Durvalumab with first-line chemotherapy in previously untreated malignant pleural mesothelioma (DREAM): a multicentre, single-arm, phase 2 trial with a safety run-in.

BACKGROUND: There is a strong unmet need to improve systemic therapy in mesothelioma. Chemotherapy with cisplatin and pemetrexed improves survival in malignant pleural mesothelioma, and immune checkpoint inhibitors are an emerging treatment in this disease. We aimed to evaluate the activity of durvalumab, an anti-PD-L1 antibody, given during and after first-line chemotherapy with cisplatin and pemetrexed in patients with advanced malignant pleural mesothelioma. METHODS: DREAM was a multicentre, single-arm, open-label, phase 2 trial done in nine hospitals in Australia. Eligible patients were aged 18 years or older and had histologically confirmed malignant pleural mesothelioma considered unsuitable for cancer-directed surgery, an Eastern Cooperative Oncology Group performance status of 0 or 1, and measurable disease as per the modified Response Evaluation Criteria in Solid Tumors version 1.0 (mRECIST) for mesothelioma that was previously untreated with systemic therapy. All histological subtypes were eligible. The first six participants were treated for two cycles in a safety run-in. All participants received cisplatin 75 mg/m2, pemetrexed 500 mg/m2, and durvalumab 1125 mg intravenously on day 1 of a 3-weekly schedule for a maximum of six cycles. Change from cisplatin to carboplatin with an area under the curve of 5 was permitted. Durvalumab was continued for a maximum of 12 months. The primary endpoint was progression-free survival at 6 months, measured according to mRECIST for malignant pleural mesothelioma and analysed in the intention-to-treat population. Safety analyses included all participants who receive at least one dose of any study drug. This study is registered with the Australia New Zealand Clinical Trials Registry, ACTRN12616001170415. FINDINGS: Between Dec 28, 2016, and Sept 27, 2017, 55 participants were enrolled. 54 patients were eligible and were followed up for a median of 28·2 months (IQR 26·5-30·2). 31 (57%; 95% CI 44-70) of 54 patients were alive and progression-free at 6 months. The most common grade 3-4 adverse events were neutropenia (seven [13%] patients), nausea (six [11%]), and anaemia (four [7%]). A total of 60 serious adverse events occurred in 29 participants, five of which were considered possibly related to durvalumab. Five patients died during the study treatment; none of these five deaths were attributed to study treatment. INTERPRETATION: The combination of durvalumab, cisplatin, and pemetrexed has promising activity and an acceptable safety profile that warrants further investigation in a randomised phase 3 trial. FUNDING: AstraZeneca.

Immune marker expression of irradiated mesothelioma cell lines.

Background: Though immune checkpoint inhibition has recently shown encouraging clinical efficacy in mesothelioma, most patients do not respond. Combining immune checkpoint inhibition with radiotherapy presents an attractive option for improving treatment responses owing to the various immunomodulatory effects of radiation on tumors. However, the ideal dosing and scheduling of combined treatment remains elusive, as it is poorly studied in mesothelioma. The present study characterizes the dose- and time-dependent changes to expression of various immune markers and cytokines important to antitumor responses following irradiation of mesothelioma cell lines. Methods: Two murine (AB1, AE17) and two human (BYE, JU77) mesothelioma cell lines were treated with titrated gamma-radiation doses (1-8 Gy) and the expression of MHC class-I, MHC class-II and PD-L1 was measured over a series of post-irradiation timepoints (1-72 hours) by flow cytometry. Levels of cytokines IL-1α, IL-1ß, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN-ß, IFN-γ, TNF-α, and GM-CSF were measured by multiplex immunoassay in murine cell lines following 8 Gy radiation. Results: Following irradiation, a dose-dependent upregulation of MHC-I and PD-L1 was observed on three of the four cell lines studied to varying extents. For all cell lines, the increase in marker expression was most pronounced 72 hours after radiation. At this timepoint, increases in levels of cytokines IFN-ß, MCP-1 and IL-6 were observed following irradiation with 8 Gy in AB1 but not AE17, reflecting patterns in marker expression. Conclusions: Overall, this study establishes the dose- and time-dependent changes in immune marker expression of commonly studied mesothelioma cell lines following radiation and will inform future study into optimal dosing and scheduling of combined radiotherapy and immune checkpoint inhibition for mesothelioma.

Effects of Photon Radiation on DNA Damage, Cell Proliferation, Cell Survival, and Apoptosis of Murine and Human Mesothelioma Cell Lines.

Purpose: To characterize the cellular responses of murine and human mesothelioma cell lines to different doses of photon radiation with a long-term aim of optimizing a clinically relevant in vivo model in which to study the interaction of radiation therapy and immunotherapy combinations. Methods and Materials: Two murine mesothelioma cell lines (AB1 and AE17) and 3 human cell lines (BYE, MC, and JU) were used in the study. Cells were treated with increasing doses of photon radiation. DNA damage, DNA repair, cell proliferation, and apoptosis at different time points after irradiation were quantified by flow cytometry, and cell survival probability was examined using clonogenic survival assay. Results: DNA damage increased with escalating dose in all cell lines. Evident G2/M arrest and reduced cell proliferation were observed after irradiation with 8 Gy. DNA repair was uniformly less efficient at higher compared with lower radiation-fraction doses. The apoptosis dose response varied between cell lines, with greater apoptosis observed at 16 Gy with human BYE and murine AB1 cell lines but less for other studied cell lines, regardless of dose and time. The α/ß ratio from the cell survival fraction of human mesothelioma cell lines was smaller than from murine ones, suggesting human cell lines in our study were more sensitive to a change of dose per fraction than were murine mesothelioma cell lines. However, in all studied cell lines, colony formation was completely inhibited at 8 Gy. Conclusions: A threshold dose of 8 Gy appeared to be appropriate for hypofractionated radiation therapy. However, the radiation therapy doses between 4 and 8 Gy remain to be systematically analyzed. These observations provide an accurate picture of the in vitro response of mesothelioma cell lines to photon irradiation and characterize the heterogeneity between human and murine cell lines. This information may guide in vivo experiments and the strengths and limitations of extrapolation from murine experimentation to potential human translation.

In vivo noninvasive preclinical tumor hypoxia imaging methods: a review.

Tumors exhibit areas of decreased oxygenation due to malformed blood vessels. This low oxygen concentration decreases the effectiveness of radiation therapy, and the resulting poor perfusion can prevent drugs from reaching areas of the tumor. Tumor hypoxia is associated with poorer prognosis and disease progression, and is therefore of interest to preclinical researchers. Although there are multiple different ways to measure tumor hypoxia and related factors, there is no standard for quantifying spatial and temporal tumor hypoxia distributions in preclinical research or in the clinic. This review compares imaging methods utilized for the purpose of assessing spatio-temporal patterns of hypoxia in the preclinical setting. Imaging methods provide varying levels of spatial and temporal resolution regarding different aspects of hypoxia, and with varying advantages and disadvantages. The choice of modality requires consideration of the specific experimental model, the nature of the required characterization and the availability of complementary modalities as well as immunohistochemistry.

A phase 1b clinical trial optimizing regulatory T cell depletion in combination with platinum-based chemotherapy in thoracic cancers.

Background: Single-agent cyclophosphamide can deplete regulatory T-cells (Treg). We aimed to determine optimal dosing and scheduling of oral cyclophosphamide, alongside pemetrexed-based chemotherapy, to deplete Treg in mesothelioma or non-small-cell lung cancer patients.Methods: 31 Patients received pemetrexed ± cisplatin or carboplatin on day 1 of a 21-day cycle (maximum 6 cycles). From cycle two, patients received cyclophosphamide, 50 mg/day, with intrapatient escalation to maximum 100/150 mg/day alternately. Immunological changes were examined by flow cytometry. Primary endpoint was Treg proportion of CD4+ T-cells, with doses tailored to target Treg nadir <4%.Results: Reduction in Treg proportion was observed on day 8 of all cycles, and was not augmented by cyclophosphamide. Few patients achieved the <4% Treg target. Treg proliferation reached nadir one week after chemotherapy, and peaked on day 1 of the subsequent cycle. Efficacy parameters were similar to chemotherapy alone. Seventeen percent of patients ceased cyclophosphamide due to toxicity.Conclusions: Specific Treg depletion to the degree seen with single-agent cyclophosphamide was not observed during pemetrexed-based chemotherapy. This study highlights the poor evidence basis for use of cyclophosphamide as an immunotherapeutic in combination with chemotherapy, and the importance of detailed flow cytometry studies.Trial registration: Clinical trial registration: www.anzctr.org.au identifier is ACTRN12609000260224.

Changes in expression of PD-L1 on peripheral T cells in patients with melanoma and lung cancer treated with PD-1 inhibitors.

Advances in cancer immunology have increased the use of immune checkpoint inhibitors in clinical practice, however not all patients respond, and treatment can have severe side-effects. Blood-based immunological biomarkers are an attractive method for predicting which patients will respond to therapy, however, reliable biomarkers for immune checkpoint blockade are lacking. This study aimed to identify patients before or early in treatment who would best respond to PD-1 inhibitors. We hypothesised that higher baseline PD-L1 and/or PD-1 on peripheral blood T cells could predict radiological response to PD-1 inhibitors. This pilot prospective cohort study assessed 26 patients with melanoma or non-small cell lung cancer, treated with pembrolizumab, nivolumab, or nivolumab/ipilimumab combined. Response was assessed by RECIST 1.1. Peripheral blood lymphocytes collected at baseline, after one cycle, 10 weeks and at discontinuation of therapy were analysed by flow cytometry. Patients with a higher proportion of PD-L1+ T cells at baseline had improved objective response to PD-1 inhibitor therapy, and patients with a lower proportion of regulatory T cells at baseline experienced more immune-related adverse events. These findings may prove useful to assist in clinical decision making. Further studies with larger cohorts are required to validate these findings.

Enhancing the efficacy of immunotherapy using radiotherapy.

Recent clinical breakthroughs in cancer immunotherapy, especially with immune checkpoint blockade, offer great hope for cancer sufferers - and have greatly changed the landscape of cancer treatment. However, whilst many patients achieve clinical responses, others experience minimal benefit or do not respond to immune checkpoint blockade at all. Researchers are therefore exploring multimodal approaches by combining immune checkpoint blockade with conventional cancer therapies to enhance the efficacy of treatment. A growing body of evidence from both preclinical studies and clinical observations indicates that radiotherapy could be a powerful driver to augment the efficacy of immune checkpoint blockade, because of its ability to activate the antitumor immune response and potentially overcome resistance. In this review, we describe how radiotherapy induces DNA damage and apoptosis, generates immunogenic cell death and alters the characteristics of key immune cells in the tumor microenvironment. We also discuss recent preclinical work and clinical trials combining radiotherapy and immune checkpoint blockade in thoracic and other cancers. Finally, we discuss the scheduling of immune checkpoint blockade and radiotherapy, biomarkers predicting responses to combination therapy, and how these novel data may be translated into the clinic.

Dexamethasone differentially depletes tumour and peripheral blood lymphocytes and can impact the efficacy of chemotherapy/checkpoint blockade combination treatment.

Dexamethasone is a synthetic glucocorticoid commonly used for the prevention and management of side effects in cancer patients undergoing chemotherapy. While it is effective as an anti-emetic and in preventing hypersensitivity reactions, dexamethasone depletes peripheral blood lymphocytes and impacts immune responses. The effect of dexamethasone on the number and quality of tumour-infiltrating leukocytes has not been reported. To address this, we calibrated the dose in two different strains of mice to achieve the same extent of peripheral blood lymphocyte depletion observed in patients with cancer. Doses that caused analogous depletion of T and B lymphocytes and NK cells from the peripheral blood, elicited no change in these populations within the tumour. The expression of immune checkpoint molecules PD-1, OX40, GITR and TIM3 on tumour-infiltrating lymphocytes was not altered. We found that dexamethasone had a small but significant deleterious impact on weakly efficacious chemoimmunotherapy but had no effect when the protocol was highly efficacious. Based on these results, we predict that dexamethasone will have a modest negative influence on the overall effectiveness of chemoimmunotherapy treatment.

Tumour associated lymphocytes in the pleural effusions of patients with mesothelioma express high levels of inhibitory receptors.

OBJECTIVE: Pleural effusion (PE) is a common feature of malignant pleural mesothelioma. These effusions typically contain lymphocytes and malignant cells. We postulated that the PE would be a source of lymphocytes for analysis of tumor immune milieu. The aim of this study was to compare the phenotype and T cell receptor usage of pleural effusion T cells with paired concurrently drawn peripheral blood lymphocytes. We used multi-parameter flow cytometry and high-throughput T cell receptor sequencing to analyse peripheral blood and pleural effusion mononuclear cells. RESULTS: Both CD8+ and CD4+ T cells from effusion showed increased expression of T cell inhibitory receptors PD-1, LAG-3 and Tim-3 compared to blood. Comprehensive T cell receptor sequencing on one of the patients showed a discordant distribution of clonotypes in the antigen-experienced (PD-1+) compartment between effusion and blood, suggesting an enrichment of antigen specific clonotypes in the effusion, with potential as an immunological response biomarker.

Dexamethasone co-medication in cancer patients undergoing chemotherapy causes substantial immunomodulatory effects with implications for chemo-immunotherapy strategies.

The glucocorticoid (GC) steroid dexamethasone (Dex) is used as a supportive care co-medication for cancer patients undergoing standard care pemetrexed/platinum doublet chemotherapy. As trials for new cancer immunotherapy treatments increase in prevalence, it is important to track the immunological changes induced by co-medications commonly used in the clinic, but not specifically included in trial design or in pre-clinical models. Here, we document a number of Dex -induced immunological effects, including a large-scale lymphodepletive effect particularly affecting CD4+ T cells but also CD8+ T cells. The proportion of regulatory T cells within the CD4+ compartment did not change after Dex was administered, however a significant increase in proliferation and activation of regulatory T cells was observed. We also noted Dex -induced proportional changes in dendritic cell (DC) subtypes. We discuss these immunological effects in the context of chemoimmunotherapy strategies, and suggest a number of considerations to be taken into account when designing future studies where Dex and other GCs may be in use.

Chemotherapy and immunotherapy: mapping the road ahead.

Cancer immunotherapy, and in particular checkpoint blockade, is now standard clinical care for a growing number of cancers. Cytotoxic drugs have been the primary weapon against cancer for a long time and have typically been understood because of their capacity to directly kill tumour cells. It is now clear that these drugs are potential partners for checkpoint blockade and different drugs can influence the immune response to cancer through a wide variety of mechanisms. Some of these relate to immunogenic cell death, whilst others relate to changes in antigen-presentation, tumour cell targeting, or depletion of immunosuppressive cells. Here, we review some recent advances in our understanding of the immunological changes associated with chemotherapy, discuss progress in combining chemotherapy with checkpoint blockade, and comment on the difficulties encountered in translating promising preclinical data into successful treatments for cancer patients.

Identification of a Bipotent Epithelial Progenitor Population in the Adult Thymus.

Thymic epithelial cells (TECs) are critically required for T cell development, but the cellular mechanisms that maintain adult TECs are poorly understood. Here, we show that a previously unidentified subpopulation, EpCam(+)UEA1(-)Ly-51(+)PLET1(+)MHC class II(hi), which comprises <0.5% of adult TECs, contains bipotent TEC progenitors that can efficiently generate both cortical (c) TECs and medullary (m) TECs. No other adult TEC population tested in this study contains this activity. We demonstrate persistence of PLET1(+)Ly-51(+) TEC-derived cells for 9 months in vivo, suggesting the presence of thymic epithelial stem cells. Additionally, we identify cTEC-restricted short-term progenitor activity but fail to detect high efficiency mTEC-restricted progenitors in the adult thymus. Our data provide a phenotypically defined adult thymic epithelial progenitor/stem cell that is able to generate both cTECs and mTECs, opening avenues for improving thymus function in patients.