CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Differential Interleukin-8 thresholds for chemotaxis and netosis in human neutrophils.

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.

Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL-8.

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8+ T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8+ T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20) and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8+ T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8+ tumour-infiltrating lymphocytes. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Performance comparison of two next-generation sequencing panels to detect actionable mutations in cell-free DNA in cancer patients.

Background Genomic alterations studies in cell-free DNA (cfDNA) have increasing clinical use in oncology. Next-generation sequencing (NGS) technology provides the most complete mutational analysis, but nowadays limited data are available related to the comparison of results reported by different platforms. Here we compare two NGS panels for cfDNA: Oncomine™ Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific), suitable for clinical laboratories, and Guardant360® (GuardantHealth), with more genes targeted but only available in an outsourcing laboratory. Methods Peripheral blood was obtained from 16 advanced cancer patients in which Guardant360® (G360) was requested as part of their clinical assistance. Blood samples were sent to be analyzed with G360 panel, and an additional blood sample was drawn to obtain and analyze cfDNA with Oncomine™ Pan-Cancer (OM) panel in an Ion GeneStudio S5™ System. Results cfDNA analysis globally rendered 101 mutations. Regarding the 55/101 mutations claimed to be included by manufacturers in both panels, 17 mutations were reported only by G360, 10 only by OM and 28 by both. In those coincident cases, there was a high correlation between the variant allele fractions (VAFs) calculated with each panel (r = 0.979, p < 0.01). Regarding the six actionable mutations with an FDA-approved therapy reported by G360, one was missed with OM. Also, 12 mutations with clinical trials available were reported by G360 but not by OM. Conclusions In summary, G360 and OM can produce different mutational profile in the same sample, even in genes included in both panels, which is especially important if these mutations are potentially druggable.

Radium-223 international early access program: results from the Spanish subset.

AIM: To report results from the Spanish subset included in the radium-223 international early access program (iEAP). PATIENTS & METHODS: Ninety patients with castration-resistant prostate cancer and bone metastases received radium-223 55 kBq/kg every 4 weeks for six cycles. RESULTS: The median time to disease progression was 8 months and to prostate-specific antigen progression was 4 months. The percentage of patients with ≥50% confirmed declines in prostate-specific antigen was 9%. The median overall survival was 14 months. Grade 3 or 4 treatment emergent adverse events (TEAEs) occurred in 34% of patients (serious TEAEs 28%, TEAEs leading to discontinuation 27%). CONCLUSION: Outcomes of the Spanish subset are consistent with the iEAP. Radium-223 was generally well tolerated with no safety concerns.

Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial.

BACKGROUND: Despite recent advances in the treatment of advanced non-small-cell lung cancer, there remains a need for effective treatments for progressive disease. We assessed the efficacy of pembrolizumab for patients with previously treated, PD-L1-positive, advanced non-small-cell lung cancer. METHODS: We did this randomised, open-label, phase 2/3 study at 202 academic medical centres in 24 countries. Patients with previously treated non-small-cell lung cancer with PD-L1 expression on at least 1% of tumour cells were randomly assigned (1:1:1) in blocks of six per stratum with an interactive voice-response system to receive pembrolizumab 2 mg/kg, pembrolizumab 10 mg/kg, or docetaxel 75 mg/m(2) every 3 weeks. The primary endpoints were overall survival and progression-free survival both in the total population and in patients with PD-L1 expression on at least 50% of tumour cells. We used a threshold for significance of p<0.00825 (one-sided) for the analysis of overall survival and a threshold of p<0.001 for progression-free survival. This trial is registered at ClinicalTrials.gov, number NCT01905657. FINDINGS: Between Aug 28, 2013, and Feb 27, 2015, we enrolled 1034 patients: 345 allocated to pembrolizumab 2 mg/kg, 346 allocated to pembrolizumab 10 mg/kg, and 343 allocated to docetaxel. By Sept 30, 2015, 521 patients had died. In the total population, median overall survival was 10.4 months with pembrolizumab 2 mg/kg, 12.7 months with pembrolizumab 10 mg/kg, and 8.5 months with docetaxel. Overall survival was significantly longer for pembrolizumab 2 mg/kg versus docetaxel (hazard ratio [HR] 0.71, 95% CI 0.58-0.88; p=0.0008) and for pembrolizumab 10 mg/kg versus docetaxel (0.61, 0.49-0.75; p<0.0001). Median progression-free survival was 3.9 months with pembrolizumab 2 mg/kg, 4.0 months with pembrolizumab 10 mg/kg, and 4.0 months with docetaxel, with no significant difference for pembrolizumab 2 mg/kg versus docetaxel (0.88, 0.74-1.05; p=0.07) or for pembrolizumab 10 mg/kg versus docetaxel (HR 0.79, 95% CI 0.66-0.94; p=0.004). Among patients with at least 50% of tumour cells expressing PD-L1, overall survival was significantly longer with pembrolizumab 2 mg/kg than with docetaxel (median 14.9 months vs 8.2 months; HR 0.54, 95% CI 0.38-0.77; p=0.0002) and with pembrolizumab 10 mg/kg than with docetaxel (17.3 months vs 8.2 months; 0.50, 0.36-0.70; p<0.0001). Likewise, for this patient population, progression-free survival was significantly longer with pembrolizumab 2 mg/kg than with docetaxel (median 5.0 months vs 4.1 months; HR 0.59, 95% CI 0.44-0.78; p=0.0001) and with pembrolizumab 10 mg/kg than with docetaxel (5.2 months vs 4.1 months; 0.59, 0.45-0.78; p<0.0001). Grade 3-5 treatment-related adverse events were less common with pembrolizumab than with docetaxel (43 [13%] of 339 patients given 2 mg/kg, 55 [16%] of 343 given 10 mg/kg, and 109 [35%] of 309 given docetaxel). INTERPRETATION: Pembrolizumab prolongs overall survival and has a favourable benefit-to-risk profile in patients with previously treated, PD-L1-positive, advanced non-small-cell lung cancer. These data establish pembrolizumab as a new treatment option for this population and validate the use of PD-L1 selection. FUNDING: Merck & Co.

Dendritic Cells in Cancer Immunology and Immunotherapy.

Cancer immunotherapy modulates the immune system, overcomes immune escape and stimulates immune defenses against tumors. Dendritic cells (DCs) are professional promoters of immune responses against tumor antigens with the outstanding ability to coordinate the innate and adaptive immune systems. Evidence suggests that there is a decrease in both the number and function of DCs in cancer patients. Therefore, they represent a strong scaffold for therapeutic interventions. DC vaccination (DCV) is safe, and the antitumoral responses induced are well established in solid tumors. Although the addition of checkpoint inhibitors (CPIs) to chemotherapy has provided new options in the treatment of cancer, they have shown no clinical benefit in immune desert tumors or in those tumors with dysfunctional or exhausted T-cells. In this way, DC-based therapy has demonstrated the ability to modify the tumor microenvironment for immune enriched tumors and to potentiate systemic host immune responses as an active approach to treating cancer patients. Application of DCV in cancer seeks to obtain long-term antitumor responses through an improved T-cell priming by enhancing previous or generating de novo immune responses. To date, DCV has induced immune responses in the peripheral blood of patients without a significant clinical impact on outcome. Thus, improvements in vaccines formulations, selection of patients based on biomarkers and combinations with other antitumoral therapies are needed to enhance patient survival. In this work, we review the role of DCV in different solid tumors with their strengths and weaknesses, and we finally mention new trends to improve the efficacy of this immune strategy.

Low-Dose Ionizing γ-Radiation Elicits the Extrusion of Neutrophil Extracellular Traps.

PURPOSE: Patients with cancer frequently undergo radiotherapy in their clinical management with unintended irradiation of blood vessels and copiously irrigated organs in which polymorphonuclear leukocytes circulate. Following the observation that such low doses of ionizing radiation are able to induce neutrophils to extrude neutrophil extracellular traps (NET), we have investigated the mechanisms, consequences, and occurrence of such phenomena in patients undergoing radiotherapy. EXPERIMENTAL DESIGN: NETosis was analyzed in cultures of neutrophils isolated from healthy donors, patients with cancer, and cancer-bearing mice under confocal microscopy. Cocultures of radiation-induced NETs, immune effector lymphocytes, and tumor cells were used to study the effects of irradiation-induced NETs on immune cytotoxicity. Radiation-induced NETs were intravenously injected to mice for assessing their effects on metastasis. Circulating NETs in irradiated patients with cancer were measured using ELISA methods for detecting MPO-DNA complexes and citrullinated histone 3. RESULTS: Irradiation of neutrophils with very low γ-radiation doses (0.5-1 Gy) elicits NET formation in a manner dependent on oxidative stress, NADPH oxidase activity, and autocrine IL8. Radiation-induced NETs interfere with NK cell and T-cell cytotoxicity. As a consequence, preinjection of irradiation-induced NETs increases the number of successful metastases in mouse tumor models. Increases in circulating NETs were readily detected in two prospective series of patients following the first fraction of their radiotherapy courses. CONCLUSIONS: NETosis is induced by low-dose ionizing irradiation in a neutrophil-intrinsic fashion, and radiation-induced NETs are able to interfere with immune-mediated cytotoxicity. Radiation-induced NETs foster metastasis in mouse models and can be detected in the circulation of patients undergoing conventional radiotherapy treatments. See related commentary by Mowery and Luke, p. 3965.

A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C.

BACKGROUND & AIMS: Tremelimumab is a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), an inhibitory co-receptor that interferes with T cell activation and proliferation. The purpose of this pilot clinical trial was to test the antitumor and antiviral effect of tremelimumab in patients with hepatocellular carcinoma (HCC) and chronic hepatitis C virus (HCV) infection; and to study the safety of its administration to cirrhotic patients. METHODS: Tremelimumab at a dose of 15 mg/kg IV every 90 days was administered until tumor progression or severe toxicity. Twenty patients were assessable for toxicity and viral response and 17 were assessable for tumor response. Most patients were in the advanced stage and 43% had an altered liver function (Child-Pugh class B). RESULTS: A good safety profile was recorded and no patient needed steroids because of severe immune-mediated adverse events. Some patients had a transient albeit intense elevation of transaminases after the first dose, but not following subsequent cycles. Partial response rate was 17.6% and disease control rate was 76.4%. Time to progression was 6.48 months (95% CI 3.95-9.14). A significant drop in viral load was observed while new emerging variants of the hypervariable region 1 of HCV replaced the predominant variants present before therapy, particularly in those patients with a more prominent drop in viral load. This antiviral effect was associated with an enhanced specific anti-HCV immune response. CONCLUSIONS: Tremelimumab safety profile and antitumor and antiviral activity, in patients with advanced HCC developed on HCV-induced liver cirrhosis, support further investigation.

Pilot clinical trial of type 1 dendritic cells loaded with autologous tumor lysates combined with GM-CSF, pegylated IFN, and cyclophosphamide for metastatic cancer patients.

Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-α, TNF-α, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day -7), GM-CSF (days 1-4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-γ-ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [(111)In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.

PSA reactivity in extracellular microvesicles to commercial immunoassays.

AIMS: Characterization of PSA in extracellular microvesicles (EVs) and its reactivity to commercial methods. MATERIALS AND METHODS: EVs derived from serum of 47 prostate cancer (PCa) patients, 27 benign prostatic hyperplasia (BPH) patients and 42 healthy controls were analyzed. EVs isolation and quantification of PSA immunoreactive to total (ev-T-PSA) or free (ev-F-PSA) PSA immunoassays, were performed using commercial assays. PSA in CD81+ or CD63+ EVs was determined directly in serum by an immunocapture-ELISA (IC-ELISA). RESULTS: Ev-T-PSA immunoreactive to Elecsys assay was detected in all samples. Median T-PSA ev/srm ratio was 2.20 % (Q1-Q3: 0.80-4.00 %), although in some samples this ratio reached 59 %. T-PSA ev/srm ratio was higher in those samples with serum T-PSA below 4 µg/L than in those exceeding that cut-off (p < 0.001). T-PSA ev/srm ratio was lower in PCa patients compared to healthy controls and BPH patients (p < 0.001). Elecsys immunoassays detected higher concentrations of ev-T-PSA and ev-F-PSA than Immulite (p < 0.001). PSA was detected by IC-ELISA more intensely in CD81+ EVs than in CD63+ EVs, and ev-T-PSA correlated with PSA+ CD63+ (p < 0.001) but not with PSA+ CD81+. CONCLUSION: EVs-bound PSA is another form of circulating PSA whose measurement could be easily performed in clinical laboratories by automated immunoassays.

CD137 (4-1BB) requires physically associated cIAPs for signal transduction and antitumor effects.

CD137 (4-1BB) is a member of the TNFR family that mediates potent T cell costimulatory signals upon ligation by CD137L or agonist monoclonal antibodies (mAbs). CD137 agonists attain immunotherapeutic antitumor effects in cancer mouse models, and multiple agents of this kind are undergoing clinical trials. We show that cIAP1 and cIAP2 are physically associated with the CD137 signaling complex. Moreover, cIAPs are required for CD137 signaling toward the NF-κB and MAPK pathways and for costimulation of human and mouse T lymphocytes. Functional evidence was substantiated with SMAC mimetics that trigger cIAP degradation and by transfecting cIAP dominant-negative variants. Antitumor effects of agonist anti-CD137 mAbs are critically dependent on the integrity of cIAPs in cancer mouse models, and cIAPs are also required for signaling from CARs encompassing CD137's cytoplasmic tail.

Synergistic effects of CTLA-4 blockade with tremelimumab and elimination of regulatory T lymphocytes in vitro and in vivo.

Anti-CTLA-4 monoclonal antibodies (mAb) that block the interaction of CTLA-4 with CD80 and CD86 such as tremelimumab and ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de-repress tumor-specific cellular immunity. Addition of the fully human anti-CTLA-4 (tremelimumab) to cultures of human T cells with allogenic dendritic cells (DCs) did not increase proliferation. Magnetic bead-mediated elimination of CD4(+) CD25(+) regulatory T cells (T(reg)) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast, predepletion of CD4(+) CD25(+) T(reg) and culture in the presence of tremelimumab synergistically resulted in increased proliferation and DC:T-cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. The synergy mechanism can be traced to enhanced CTLA-4 expression in effector cells as a result of T(reg) elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2(-/-) IL-2Rγ(-/-) mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T(reg) cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T-cell compartment. In addition, T(reg) depletion and CTLA-4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV-transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T(reg) depletion.

Activity of a multitargeted chemo-switch regimen (sorafenib, gemcitabine, and metronomic capecitabine) in metastatic renal-cell carcinoma: a phase 2 study (SOGUG-02-06).

BACKGROUND: Maximum tolerated dose (MTD) chemotherapy followed by metronomic chemotherapy (low doses given on a frequent schedule) acts on tumour vascular endothelial cells by increasing the anti-tumour effect of anti-angiogenic agents. This multicentre, phase 2 study investigated the effectiveness of MTD gemcitabine combined with metronomic capecitabine plus the multikinase inhibitor sorafenib for the treatment of metastatic renal-cell carcinoma (RCC). METHODS: Patients were enrolled at eight centres across Spain between Dec 13, 2006, and April 17, 2008. Patients were aged 18 years or older, had confirmed metastatic RCC with clear-cell histology, had an Eastern Cooperative Oncology Group performance status of 0 or 1, had not undergone previous therapy, and were unsuitable for, or intolerant to, immunotherapy. Treatment consisted of intravenous gemcitabine 1000 mg/m(2) (days 1 and 8), oral capecitabine 500 mg/m(2) twice a day (final dose after adjustment, days 1-14), and oral sorafenib 400 mg twice a day (days 1-21), for six cycles, followed by sorafenib monotherapy (at the investigator's discretion if clinical benefit was maintained). The primary endpoint was median progression-free survival (PFS) analysed in a population of all patients who received treatment. The trial is registered with ClinicalTrials.gov, number NCT00496301. FINDINGS: 44 patients enrolled in the study, 40 of whom received treatment. Median PFS for these patients was 11.1 months (95% CI 7.9-17.1). A partial response was achieved in 20 patients, and stable disease in 17 patients. Most adverse events were grade 1 or 2. Grade 3 adverse events were fatigue or asthenia (n=9), hand-foot skin reaction (n=11), mucositis (n=3), diarrhoea (n=2), infection (n=2), and allergic reaction, hypertension, and rash (all n=1). Grade 3 haematological toxicity was noted in nine patients. One death due to pulmonary embolism was reported as grade 5 dyspnoea possibly related to study drug. INTERPRETATION: PFS and response rates were greater than those previously observed with gemcitabine and capecitabine or sorafenib monotherapy in patients with metastatic RCC. Adverse events were manageable in most patients. These findings provide preliminary confirmation of the synergistic activity of the chemo-switch concept seen in preclinical studies, and merit further exploration. FUNDING: Spanish Oncology Genitourinary Group (SOGUG).

Paradigms on Immunotherapy Combinations with Chemotherapy.

Checkpoint inhibitors are being added to standard-of-care chemotherapy in multiple clinical trials. Success has been reported in non-small and small cell lung carcinomas and urothelial, head and neck, gastric, and esophageal cancers, and promising results are already available in triple-negative breast and pancreatic malignancies. The potential mechanisms of synergy include immunogenic tumor cell death, antiangiogenesis, selective depletion of myeloid immunosuppressive cells, and lymphopenia, which reduces regulatory T cells and makes room for proliferation of effector T cells. However, chemotherapy regimens have not been optimized for such combinations, perhaps explaining some recent clinical trial disappointments. Approaches to make the most of chemoimmunotherapy include neoadjuvant and adjuvant schemes.Significance: Immunotherapy of cancer based on PD-1/PD-L1 blockade has prompted a revolution in cancer clinical management. Evidence in phase III clinical trials already supports combinations of immunotherapy with standard-of-care chemotherapy for a number of malignant diseases. This review focuses on such evidence and provides an overview of the potential synergistic mechanisms of action and the opportunities to optimize chemoimmunotherapy regimens.

Five Year Survival Update From KEYNOTE-010: Pembrolizumab Versus Docetaxel for Previously Treated, Programmed Death-Ligand 1-Positive Advanced NSCLC.

INTRODUCTION: In the KEYNOTE-010 study, pembrolizumab improved overall survival (OS) versus docetaxel in patients with previously treated, advanced NSCLC with programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) ≥50% and ≥1%. We report 5-year efficacy and safety follow-up for the KEYNOTE-010 study. METHODS: Patients were randomized to pembrolizumab 2 mg/kg or 10 mg/kg once every 3 weeks or docetaxel 75 mg/m2 once every 3 weeks for up to 35 cycles (2 y). Patients who completed pembrolizumab treatment and subsequently had recurrence could receive second-course pembrolizumab for up to 17 cycles (1 y). Pembrolizumab doses were pooled in this analysis. RESULTS: A total of 1034 patients were randomized (pembrolizumab, n = 691; docetaxel, n = 343). Median study follow-up was 67.4 months (range: 60.0‒77.9). The hazard ratio (95% confidence interval) for OS was 0.55 (0.44‒0.69) for patients with PD-L1 TPS ≥50% and 0.70 (0.61‒0.80) with PD-L1 TPS ≥1%. The 5-year OS rates for pembrolizumab versus docetaxel were 25.0% versus 8.2% in patients with PD-L1 TPS ≥50% and 15.6% versus 6.5% with PD-L1 TPS ≥1%. Among 79 patients who completed 35 cycles/2 years of pembrolizumab, the OS rate 3 years after completion (∼5 y from randomization) was 83.0%. A total of 21 patients received second-course pembrolizumab; 11 (52.4%) had an objective response after starting the second course and 15 (71.4%) were alive at data cutoff. Exploratory biomarker analysis revealed that higher tissue tumor mutational burden (≥175 mutations per exome) was associated with improved outcomes with pembrolizumab. CONCLUSIONS: Pembrolizumab continued to provide long-term benefit than docetaxel in patients with previously treated advanced NSCLC with PD-L1 TPS ≥50% and ≥1%. Our findings confirm pembrolizumab as a standard-of-care treatment in the second-line or later setting.

Partial Response and Stable Disease Correlate with Positive Outcomes in Atezolizumab-treated Patients with Advanced Urinary Tract Carcinoma.

BACKGROUND: The value of a complete response to immune checkpoint inhibitor treatment for urothelial cancer is well recognised, but less is known about long-term outcomes in patients with a partial response or the benefit of achieving disease stabilisation. OBJECTIVE: To determine clinical outcomes in patients with a partial response or stable disease on atezolizumab therapy for advanced urinary tract carcinoma (UTC). DESIGN, SETTING, AND PARTICIPANTS: Data were extracted from three prospective trials (IMvigor210 cohort 2, SAUL, and IMvigor211) evaluating single-agent atezolizumab therapy for platinum-pretreated advanced UTC. The analysis population included 604 atezolizumab-treated and 208 chemotherapy-treated patients (229 achieving a partial response and 583 achieving stable disease). INTERVENTION: Atezolizumab 1200 mg every 3 wk until progression or unacceptable toxicity or single-agent chemotherapy for patients in the control arm of IMvigor211. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Baseline characteristics, treatment exposure, overall survival, duration of disease control. Partial response and stable disease populations were analysed separately. RESULTS AND LIMITATIONS: The population of patients with a partial response included more patients with programmed cell death ligand 1 (PD-L1) expression on ≥5% of tumour-infiltrating immune cells than the stable disease population. The median time to best response was 2.1 mo across trials and treatments, regardless of the type of response. Atezolizumab-treated patients with a partial response had sustained disease control (median overall survival not reached); durations of disease control and overall survival were longer with atezolizumab than with chemotherapy. In patients with stable disease, median overall survival was numerically longer with atezolizumab (exceeding 1 yr) than with chemotherapy. Irrespective of treatment, durations of disease control and survival were shorter in patients with stable disease than in those achieving a partial response. These analyses are limited by their post hoc exploratory nature and relatively short follow-up. CONCLUSIONS: Stable disease and partial response are meaningful clinical outcomes in atezolizumab-treated patients with advanced UTC. PATIENT SUMMARY: In this report, we looked at the outcomes in patients whose tumours responded to treatment to some extent, but the tumour did not disappear completely. We aimed to understand whether a modest response to treatment was associated with meaningful long-term outcomes for patients. We found that on average, life expectancy was >1 yr in patients whose disease was stabilised and even longer in those whose tumours showed some shrinkage in response to treatment.

In vivo depletion of DC impairs the anti-tumor effect of agonistic anti-CD137 mAb.

Anti-CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti-tumor effect involves co-stimulation of tumor-specific CD8(+) T cells. Whether antigen cross-presenting DC are required for the efficacy of anti-CD137 mAb treatment has never been examined. Here we show that the administration of anti-CD137 mAb eradicates EG7-OVA tumors by a strictly CD8beta(+) T-cell-dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross-presentation revealed that CD11c(+) cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor-draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti-OVA CTL induction. Using CD11c diphtheria toxin receptor-green fluorescent protein-->C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross-presentation in CTL induction against OVA(257-264) epitope and in the antitumor efficacy induced by anti-CD137 mAb.

Delivery of immunostimulatory monoclonal antibodies by encapsulated hybridoma cells.

Immunostimulatory monoclonal antibodies are immunoglobulins directed toward surface proteins of immune system cells that augment the immune response against cancer in a novel therapeutic fashion. Exogenous administration of the recombinant humanized immunoglobulins is being tested in clinical trials with agents of this kind directed at a variety of immune-controlling molecular targets. In this study, the encapsulation of antibody-producing hybridoma cells was tested in comparison with the systemic administration of monoclonal antibodies. Hybridomas producing anti-CD137 and anti-OX40 mAb were encapsulated in alginate to generate microcapsules containing viable cells that secrete antibody. Immobilized cells in vitro were able to release the rat immunoglobulin produced by the hybridomas into the supernatant. Microcapsules were implanted by injection into the subcutaneous tissue of mice and thereby provided a platform for viable secreting cells, which lasted for more than 1 week. The pharmacokinetic profile of the rat monoclonal antibodies following microcapsule implantation was similar to that attained following an intraperitoneal administration of the purified antibodies. The rat-mouse hybridoma cells did not engraft as tumors in immunocompetent mice, while they lethally xenografted in immunodeficient mice, if not microencapsulated. The antitumor therapeutic activity of the strategy was studied on established CT26 colon carcinomas resulting in complete tumor eradication in an elevated fraction of cases and strong tumor-specific CTL responses with either anti-CD137 or anti-OX40 producing hybridomas, thus offering proof of the concept. This form of administration permitted combinations of more than one immunostimulatory monoclonal antibody to exploit the synergistic effects such as those known to be displayed by anti-CD137 and anti-OX40 mAb.

Treatment with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor immunotherapeutic effects in this organ.

BACKGROUND/AIMS: Cancer therapy with agonist anti-CD137 mAbs has been shown to induce immune-mediated tumor rejections in mice, and equivalent agents of this kind are currently being tested in cancer patients. Previous reports indicated that CD137 stimulation induced polyclonal infiltrates of T lymphocytes in the liver. This study characterizes the liver infiltrates and the target dependency of the phenomena and addresses the question of whether tumors nested in the liver are a more favorable target for CD137-based immunotherapy. METHODS: Liver infiltrates were studied with conventional histology and multiple color flow cytometry of total liver leukocytes. CD137(-/-) mice, mice with a single rearrangement of the TCR (OT-1 mice) and Rag(-/-) mice were used to clarify molecular requirements. Mice implanted with MC38 colon carcinomas either subcutaneously or inside the liver were used for comparative studies under treatment with agonist anti-CD137 mAbs. RESULTS: CD137 treatment caused mononuclear inflammation in the portal spaces of the liver, which gave rise to moderate increases in transaminases without signs of cholestasis. Marked increases in the numbers of CD8+ T cells were observed, including CD8+ T lymphocytes co-expressing CD11c. Infiltrates were absent in CD137(-/-) mice and mitigated in mice harboring a single transgenic TCR on their CD8 T cells. Despite the tumor-independent accumulation of T cells in the liver, immunotherapeutic effects were not more prominent against tumors located in this organ. CONCLUSIONS: Target-dependent effects of CD137 stimulation lead to liver infiltration with T cells, but lymphocyte enrichment in this organ does not privilege this site for immunotherapeutic effects against transplanted tumors.