Carmen Oñate

Serum Interleukin-8 Reflects Tumor Burden and Treatment Response across Malignancies of Multiple Tissue Origins

Purpose: Interleukin-8 (IL8) is a chemokine produced by malignant cells of multiple cancer types. It exerts various functions in shaping protumoral vascularization and inflammation/immunity. We evaluated sequential levels of serum IL8 in preclinical tumor models and in patients to assess its ability to estimate tumor burden. Experimental Design: IL8 levels were monitored by sandwich ELISAs in cultured tumor cells supernatants, tumor-xenografted mice serum, and in samples from 126 patients with cancer. We correlated IL8 serum levels with baseline tumor burden and with treatment-induced changes in tumor burden, as well as with prognosis. Results: IL8 concentrations correlated with the number of IL8-producing tumor cells in culture. In xenografted neoplasms, IL8 serum levels rapidly dropped after surgical excision, indicating an accurate correlation with tumor burden. In patients with melanoma (n = 16), renal cell carcinoma (RCC; n = 23), non–small cell lung cancer (NSCLC; n = 21), or hepatocellular carcinoma (HCC; n = 30), serum IL8 concentrations correlated with tumor burden and stage, survival (melanoma, n = 16; RCC, n = 23; HCC, n = 33), and objective responses to therapy, including those to BRAF inhibitors (melanoma, n = 16) and immunomodulatory monoclonal antibodies (melanoma, n = 8). IL8 concentrations in urine (n = 18) were mainly elevated in tumors with direct contact with the urinary tract. Conclusions: IL8 levels correlate with tumor burden in preclinical models and in patients with cancer. IL8 is a potentially useful biomarker to monitor changes in tumor burden following anticancer therapy, and has prognostic significance. Clin Cancer Res; 20(22); 5697–707. ©2014 AACR.

Tumor-produced interleukin-8 attracts human myeloid-derived suppressor cells and elicits extrusion of neutrophil extracellular traps (NETs)

Purpose: Myeloid-derived suppressor cells (MDSC) are considered an important T-cell immunosuppressive component in cancer-bearing hosts. The factors that attract these cells to the tumor microenvironment are poorly understood. IL8 (CXCL8) is a potent chemotactic factor for neutrophils and monocytes. Experimental Design: MDSC were characterized and sorted by multicolor flow cytometry on ficoll-gradient isolated blood leucokytes from healthy volunteers (n = 10) and advanced cancer patients (n = 28). In chemotaxis assays, sorted granulocytic and monocytic MDSC were tested in response to recombinant IL8, IL8 derived from cancer cell lines, and patient sera. Neutrophil extracellular traps (NETs) formation was assessed by confocal microscopy, fluorimetry, and time-lapse fluorescence confocal microscopy on short-term MDSC cultures. Results: IL8 chemoattracts both granulocytic (GrMDSC) and monocytic (MoMDSC) human MDSC. Monocytic but not granulocytic MDSC exerted a suppressor activity on the proliferation of autologous T cells isolated from the circulation of cancer patients. IL8 did not modify the T-cell suppressor activity of human MDSC. However, IL8 induced the formation of NETs in the GrMDSC subset. Conclusions: IL8 derived from tumors contributes to the chemotactic recruitment of MDSC and to their functional control. Clin Cancer Res; 22(15); 3924–36. ©2016 AACR.

Nivolumab and urelumab enhance antitumor activity of human T lymphocytes engrafted in Rag2-/-IL2Rγnull immunodeficient mice

A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations.

Dendritic Cells Take up and Present Antigens from Viable and Apoptotic Polymorphonuclear Leukocytes

Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2d) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2d) are coinjected in the footpad of mice with autologous DC (H-2b). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.

Interleukin-8 in cancer pathogenesis, treatment and follow-up

Interleukin-8 (CXCL8) was originally described asa chemokine whose main function is the attraction of a polymorphonuclear inflammatory leukocyte infiltrate acting on CXCR1/2. Recently, it has been found that tumors very frequently coopt the production of this chemokine, which in this malignant context exerts different pro-tumoral functions. Reportedly, these include angiogenesis, survival signaling for cancer stem cells and attraction of myeloid cells endowed with the ability to immunosuppress and locally provide growth factors. Given the fact that in cancer patients IL-8 is mainly produced by tumor cells themselves, its serum concentration has been shown to correlate with tumor burden. Thus, IL-8 serum concentrations have been shown to be useful asa pharmacodynamic biomarker to early detect response to immunotherapy. Finally, because of the roles that IL-8 plays in favoring tumor progression, several therapeutic strategies are being developed to interfere with its functions. Such interventions hold promise, especially for therapeutic combinations in the field of cancer immunotherapy.

Combined immunotherapy encompassing intratumoral poly-ICLC, dendritic-cell vaccination and radiotherapy in advanced cancer patients

Background Combination immunotherapy has the potential to achieve additive or synergistic effects. Combined local injections of dsRNA analogues (mimicking viral RNA) and repeated vaccinations with tumor-lysate loaded dendritic cells shows efficacy against colon cancer mouse models. In the context of immunotherapy, radiotherapy can exert beneficial abscopal effects. Patients and methods In this two-cohort pilot phase I study, 15 advanced cancer patients received two 4-week cycles of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24 h with poly-ICLC (Hiltonol), TNF-α and IFN-α. On days +8 and +10 of each cycle, patients received intratumoral image-guided 0.25 mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600 mg/m2 was administered 1 week before. Six patients received stereotactic ablative radiotherapy (SABR) on selected tumor lesions, including those injected with Hiltonol. Expression of 25 immune-relevant genes was sequentially monitored by RT-PCR on circulating peripheral blood mononuclear cell (PBMCs) and serum concentrations of a cytokine panel were sequentially determined before and during treatment. Pre- and post-treatment PBMC from patients achieving durable stable disease (SD) were studied by IFNγ ELISPOT-assays responding to tumor-lysate loaded DC and by TCRβ sequencing. Results Combined treatment was, safe and well tolerated. One heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to SABR+ immunotherapy. No objective responses were observed, while nine patients presented SD (five of them in the six-patient radiotherapy cohort). Intratumoral Hiltonol increased IFN-β and IFN-α mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1β concentrations, especially in patients presenting SD. IFNγ-ELISPOT reactivity to tumor lysates was observed in two patients experiencing durable SD. Conclusions This radio-immunotherapy combination strategy, aimed at resembling viral infection in tumor tissue in combination with a dendritic-cell vaccine and SABR, is safe and shows immune-associated activity and signs of preliminary clinical efficacy.

Functional expression of CD137 (4-1BB) on T helper follicular cells

CD137 (4-1BB) is a surface protein initially discovered to mark activated T lymphocytes. However, its broader expression pattern also encompasses activated NK cells, B cells and myeloid cells, including mature dendritic cells. In this study, we have immunostained for CD137 on paraffin-embedded lymphoid tissues including tonsils, lymph nodes, ectopic tertiary lymphoid tissue in Hashimoto thyroiditis and cancer. Surprisingly, immunostaining mainly decorated intrafollicular lymphocytes in the tissues analyzed, with only scattered staining in interfollicular areas. Moreover, pathologic lymphoid follicles in follicular lymphoma and tertiary lymphoid tissue associated with non-small cell lung cancer showed a similar pattern of immunostaining. Multispectral fluorescence cytometry demonstrated that CD137 expression was restricted to CD4+ CXCR5+ follicular T helper lymphocytes (TFH cells) in tonsils and lymph nodes. Short-term culture of lymph node cell suspensions in the presence of either an agonistic anti-CD137 monoclonal antibody (mAb) or CD137-ligand stimulated the functional upregulation of TFH cells in 3 out of 6 cases, as indicated by CD40L surface expression and cytokine production. As a consequence, immunostimulatory monoclonal antibodies targeting CD137 (such as urelumab and PF-05082566) should be expected to primarily act on this lymphocyte subset, thus modifying ongoing humoral immune responses in patients with autoimmune disease and cancer.

Abstract 4012: Improving radiotherapy abscopal effects with anti-PD1 and anti-CD137-based immunotherapy

Radiotherapy is considered an efficacious local tool to erradicate or at least control cancer progression. However, recent lines of preclinical and clinical evidence indicate that proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to act both on irradiated tumor lessions and on distant, non-irradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favourable effects on distant non-irradiated tumor lesions as observed on transplanted MC38 (colorectal cancer), B16OVA (melanoma) and 4T1 (breast cancer) models. Immunotherapy and radiotherapy synergized both when irradiation was given using external beams or provided with brachytherapy. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. The irradiation regimen induced immune infiltrate changes in the irradiated and non-irradiated lesions featured by reductions in the content of effector T cells, Tregs, and myeloid-derived supresor cells (MDSC), while effector T cells were expressing more intracellular IFN gamma in both the irradiated and contralateral tumors. Importantly, 48h following irradiation CD8+ TILs showed brighter expression of CD137 and PD-1 thereby displaying more target molecules for the activity of the corresponding monoclonal antibodies. Likewise, PD-1 and CD137 were induced on tumor infiltrating lymphocytes from surgically excised of human carcinoma lessions that were irradiated ex-vivo. These findings advocate for clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompained by radiotherapy strategies on treatable lesions, even if leaving disease outside the irradiation field. Citation Format: MariaE. Rodriguez-Ruiz, Inmaculada Rodriguez, Saray Garasa, Benigno Barbes, Jose Luis Solorzano, Jose Luis Perez Gracia, Sara Labiano, Arantza Azpilikueta, Elixabet Bolanos, Alfonso R. Sanchez-Paulete, M. Angela Aznar, Ana Rouzaut, Maria Jure-Kunkel, Inaki Etxeberria, Carlos Alfaro, Carmen Onate, Mariano Ponz, Ignacio Melero. Improving radiotherapy abscopal effects with anti-PD1 and anti-CD137-based immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4012.

1076TiPRANDOMIZED PHASE II TRIAL WITH DENDRITIC CELL (DC) IMMUNOTHERAPY IN PATIENTS WITH COLORECTAL CARCINOMA AND LIVER METASTASIS FOLLOWING COMPLETE RESECTION AND ADJUVANT CHEMOTHERAPY

ABSTRACT Background: Cellular immunotherapy with DC has well-established anti-tumor activity, as confirmed by Sipuleucel-T, the first approved treatment based in this strategy. Preclinical and clinical data indicate that efficacy of DC immunotherapy is maximized when used in the setting of minimal residual disease. Our study explores the efficacy of an autologous DC vaccine loaded with self-tumor antigens that we developed in a previous study (Alfaro, J Immunology 2011) in patients with colorectal cancer that have undergone complete resection of hepatic metastasis and standard adjuvant therapy. Trial design: In this randomized phase II study, patients with colorectal carcinoma with hepatic metastasis that have undergone standard treatment, including complete surgical resection and standard adjuvant chemotherapy, are randomized to receive DC vaccine or to observation. The two-cycle vaccination protocol includes the following strategies: a) pretreatment with cyclophosphamide to decrease regulatory T cells; b) maturation and activation of DC with TNF-alpha, interferon-alpha and poly I:C, a potent inducer of type I interferon; c) use of autologous tumor from resected liver metastasis as antigenic source, to include antigens that are exclusive of tumor cells; and d) administration of daily intradermal vaccines during four consecutive days in 2 cycles every 4 weeks. Our aim is to replicate the immune response observed during an acute viral infection in terms of activation signals and persistence of antigens in lymph nodes. The main objective is progression-free survival. Thirty-six patients will be included, allowing to detect a HR = 1.75 (alpha error = 0.2, beta error = 0.35). Secondary objectives are: safety, overall survival and immunologic response (in vitro lymphocyte responses against tumor antigens; delayed hypersensitivity reactions; induction of tumor antibody responses; DC activation parameters including IL-12 and IL-6 production and expression of CD80, CD83, CD86, B7-H1, B7-H4 and B7-DC; assessment of DC maturation by expression of pro-inflammatory cytokines; and DC migration) (ClinicalTrials.gov Identifier: NCT01348256). This abstract was accepted and previously presented at the 2014 ASCO Annual Meeting Chicago, June 2014 (TPS3129). Disclosure: All authors have declared no conflicts of interest.

252PSERUM INTERLEUKIN-8 REFLECTS TUMOR BURDEN AND TREATMENT RESPONSE ACROSS MALIGNANCIES OF MULTIPLE TISSUE ORIGINS

ABSTRACT Aim: IL-8 is produced by multiple tumors and its functions include regulation of tumor angiogenesis and immune response. We evaluated the correlation of IL-8 with tumor burden and its potential role as a cancer biomarker by studying sequential levels of serum IL-8 in preclinical tumor models and in patients with multiple tumor types, at baseline and following anticancer treatment. Methods: IL-8 levels were monitored sequentially by sandwich ELISAs in three different models: a) cultured tumor cells supernatant; b) serum of tumor-xenografted mice and; c) serum and urine samples from 119 cancer patients, at baseline and following diverse anticancer treatments. We correlated IL-8 levels with tumor burden, treatment induced response and cancer prognosis. Results: IL-8 levels showed a strong correlation with tumor burden in all the models explored: a) in tumor cultures, IL-8 levels correlated accurately with the number of cancer cells; b) in tumor-xenografted mice, IL-8 serum levels correlated with tumor burden and rapidly dropped following surgical excision; and c) in patients with melanoma, renal-cell carcinoma, non-small cell lung cancer and hepatocellular carcinoma, serum IL-8 levels correlated with tumor burden and stage, survival and objective responses to therapy, including BRAF inhibitors and immunomodulatory monoclonal-antibodies. IL-8 concentrations in urine were mainly elevated in tumors with direct contact with the urinary tract. Conclusions: IL-8 levels correlate with tumor burden in preclinical models and in cancer patients and are a potentially useful biomarker to monitor response to cancer treatment. This might be particularly relevant to assess the therapeutic effects of drugs that may induce “pseudo-progressions”, such as immunomodulatory monoclonal antibodies. Disclosure: All authors have declared no conflicts of interest.