA networks control this balance in pathophysiology. Methodology: We have established a triple reporter mouse for Ifng, Il17, and Foxp3, and subjected it to experimental autoimmune encephalomyelitis (EAE). We performed miRNA-seq analysis on Th1, Th17, and Treg cells isolated from the spleen and lymph nodes (LNs) at the peak plateau stage to identify miRNA candidates specifically expressed in one of the cell populations. We have in vivo modulated their expression levels using antagomiRs observed the course of EAE progression and characterised their upstream regulation in vitro in either Th1 or Th17 differentiation conditions. Results: The miRNA-seq data has allowed the identification of 110 miRNAs differentially expressed between effector (Th1 and Th17) and regulatory (Treg) subsets. From those, 9 were specifically upregulated in one population versus the others. In vivo miRNA modulation showed that silencing miR-122 precipitated the onset of EAE, whereas overexpressing miR-1247 decreased the severity of the disease. Cytokine-regulated miR-1247 and miR-122 expression levels are inversely associated with pathogenic signatures of Th1 and Th17 cells between lymphoid and central nervous systems. Discussion: Our results suggest that miR-122 and miR-1247 act as peripheral brakes to CD4+ T cell pathogenicity that are overruled in the inflamed target organ. These findings may have important implications for autoimmune diseases.info:eu-repo/semantics/publishedVersio

Maternal γδ T cells shape offspring pulmonary type 2 immunity in a microbiota-dependent manner.

Immune development is profoundly influenced by vertically transferred cues. However, little is known about how maternal innate-like lymphocytes regulate offspring immunity. Here, we show that mice born from γδ T cell-deficient (TCRδ-/-) dams display an increase in first-breath-induced inflammation, with a pulmonary milieu selectively enriched in type 2 cytokines and type 2-polarized immune cells, when compared with the progeny of γδ T cell-sufficient dams. Upon helminth infection, mice born from TCRδ-/- dams sustain an increased type 2 inflammatory response. This is independent of the genotype of the pups. Instead, the offspring of TCRδ-/- dams harbors a distinct intestinal microbiota, acquired during birth and fostering, and decreased levels of intestinal short-chain fatty acids (SCFAs), such as pentanoate and hexanoate. Importantly, exogenous SCFA supplementation inhibits type 2 innate lymphoid cell function and suppresses first-breath- and infection-induced inflammation. Taken together, our findings unravel a maternal γδ T cell-microbiota-SCFA axis regulating neonatal lung immunity

Crosstalk between immunosuppressive myeloid and effector lymphoid cells in the tumour microenvironment

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015O sistema imunitário é constituído por estruturas organizadas e moléculas com papéis específicos na defesa contra patogénios e células transformadas, e é habitualmente segregado em dois principais componentes: o sistema inato e o adaptativo. O sistema inato está presente em todos os organismos multicelulares e é caracterizado por apresentar uma resposta rápida a padrões conservados em patogénios. O sistema adaptativo é caracterizado pela capacidade de gerar uma grande variedade de receptores de antigénios e células de memória que respondem de forma mais eficaz numa exposição repetida a um dado patogénio. O sistema inato é composto por células fagocíticas (neutrófilos, monócitos e macrófagos), células ‘’natural killer’’ (NK) e células que produzem mediadores inflamatórios como os basófilos, mastócitos e eosinófilos. O sistema adaptativo é composto por células T e B específicas para antigénios, cujos receptores são produzidos por recombinação somática. As células B, que se desenvolvem na medula óssea, após estimulação, secretam imunoglobulinas que são responsáveis pela eliminação de microorganismos extracelulares. As células T, que se desenvolvem no timo, apresentam funções auxiliadoras e citotóxicas, através da produção de citocinas e enzimas citolíticas. Os linfócitos T podem ser categorizados de acordo com as cadeias que expressam nos receptores de células T (TCR), em células Tαβ e células Tγδ. As células Tαβ ‘’naïve’’ são activadas em orgãos secundários linfóides por células apresentadoras de antigénios que processam e apresentam os antigénios conjugados com moléculas do complexo major de histocompatibilidade (MHC - major histocompatibility complex) à sua superfície. Em oposição, as células Tγδ populam os tecidos epiteliais e respondem rapidamente a sinais de stress. As células Tγδsão linfócitos que expressam TCRs compostos por uma cadeia γ e uma cadeia δ e que realizam recombinação somática para os originar. São, no entanto, consideradas células “innate-like” devido à sua capacidade de actuar como primeira linha de defesa, iniciar uma resposta inflamatória rápida e por se localizarem preferencialmente em tecidos epiteliais. São células que se caracterizam funcionalmente pela capacidade de rápida produção de interferão-gama (IFN-γ) e interleucina-17 (IL-17), em resposta a estímulos externos. Estas células são consideradas como não redundantes relativamente às células Tαβ essencialmente por apresentarem uma cinética de resposta diferente, reconhecerem antigénios distintos e apresentarem uma distribuição anatómica também ela distinta. Dependendo das suas funções efectoras, as células Tγδ podem ser categorizadas em duas populações: as produtoras de IFN-γ, que expressam o marcador de superfície CD27, e as produtoras de IL-17, que não expressam este marcador. As células Tγδ apresentam potentes propriedades antitumorais mediadas pela produção de granzimas, perforinas, FasL, TRAIL e IFN-γ, o que já foi demonstrado ter um papel importante na imunovigilância de células cancerígenas, tanto em ratinhos como em humanos. Nomeadamente, num modelo subcutâneo de melanoma (B16), estabelecido no laboratório de acolhimento, ratinhos deficientes para estas células (TCRδ-/-) apresentaram um maior desenvolvimento tumoral. Já em ratinhos da estirpe selvagem, as células Tγδ acumulam-se no tumor, onde apresentam funções citotóxicas potentes e produzem IFN-γ No sentido oposto, as células Tγδ foram recentemente associadas à promoção do desenvolvimento tumoral, em modelos de cancro do fígado e de ovário, devido à produção de IL-17. No entanto, o papel desta citocina no ambiente tumoral é muito controverso e continua a ser alvo de estudo. Neste trabalho utilizou-se uma linha celular de melanoma (B16), injectada intraperitonealmente, com o objectivo de estudar a resposta das células Tγδ durante o desenvolvimento tumoral. Observou-se que neste modelo as células Tγδ , ao contrário de outras células imunitárias como células T CD4, T CD8 e NK, não se acumulam em resposta ao estímulo da injecção tumoral. A análise funcional das células Tγδ revelou ainda que estas não são capazes de aumentar a expressão das citocinas IL-17 e IFN-γ nem de enzimas citolíticas, como granzima B e perforina. Desta análise conclui-se que as células Tγδ no ambiente tumoral deste modelo se encontram anérgicas. Em concordância com estes resultados, a análise do crescimento tumoral em ratinhos TCRδ-/- revelou que estes apresentam uma cinética semelhante aos ratinhos da estirpe selvagem. Estes resultados estabelecem que as células Tγδ apresentam um papel neutro no crescimento tumoral, não sendo responsáveis nem pela promoção nem pela eliminação da linha de melanoma B16 quando esta é injectada na cavidade peritoneal. Na tentativa de perceber se a “anergia” das células Tγδ se deve a um mecanismo de imunossupressão analisou-se a presença de células supressoras no ambiente tumoral. Nessa análise, verificou-se que a frequência de células T reguladoras, caracterizadas pela expressão dos marcadores de superfície CD4 e CD25, pelo factor de transcrição FoxP3 e pelas suas potentes capacidades de supressão de células T, está diminuida. Por esse motivo, estas são improváveis mediadores da supressão das células Tγδ. Pelo contrário, detectámos uma frequência aumentada de células supressoras de origem mielóide (MDSC, myeloid derived suppressor cells). As MDSC são uma população heterogénea de progenitores mielóides e células mielóides imaturas que são induzidas em situações patológicas, incluindo no desenvolvimento tumoral. Estas células são caracterizadas pela co-expressão dos marcadores de superfície CD11b e Gr-1. Em ratinho, as MDSC podem ser divididas em duas principais subpopulações, as células monocíticas supressoras de origem mielóide (Mo-MDSC, monocytic myeloid–derived suppressor cells) e as células polimorfonucleares supressoras de origem mielóide (PMN-MDSC, polymorphonuclear myeloid-derived suppressor cells). As Mo-MDSCs apresentam o fenótipo CD11b+Gr-1intLy6G-Ly6C+ enquanto as PMN-MDSC são caracterizadas por serem CD11b+Gr-1highLy6G+Ly6Cint. As MDSC têm a capacidade de suprimir as respostas de linfócitos T, tanto in vitro como in vivo, através de vários mecanismos conhecidos, como a produção de óxido nítrico e a diminuição da arginina disponível no meio. Sabe-se que as MDSC suprimem as células T CD8 citotóxicas e induzem o desenvolvimento de células T reguladoras, contribuindo para um desequilíbrio da resposta imunitária, no sentido de promover a tolerância e assim contribuindo para o desenvolvimento tumoral. No entanto, pouco se sabe sobre a interação entre as MDSC e as células Tγδ No nosso modelo (melanoma B16 intraperitoneal), observou-se um aumento tanto da subpopulação monocítica como da polimorfonuclear de MDSC, em relação a ratinhos sem tumor. Para perceber se as MDSC poderiam ter impacto na resposta das células Tγδ, recorremos ao anticorpo monoclonal anti-Gr-1 para as depletar. A injecção deste anticorpo em ratinhos com tumor levou à depleção específica de PMN-MDSC, visto que estas expressam níveis mais elevados do marcador de superfície Gr-1, relativamente às Mo-MDSCs. Esta depleção resultou na acumulação selectiva de células Tγδ no ambiente tumoral, sem alteração da frequência de células T CD4, T CD8 e NK. As células Tγδ que se acumularam em consequência da depleção das PMN-MDSC expressam a cadeia Vγ6 do receptor de células T e não expressam o marcador de superfície CD27. Em concordância com a falta de expressão desse marcador, estas células são produtoras de IL-17. Visto a IL-17 ter um papel controverso no contexto tumoral, avaliou-se qual o papel desta citocina e das células Tγδ no desenvolvimento tumoral, após depleção das PMN-MDSC. Observou-se que tanto os ratinhos IL-17-/- como os ratinhos TCRδ-/- apresentam um crescimento tumoral diminuido em relação aos ratinhos da estirpe selvagem. Esta observação implica que tanto as células Tγδ como a citocina IL-17 desempenham um papel pro-tumoral neste modelo. Os resultados apresentados nesta tese mostram, pela primeira vez, que as células Tγδ podem ser alvo de inibição pelas PMN-MDSC e que, contrariamente ao seu conhecido papel pro-tumoral, esta população mielóide pode mediar a protecção do hospedeiro, no contexto tumoral.The tumour microenvironment contains a variety of immune cells that can either inhibit or promote tumour growth. These immune populations comprise γδ T cells, which have been described to provide large amounts of anti-tumour molecules, such as interferon-γ (IFN-γ) and cytolytic enzymes, but can also produce interleukin-17 (IL-17) that supports tumour growth. By contrast, in this thesis we report that, in a peritoneal melanoma B16 tumour model, γδ T cells are unresponsive and thus neutral to tumour growth. Upon tumour challenge, γδ T cells were unable to accumulate and to upregulate IL-17, IFN-γ, granzyme B and perforin expression, in comparison to steady-state. Consistent with this, TCRδ-/- mice displayed similar tumour growth kinetics to wild-type (WT) controls, further corroborating the inability of γδ T cells to respond to tumour challenge. Concomitantly, we observed an accumulation of myeloid-derived suppressor cells (MDSC), both of monocytic (Mo-MDSC) and polymorphonuclear (PMN-MDSC) origin, which are known to suppress tumour immunity. Hence, we hypothesized that MDSCs could prevent γδ T cell response in this model. To modulate MDSC we employed an anti-Gr-1 monoclonal antibody (mAb), which led to specific depletion of PMN-MDSC. Such manipulation resulted in γδ T cell-specific accumulation and increased proportion of IL-17-producing γδ T cells. These γδ T cells were characterized by the expression of a Vγ6+ T cell receptor, and by lack of CD27 expression. Analysis of TCRδ-/- and IL17-/- mice upon PMN-MDSC depletion revealed, by comparison with WT controls that both γδ T cells and IL-17 contributed to tumour growth. Collectively, the work presented in this thesis showed, for the first time, that γδ T cells can be targeted by PMN-MDSCs and that, contrary to their well-established pro-tumour role, this myeloid subset can also mediate host protection in the tumour context

Regulation of T cell fitness and functions in tumour responses

The immune system, mainly through the action of T cells (and NK), patrols nascent tumours and controls their progression, in a process called immunosurveillance. However, tumours still develop. The failure of tumour control by the immune system is mediated by several escape mechanisms, and the purpose of immunotherapy is to reverse that escape. Substantial advances had been made in this field with the discovery and therapeutic manipulation of immune checkpoints, which are negative regulators of T cell responses. Their discovery was so important that the two pioneer investigators, Tasuku Honjo and James Allison, received the Nobel Prize of Physiology or Medicine in 2018. Clinical blockade of these pathways has shifted the paradigm in the treatment of several types of cancer; however, the efficacy of this strategy is limited to some types of tumours and to an overall minority of patients. This limited efficacy highlights the need of unravelling new mechanisms responsible for T cell regulation in the tumour microenvironment (TME), which we undertook in this thesis. First, we studied the negative regulation of IL-17-producing γδ (γδ17) T cells, which we and others had previously shown to play paradoxical tumour-promoting roles. We used two murine cancer models consisting in the injection of a melanoma (B16) or a hepatoma (Hepa 1-6) cell line, in the peritoneal cavity or liver, respectively. These anatomic locations are favourable to the induction of γδ17 T cell responses. However, to our surprise, these cells did not expand upon tumour challenge, in comparison to tumour-free controls. Concomitantly to the absence of a γδ17 T cell response, we observed a striking accumulation of neutrophils, leading us to hypothesize that neutrophils could suppress γδ17 T cell responses. Genetic approaches, depletion strategies and in vitro studies collectively demonstrated that tumour-associated neutrophils inhibited γδ17 T cell proliferation, through the production of reactive oxygen species (ROS). Interestingly, γδ17 T cells expressed low levels of antioxidants, particularly glutathione, which rendered them particularly susceptible to oxidative stress. In sum, we unraveled a new mechanism responsible for the regulation of a critical pro-tumoural T cell subset in the tumour microenvironment. On the other hand, we also investigated mechanisms of positive regulation of T cells, towards the potentiation of their anti-tumour functions. Given that the essential amino acid, methionine, had previously been shown to be required for T cell proliferation and effector function, we tested the effect of its supplementation in tumour-bearing mice. We used an orthotopic murine triple-negative breast cancer (TNBC) model consisting in the injection of the E0771 cell line in the mammary fat pad. We found that methionine supplementation, while not having a direct effect on tumour progression, potentiated the efficacy of suboptimal anti-PD-1 treatment. The combination of anti-PD-1 with methionine led to an increased infiltration of IFNγ-producing γδ (γδIFNγ) T cells and cytotoxic CD8+ T cells in the tumour bed. Both these subsets were required for the increased efficacy of the combination therapy, with γδ T cells seemingly acting upstream of CD8+ T cells, and the latter becoming more effective tumour killers upon combined therapy. While the molecular mediators underlying the increased T cell infiltration and effector function are still to be determined, our data reveal a new avenue to potentiate the anti-tumour function of γδ and CD8+ T cells, through the combination of anti-PD-1 and methionine. In conclusion, the work presented in this thesis unravels two new mechanisms of T cell regulation in tumours: one that inhibits pro-tumoural T cells and another that potentiates anti-tumour T cell responses. We hope our findings may contribute to the (re)design of more efficacious cancer immunotherapy strategies

γδ T cells maintain sensitivity to immunotherapy in MHC-I-deficient tumors

Copyright © 2023, Springer Nature America, Inc.Loss of human leukocyte antigen (HLA) class I expression is a major immune evasion mechanism, given the strict requirement of HLA-class-I-mediated antigen presentation for cytotoxic CD8+ T cell activation and tumor cell recognition. In that context, how tumors may remain sensitive to immunotherapy, namely immune checkpoint blockade (ICB), is a highly relevant biological and medical question. A recent study in Nature by de Vries et al. focused on a subgroup of patients with colorectal cancer (CRC) and found that HLA-class-I-deficient tumors retain responsiveness to ICB based on the expansion and effector functions of γδ T cells.We acknowledge funding from Fundação para a Ciência e Tecnologia of Portugal’s Ministério da Ciência, Tecnologia e Ensino Superior (PTDC/MED-ONC/6829/2020 to B.S.-S. and 2021.01953.CEECIND to S.M.)info:eu-repo/semantics/publishedVersio

γδ T cells: pleiotropic immune effectors with therapeutic potential in cancer

Copyright © 2019, The PublisherThe potential of cancer immunotherapy relies on the mobilization of immune cells capable of producing antitumour cytokines and effectively killing tumour cells. These are major attributes of γδ T cells, a lymphoid lineage that is often underestimated despite its major role in tumour immune surveillance, which has been established in a variety of preclinical cancer models. This situation notwithstanding, in particular instances the tumour microenvironment seemingly mobilizes γδ T cells with immunosuppressive or tumour-promoting functions, thus emphasizing the importance of regulating γδ T cell responses in order to realize their translation into effective cancer immunotherapies. In this Review we outline both seminal work and recent advances in our understanding of how γδ T cells participate in tumour immunity and how their functions are regulated in experimental models of cancer. We also discuss the current strategies aimed at maximizing the therapeutic potential of human γδ T cells, on the eve of their exploration in cancer clinical trials that may position them as key players in cancer immunotherapy.We acknowledge funding from the European Research Council (CoG_646701 to B.S.-S.), Cancer Research UK Glasgow Centre (A25142 to S.B.C.), Breast Cancer Now (2018JulPR1101 to S.B.C.), Wellcome Trust (208990/Z/17/Z to S.B.C.), Tenovus Scotland (Project S17-17 to S.B.C.) and Fundação para a Ciência e a Tecnologia / Ministério da Ciência, Tecnologia e Ensino Superior through Fundos do Orçamento de Estado (refs. UID/BIM/50005/2019 and PD/BD/114099/2015).info:eu-repo/semantics/publishedVersio

Primary tumors limit metastasis formation through induction of IL15-mediated cross-talk between patrolling monocytes and NK cells

© 2017 American Association for Cancer ResearchMetastases are responsible for the vast majority of cancer-related deaths. Although tumor cells can become invasive early during cancer progression, metastases formation typically occurs as a late event. How the immune response to primary tumors may dictate this outcome remains poorly understood, which hampers our capacity to manipulate it therapeutically. Here, we used a two-step experimental model, based on the highly aggressive B16F10 melanoma, that temporally segregates the establishment of primary tumors (subcutaneously) and the formation of lung metastases (from intravenous injection). This allowed us to identify a protective innate immune response induced by primary tumors that inhibits experimental metastasis. We found that in the presence of primary tumors, increased numbers of natural killer (NK) cells with enhanced IFNγ, granzyme B, and perforin production were recruited to the lung upon metastasis induction. These changes were mirrored by a local accumulation of patrolling monocytes and macrophages with high expression of MHC class II and NOS2. Critically, the protective effect on metastasis was lost upon patrolling monocyte or NK cell depletion, IL15 neutralization, or IFNγ ablation. The combined analysis of these approaches allowed us to establish a hierarchy in which patrolling monocytes, making IL15 in response to primary tumors, activate NK cells and IFNγ production that then inhibit lung metastasis formation. This work identifies an innate cell network and the molecular determinants responsible for "metastasis immunosurveillance," providing support for using the key molecular mediator, IL15, to improve immunotherapeutic outcomes.This work was funded by the European Research Council (CoG_646701 to B. Silva-Santos). S. Mensurado (PD/BD/114099/2015) and K. Serre (IF/00004/2014) acknowledge their individual funding from Fundação para a Ciência e Tecnologia.info:eu-repo/semantics/publishedVersio

Low-Density Lipoprotein Uptake Inhibits the Activation and Antitumor Functions of Human Vγ9Vδ2 T Cells

Abstract Vγ9Vδ2 T cells, the main subset of γδ T lymphocytes in human peripheral blood, are endowed with antitumor functions such as cytotoxicity and IFNγ production. These functions are triggered upon T-cell receptor–dependent activation by non-peptidic prenyl pyrophosphates (“phosphoantigens”) that are selective agonists of Vγ9Vδ2 T cells, and which have been evaluated in clinical studies. Because phosphoantigens have shown interindividual variation in Vγ9Vδ2 T-cell activities, we asked whether metabolic resources, namely lipids such as cholesterol, could affect phosphoantigen-mediated Vγ9Vδ2 T-cell activation and function. We show here that Vγ9Vδ2 T cells express the LDL receptor upon activation and take up LDL cholesterol. Resulting changes, such as decreased mitochondrial mass and reduced ATP production, correlate with downregulation of Vγ9Vδ2 T-cell activation and functionality. In particular, the expression of IFNγ, NKG2D, and DNAM-1 were reduced upon LDL cholesterol treatment of phosphoantigen-expanded Vγ9Vδ2 T cells. As a result, their capacity to target breast cancer cells was compromised both in vitro and in an in vivo xenograft mouse model. Thus, this study describes the role of LDL cholesterol as an inhibitor of the antitumor functions of phosphoantigen-activated Vγ9Vδ2 T cells. Our observations have implications for therapeutic applications dependent on Vγ9Vδ2 T cells. Cancer Immunol Res; 6(4); 448–57. ©2018 AACR.</jats:p