Maria Libera Ascierto

Melanoma: From Incurable Beast to a Curable Bet. The Success of Immunotherapy.

After Coley’s observation in 1891 of tumor regression in a patient who developed a postoperative infection, the field of immunotherapy is finally reborn. Avoiding immune destruction is now considered a hallmark of cancer, and the immunotherapy arena has exploded with the recent advances demonstrating an improvement in survival and a durability of response in patients with different cancer types, which translates into improved overall survival benefit. Here, we provide an overview of the main immune-oncology treatment strategies that, either alone or in combination, are undergoing clinical development. Namely, we will refer to those immunotherapeutic strategies that include adoptive transfer of ex vivo activated T cells, immunomodulatory monoclonal antibodies, and cancer vaccines. Our major focus will be to describe these approaches in melanoma, a cancer type transformed by immunotherapy into a potentially curable disease.

Baseline and Dynamic Expression of Activating NK Cell Receptors in the Control of Chronic Viral Infections: The Paradigm of HIV-1 and HCV

Natural killer (NK) cell function is regulated by a balance between the triggering of activating and inhibitory receptors expressed on their surface. A relevant effort has been focused so far on the study of KIR carriage/expression setting the basis for NK cell education and self-tolerance. Focus on the evolution and regulation of activating NK receptors has lagged behind so far. Our understanding of activating receptor expression and regulation has recently improved by evidences derived from in vitro and in vivo studies. Virus infection – either acute or chronic – determines preferential expansion of NK cells with specific phenotype, activating receptors, and with recall-like functional activity. Studies on patients with viral infection (HIV and HCV) and specific diverging clinical courses confirm that inter-individual differences may exist in baseline expression of natural cytotoxicity receptors (NKp46 and NKp30). The findings that patients with divergent clinical courses have different kinetics of activating receptor density expression upon NK cell activation in vitro provide an additional, time-dependent, functional parameter. Kinetic changes in receptor expression thus represent an additional parameter to basal receptor density expression. Different expression and inducibilities of activating receptors on NK cells contribute to the high diversity of NK cell populations and may help our understanding of the inter-individual differences in innate responses that underlie divergent disease courses.

Control of the HIV-1 DNA Reservoir Is Associated In Vivo and In Vitro with NKp46/NKp30 (CD335 CD337) Inducibility and Interferon Gamma Production by Transcriptionally Unique NK Cells

ABSTRACT The size of lentiviral DNA reservoirs reflects the effectiveness of immune responses against lentiviruses. So far, abundant information has been gathered on the control of HIV-1 replication. Understanding the innate mechanisms contributing to containment of the HIV DNA reservoir, however, are only partly clarified and are relevant to guiding interventions for reservoir containment or eradication. We studied the contribution of natural killer (NK) cell functional features in HIV patients controlling replication either spontaneously (HIV controllers [HIC]) or after progression and antiretroviral treatment (progressor patients [PP]). An inverse correlation between HIV DNA copy numbers (either total or integrated) in circulating CD4+ cells and NK cell function was observed. Induced interferon gamma (IFN-γ) production and NKp46/NKp30 activating receptor-induced expression correlated inversely with reservoir size. The correlation was present not only for a homogeneous cohort of HIC patients but also when PP were included in the analysis. Adaptive (NKG2C+ CD57+) NK cell features were not associated with reservoir size. However, a distinct set of 370 differentially expressed transcripts was found to underlie functional differences in NK cells controlling HIV DNA reservoir size. In proof-of-principle in vitro experiments of CD4+ cell infection with HIV-1, purified NK cells with the above-mentioned functional/transcriptional features displayed 10- and 30-fold higher abilities to control HIV replication and DNA burdens in vitro, respectively, than those of other NK cells. Thus, NK cells with a specific functional and transcriptional signature contribute to control of the HIV reservoir in CD4+ cells. Their selection, expansion, and/or adoptive transfer may support strategies to eradicate HIV-1 infection or to safely deescalate antiretroviral treatment. IMPORTANCE The most relevant feature of HIV-1 infection is represented by its DNA reservoir size in the body, which guarantees lifelong infection and resumption of virus replication after antiretroviral treatment interruption. So far, there has been little success in the identification of factors contributing to HIV-1 reservoir containment. In this study, by studying quantitative total and integrated HIV-1 DNA levels and NK cells in HIV-1 patients with either progressive or nonprogressive disease, we observed that inducible IFN-γ and natural cytotoxicity receptor (NCR) expression in a specific subset of NK cells with a characteristic transcriptional signature represents a correlate for HIV-1 reservoir control. This represents an advance in our understanding of the mechanism(s) that controls the lentivirus reservoir. Monitoring, selection, expansion, and adoptive transfer of these NK cells may allow monitoring of treatment efficacy and the likelihood of reservoir control and may support protocols for HIV-1 eradication.

Th17 immune microenvironment in Epstein-Barr virus–negative Hodgkin lymphoma: implications for immunotherapy

Classical Hodgkin lymphoma (CHL) is a neoplasm characterized by robust inflammatory infiltrates and heightened expression of the immunosuppressive PD-1/PD-L1 pathway. Although anti-PD-1 therapy can be effective in >60% of patients with refractory CHL, improved treatment options are needed for CHLs which are resistant to anti-PD-1 or relapse after this form of immunotherapy. A deeper understanding of immunologic factors in the CHL microenvironment might support the design of more effective treatment combinations based on anti-PD-1. In addition, because the Epstein-Barr virus (EBV) residing in some CHL tumors is strongly immunogenic, we hypothesized that characteristics of the tumor immune microenvironment in EBV+ CHL would be distinct from EBV- CHL, with specific implications for designing combination treatment regimens. Employing immunohistochemistry for immune cell subsets and checkpoint molecules, as well as gene expression profiling, we characterized 32 CHLs from the Johns Hopkins archives, including 12 EBV+ and 20 EBV- tumors. Our results revealed a dichotomous cellular and cytokine immune milieu in EBV+ vs EBV- CHL. EBV+ tumors displayed a T helper 1 (Th1) profile typical of effective antitumor immunity, with increased infiltration of CD8+ T cells and coordinate expression of the canonical Th1 transcription factor Tbet (TBX21), interferon-γ (IFNG), and the IFN-γ-inducible immunosuppressive enzyme indoleamine 2,3-dioxygenase. In contrast, EBV- tumors manifested a pathogenic Th17 profile and ongoing engagement of the interleukin-23 (IL-23)/IL-17 axis, with heightened phosphorylated signal transducer and activator of transcription 3 expression in infiltrating lymphocytes. These findings suggest that drugs blocking the IL-23/IL-17 axis, which are already in the clinic for treating certain autoimmune disorders, may enhance the therapeutic impact of anti-PD-1 therapy in EBV- CHL.

‘Emergency exit’ of bone-marrow-resident CD34 + DNAM-1 bright CXCR4 + -committed lymphoid precursors during chronic infection and inflammation

During chronic inflammatory disorders, a persistent natural killer (NK) cell derangement is observed. While increased cell turnover is expected, little is known about whether and how NK-cell homeostatic balance is maintained. Here, flow cytometric analysis of peripheral blood mononuclear cells in chronic inflammatory disorders, both infectious and non-infectious, reveals the presence of a CD34+CD226(DNAM-1)brightCXCR4+ cell population displaying transcriptional signatures typical of common lymphocyte precursors and giving rise to NK-cell progenies with high expression of activating receptors and mature function and even to α/β T lymphocytes. CD34+CD226brightCXCR4+ cells reside in bone marrow, hardly circulate in healthy donors and are absent in cord blood. Their proportion correlates with the degree of inflammation, reflecting lymphoid cell turnover/reconstitution during chronic inflammation. These findings provide insight on intermediate stages of NK-cell development, a view of emergency recruitment of cell precursors, and upgrade our understanding and monitoring of chronic inflammatory conditions.

Inherent transcriptional signatures of NK cells are associated with response to IFNα + rivabirin therapy in patients with Hepatitis C Virus

BackgroundDifferences in the expression of Natural Killer cell receptors have been reported to reflect divergent clinical courses in patients with chronic infections or tumors. However, extensive molecular characterization at the transcriptional level to support this view is lacking. The aim of this work was to characterize baseline differences in purified NK cell transcriptional activity stratified by response to treatment with PEG-IFNα/RBV in patients chronically infected with HCV.MethodsTo this end we here studied by flow cytometer and gene expression profile, phenotypic and transcriptional characteristics of purified NK cells in patients chronically infected with HCV genotype-1 virus who were subsequently treated with PEG-IFNα/RBV. Results were further correlated with divergent clinical response obtained after treatment.ResultsThe pre-treatment transcriptional patterns of purified NK cells from patients subsequently undergoing a sustained virologic response (SVR) clearly segregated from those of non-responder (NR) patients. A set of 476 transcripts, including molecules involved in RNA processing, ubiquitination pathways as well as HLA class II signalling were differently expressed among divergent patients. In addition, treatment outcome was associated with differences in surface expression of NKp30 and NKG2D. A complex relationship was observed that suggested for extensive post-transcriptional editing. Only a small number of the NK cell transcripts identified were correlated with chronic HCV infection/replication indicating that inherent transcriptional activity prevails over environment effects such as viral infection.ConclusionsCollectively, inherent/genetic modulation of NK cell transcription is involved in setting the path to divergent treatment outcomes and could become useful to therapeutic advantage.

Perspectives in immunotherapy: meeting report from the “Immunotherapy Bridge”, Napoli, December 5 th 2015

Harnessing the immune system and preventing immune escape, the immunotherapy of cancer provides great potential for clinical application, in broad patient populations, achieving both conventional and unconventional clinical responses. After the substantial advances in melanoma, the focus of cancer immunotherapy has expanded to include many other cancers. Targeting immune checkpoints and further mechanisms used by tumors to avoid anticancer immunity, different approaches are under evaluation, including combination therapies.The first Immunotherapy Bridge meeting focused on various cancer types including melanoma, non-small cell lung cancer, renal cell, breast and ovarian carcinoma, and discussed mechanisms of action of single agents and combination strategies, and the prediction of clinical responses.

Enhancing T Cell Performance Against Cancer in Combination Treatment Strategies

Increasing evidence suggests that some patients with cancer can mount an antitumor immune response capable of controlling cancer. Although both innate (natural killer cells, dendritic cells) and adaptive (T cells) immunity play important roles in cancer immune surveillance, most emphasis has been placed on the exploitation of adaptive immune responses for cancer immunotherapy. In particular, several preclinical and clinical reports have shown that both cytotoxic CD8+ T cells (CTLs) and CD4+ Th1 cells are able to control and even completely reject tumors through the secretion of cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor alfa (TNF-α). These observations have spurred a revolution in cancer therapeutics, as several treatment strategies are clinically tested and developed in an iterative fashion. In melanoma, multiple immunotherapeutic approaches aimed at manipulating and optimizing the activation of CTLs and CD4+ Th1 cells and their recruitment to malignant tissue are being assessed. Here, we provide an overview of the main immune-oncology treatment strategies that, either alone or in combination, are undergoing clinical development at different stages. Namely, we will refer to those immunotherapeutic strategies that include cancer vaccines, adoptive transfer of ex vivo activated T cells and immunomodulatory monoclonal antibodies.

Abstract 1312: Transcriptional signatures associated with lack of response to anti-PD-1 therapy in patients with renal cell carcinoma

Background: The PD-1/PD-L1 immune checkpoint pathway limits host immune responses to cancer in the local tumor microenvironment. Monoclonal antibodies blocking PD-1 or PD-L1 have shown promising clinical results in a variety of advanced human cancers including renal cell carcinoma (RCC). We previously reported that response to anti-PD-1 therapy correlates with PD-L1 expression by tumor cells in pre-treatment biopsies. Although 20-30% of patients with metastatic RCC respond to anti-PD-1 therapy, many patients with PD-L1+ tumors still do not respond. The current study was undertaken to understand mechanisms underlying the failure of anti-PD-1 targeted therapies in patients with PD-L1+ RCC. Methods: The specimen cohort included formalin-fixed, paraffin-embedded (FFPE) pre-treatment tumor biopsies expressing PD-L1, derived from 13 RCC patients treated with nivolumab (anti-PD-1) at a single institution [4 responders (R), 9 non-responders (NR); RECIST]. PD-L1+ specimens were defined as those having ≥5% of tumor cells with cell surface PD-L1 expression by immunohistochemistry (IHC). RNA was isolated from PD-L1+ regions on FFPE slides. Whole genome microarray profiling with cDNA-mediated Annealing, Selection, extension and Ligation (DASL) was performed. Global gene expression analysis was profiled using BRBArrayTools. Multiplex quantitative (q)RT-PCR was used to validate differential expression of genes of interest, and IHC was used to validate protein expression from select genes, in R vs. NR. Results: Whole genome analysis revealed 234 transcripts that were differentially expressed in R vs. NR (p value ≤ 0.01, fold change ≥1.5). Ingenuity Pathway Analysis (IPA) of these transcripts showed the involvement of metabolic and immune pathways as well as genes encoding oxidation stress response molecules. Multiplex qRT-PCR for a subset of 60 differentially expressed genes validated significant over-expression of genes with metabolic functions, such as drug glucuronidation (UGT1A6/A1/A3), glucose transport (SLC23A1), and mitochondrial oxidation (AKR1C3) in NR vs. R. Conversely, R were found to overexpress immune markers such as BMP1, which has been shown to positively regulate PD-L1 expression, and CCL3 involved in leukocyte migration. Conclusions: Although tumor PD-L1 expression is associated with an increased likelihood of response to anti-PD-1/PD-L1 therapy, tumor cell-intrinsic metabolism may contribute to treatment resistance in PD-L1+ patients. Our data suggest that overexpression of certain metabolic factors may contribute to the failure of PD-L1+ RCC to respond to PD-1 pathway blockade, while immune factors in the tumor immune microenvironment may contribute to success. Treatment strategies that co-target these factors may be needed to enhance responses to anti-PD-1 immunotherapy in RCC. Supported by grants from Bristol-Myers Squibb and Stand Up to Cancer Citation Format: Maria Libera Ascierto, Tracee McMiller, Alan Berger, Robert A. Anders, Chris Cheadle, Haiying Hu, Charles Drake, Drew Pardoll, Janis Taube, Suzanne L. Topalian. Transcriptional signatures associated with lack of response to anti-PD-1 therapy in patients with renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1312. doi:10.1158/1538-7445.AM2015-1312