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The discovery that anti-programmed death-1 antibody (anti-PD-1) immunotherapy can cure patients with multidrug-resistant gestational trophoblastic neoplasia provides a new powerful and low toxicity treatment. This heralds an era within which the majority of patients, including those with previously difficult to treat disease, can expect to achieve long-term remission. This development should prompt a rethink of how patients with this rare disease are managed, focusing on maximizing cure rate with minimal exposure to toxic chemotherapy.

Purpose:Immune dysregulation is described in multiple myeloma. While preclinical models suggest a role for altered T-cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterize marrow-infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first-line therapy.Experimental Design:We undertook detailed characterization of T cells from bone marrow (BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression-free survival.Results:We found that patients with multiple myeloma had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared with healthy donors. Patients with higher frequencies of Tregs had shorter PFS and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared with patients with lower frequencies of Tregs. Analysis of CD4 and CD8 effectors revealed that low CD4effector (CD4eff):Treg ratio and increased frequency of PD-1–expressing CD4eff cells were independent predictors of early relapse over and above conventional risk factors, such as genetic risk and depth of response. Ex vivo functional analysis and RNA sequencing revealed that CD4 and CD8 cells from patients with greater abundance of CD4effPD-1+ cells displayed transcriptional and secretory features of dysfunction.Conclusions:BM-infiltrating T-cell subsets, specifically Tregs and PD-1–expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies.

Supplementary tables and figures

Intratumoral regulatory T (Treg) cell abundance associates with diminished antitumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high-affinity receptor subunit for interleukin (IL)-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their antitumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Treg cells, while preserving IL-2-STAT5 signaling on effector T cells and show synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both nonhuman primates and humanized mouse models, efficient Treg cell depletion with no overt immune-related toxicities. Our data support the clinical development of RG6292 and evaluation of new combination therapies incorporating non-IL-2-blocking anti-CD25 antibodies in clinical studies. Quezada and colleagues develop improved anti-CD25 antibodies that preserve IL-2 signaling and enhance single-agent antitumor immunity and immunotherapy through specific and efficient Treg cell depletion.

Purpose: Immune dysregulation is described in multiple myeloma. While preclinical models suggest a role for altered T-cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterize marrow-infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first-line therapy. Experimental Design: We undertook detailed characterization of T cells from bone marrow (BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression-free survival. Results: We found that patients with multiple myeloma had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared with healthy donors. Patients with higher frequencies of Tregs had shorter PFS and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared with patients with lower frequencies of Tregs. Analysis of CD4 and CD8 effectors revealed that low CD4effector (CD4eff):Treg ratio and increased frequency of PD-1–expressing CD4eff cells were independent predictors of early relapse over and above conventional risk factors, such as genetic risk and depth of response. Ex vivo functional analysis and RNA sequencing revealed that CD4 and CD8 cells from patients with greater abundance of CD4effPD-1+ cells displayed transcriptional and secretory features of dysfunction. Conclusions: BM-infiltrating T-cell subsets, specifically Tregs and PD-1–expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies.

Immune checkpoint inhibitors (ICI) have revolutionised treating advanced cancers. ICI are administered intravenously every 2-6 weeks for up to 2 years, until cancer progression/unacceptable toxicity. Physiological efficacy is observed at lower doses than those used as standard of care (SOC). Pharmacodynamic studies indicate sustained target occupancy, despite a pharmacological half-life of 2-3 weeks. Reducing frequency of administration may be possible without compromising outcomes. The REFINE trial aims to limit individual patient exposure to ICI whilst maintaining efficacy, with potential benefits in quality of life and reduced drug treatment/attendance costs.REFINE is a randomised phase II, multi-arm, multi-stage (MAMS) adaptive basket trial investigating extended interval administration of ICIs. Eligible patients are those responding to conventionally dosed ICI at 12 weeks. In stage I, patients (n = 160 per tumour-specific cohort) will be randomly allocated (1:1) to receive maintenance ICI at SOC vs extended dose interval. REFINE is currently recruiting UK patients with locally advanced or metastatic renal cell carcinoma (RCC) who have tolerated and responded to initial nivolumab/ipilimumab, randomised to receive maintenance nivolumab SOC (480 mg 4 weekly) vs extended interval (480 mg 8 weekly). Additional tumour cohorts are planned. Subject to satisfactory outcomes (progression-free survival) stage II will investigate up to 5 different treatment intervals. Secondary outcome measures include overall survival, quality-of-life, treatment-related toxicity, mean incremental pathway costs and quality-adjusted life-years per patient. REFINE is funded by the Jon Moulton Charity Trust and Medical Research Council, sponsored by University College London (UCL), and coordinated by the MRC CTU at UCL. Trial Registration ISRCTN79455488. NCT04913025 EUDRACT #: 2021-002060-47. CTA 31330/0008/001-0001; MREC approval: 21/LO/0593. REFINE Protocol version 4.0.

Objective To determine the optimal duration of human chorionic gonadotrophin (hCG) surveillance following treatment for low and high risk gestational trophoblastic neoplasia (GTN) and establish whether the current surveillance protocol that recommends life-long hCG monitoring requires revision. Methods A population-based cohort study was undertaken using a national registry, comprising patients from both tertiary trophoblastic disease treatment units in the UK (London and Sheffield). All patients who received chemotherapy for low or high risk GTN in the UK between 1958 and 2014 in London and 1973 and 2015 in Sheffield (n = 4201) were included in the study. Patients with placental site trophoblastic tumours and epithelioid trophoblastic tumours were excluded due to their distinct clinical behavior, treatment and follow-up requirements. The risk of recurrence with time following completion of chemotherapy for low or high risk GTN was measured. Results The overall risk of relapse in this national cohort of 4201 patients was 4.7% (198/4201) with a median time to recurrence of 117.5 days (range 9 days to 6.54 years). The greatest risk of recurrence occurred in the first year after completing treatment for either low or high risk GTN measuring 72.7% (n = 112) or 86.4% (n = 38), respectively. The subsequent recurrence risk reduced over time with none observed beyond 7 years. Conclusions The absence of any recurrences beyond seven years following completion of chemotherapy for GTN indicates that the UK policy of life-long hCG surveillance is unnecessary. Our revised conservative protocol recommends stopping after 10 years.

Considerable differences exist in the management of gestational trophoblastic disease (GTD) both nationally and internationally despite numerous efforts to harmonise patient care. This partly reflects differences in healthcare systems and availability of resources. However, even in first world equivalent economies with similar healthcare systems differences remain, which appear to impact survival. Recently, new international guidelines have been established in Europe through a series of consensus meetings. Improvement of outcomes will depend on the establishment of dedicated centres with appropriate patient pathways according to these guidelines. This review will highlight some of the differences and efforts to unify the management of GTD across the globe and discuss areas for future development.

Highlights • Placental site trophoblastic tumor can be resistant to chemotherapy. • Multidisciplinary care is required for management of advanced disease. • Increased PD-L1 expression can help guide use of immunotherapies. • Complete responses are possible with aggressive multidisciplinary management., Placental Site Trophoblastic Tumor (PSTT) is a rare malignancy that often presents with extensive disease and can be resistant to traditional treatments. We present the case of a woman with stage IV PSTT who was initially managed with neoadjuvant chemotherapy followed by tumor debulking. Adjuvant therapy was guided by further pathologic analysis that revealed high levels of staining for PD-L1 as well as the presence of tumor infiltrating lymphocytes (TILs). Subsequently, the patient was treated with traditional chemotherapy with the EP/EMA regimen with the addition of pembrolizumab. The patient’s treatment course was complicated by the development of pulmonary arteriovenous malformations, autoimmune thyroiditis thought to be secondary to immunotherapy, and significant tinnitus secondary to platinum agents. Currently the patient is in follow up and remains in a complete remission.

SummaryAntigen recognition and T-cell mediated cytotoxicity in clear-cell renal cell carcinoma (ccRCC) remains incompletely understood. To address this knowledge gap, we analysed 115 multiregion tumour samples collected from 15 treatment-naïve patients pre- and post-nivolumab therapy, and at autopsy in three patients. We performed whole-exome sequencing, RNAseq, TCRseq, multiplex immunofluorescence and flow cytometry analyses and correlated with clinical response. We observed pre-treatment intratumoural TCR clonal expansions suggesting pre-existing immunity. Nivolumab maintained pre-treatment expanded, clustered TCR clones in responders, suggesting ongoing antigen-driven stimulation of T-cells. T-cells in responders were enriched for expanded TCF7+CD8+T-cells and upregulated GZMK/B upon nivolumab-binding. By contrast, nivolumab promoted accumulation of new TCR clones in non-responders, replacing pre-treatment expanded clonotypes. In this dataset, mutational features did not correlate with response to nivolumab and human endogenous retrovirus expression correlated indirectly. Our data suggests that nivolumab potentiates clinical responses in ccRCC by binding pre-existing expanded CD8+T-cells to enhance cytotoxicity.

Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.

Antigen recognition and T-cell mediated cytotoxicity in clear-cell renal cell carcinoma (ccRCC) remains incompletely understood. To address this knowledge gap, we analysed 115 multiregion tumour samples collected from 15 treatment-naive patients pre- and post-nivolumab therapy, and at autopsy in three patients. We performed whole-exome sequencing, RNAseq, TCRseq, multiplex immunofluorescence and flow cytometry analyses and correlated with clinical response. We observed pre-treatment intratumoural TCR clonal expansions suggesting pre-existing immunity. Nivolumab maintained pre-treatment expanded, clustered TCR clones in responders, suggesting ongoing antigen-driven stimulation of T-cells. T-cells in responders were enriched for expanded TCF7+CD8+ T-cells and upregulated GZMK/B upon nivolumab-binding. By contrast, nivolumab promoted accumulation of new TCR clones in non-responders, replacing pre-treatment expanded clonotypes. In this dataset, mutational features did not correlate with response to nivolumab and human endogenous retrovirus expression correlated indirectly. Our data suggests that nivolumab potentiates clinical responses in ccRCC by binding pre-existing expanded CD8+ T-cells to enhance cytotoxicity.

ADAPTeR is a prospective, phase II study of nivolumab (anti-PD-1) in 15 treatment-naive patients (115 multiregion tumor samples) with metastatic clear cell renal cell carcinoma (ccRCC) aiming to understand the mechanism underpinning therapeutic response. Genomic analyses show no correlation between tumor molecular features and response, whereas ccRCC-specific human endogenous retrovirus expression indirectly correlates with clinical response. T cell receptor (TCR) analysis reveals a significantly higher number of expanded TCR clones pre-treatment in responders suggesting pre-existing immunity. Maintenance of highly similar clusters of TCRs post-treatment predict response, suggesting ongoing antigen engagement and survival of families of T cells likely recognizing the same antigens. In responders, nivolumab-bound CD8+ T cells are expanded and express GZMK/B. Our data suggest nivolumab drives both maintenance and replacement of previously expanded T cell clones, but only maintenance correlates with response. We hypothesize that maintenance and boosting of a pre-existing response is a key element of anti-PD-1 mode of action.

Background Regulatory T cell (Treg) depletion with antibodies against CD25 is effective in tumor models but response rates are low in poorly infiltrated B16 melanomas. Combination with a tumor vaccine enhances efficacy, but relapse usually occurs following partial control, similar to what is seen clinically. How resistance develops is unknown. Materials and Methods C57BL/6 mice were injected subcutaneously with B16 cells. Treatments included a depleting mouse IgG2a αCD25 antibody and/or a genetically modified, granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting whole B16 tumor vaccine (Gvax). Changes in the immune landscape were assessed with high dimensional flow cytometry. Results Compared to monotherapies, combined Gvax/αCD25 significantly delayed tumour growth and prolonged survival, in association with enhanced infiltration of T cells with an activated phenotype. Approximately 50% of mice achieved partial response with relapse at day 35–45 post tumor injection. To characterize immune evolution prior to relapse, we analysed stable, partially responding tumors and paired draining lymph nodes (DLNs). Over time, activated PD-1+ICOS+TCF7- T cells with an effector memory (CD44+CD62L-) phenotype fell from 30% to 10% whilst resting, TCF7+ early differentiated cells rose in abundance towards levels seen in untreated tumors. Abundance of Ki67-, resting Tregs also recovered. Similar results were obtained in analysis of DLNs. Conclusions Combined Treg depletion/whole tumor vaccination therapy is effective in a poorly infiltrated B16 melanoma model. Combined treatment promotes T cell infiltration and activation. In mice achieving a partial response, treatment effects on the immune landscape were observed to decay over time suggesting a return to immune equilibrium. Further studies to explore the mechanistic basis of this observation are underway. Disclosure Information C. Qing: None. E. Ghorani: None. I. Solomon: None. F. Galvez-Cancino: None. F. Vargas: None. K. Peggs: None. S. Quezada: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; F. HOFFMANN-LA ROCHE LTD.

Background Effective anti-tumour immunity requires cancer antigen expression, but persistent antigen exposure in chronic viral infections and autoimmunity has a detrimental effect on immune function. This is associated with a decline of early differentiated T cell populations in favour of later differentiated, dysfunctional subsets, resulting in an unfavourable skewing of the immune landscape. It is unknown whether this occurs locally within the antigen rich tumour microenvironment, driving immune failure. Materials and Methods We combined tumour infiltrating lymphocyte (TIL) high dimensional flow cytometry, bulk exome and RNA sequencing data from multiregional samples obtained from surgically resected tumours of treatment naive patients with non-small cell lung cancer (NSCLC) amongst the first 100 recruited to the prospective, UK-wide lung TRACERx study. Clonal relationship between T cell populations was determined by T cell receptor (TCR) sequencing. We additionally analysed publically available single T cell RNA sequencing data and bulk RNA sequencing data within TCGA. Results T cell differentiation skewing (TDS) occurred amongst TILs in association with tumour mutational burden (TMB). Surprisingly, this was most evident within the CD4 compartment that had a greater abundance of central memory cells expressing the key transcription factor TCF7. Amongst CD4 cells, loss of a PD1-CCR7+ T central memory population was accompanied by gain in abundance of PD1+ populations with exhausted (CD57-ICOShiCTLA4hi) and terminally differentiated effector (CD57+Eomes+) features. TCR sequencing revealed early and dysfunctional differentiated populations to be clonally related and CDR3 clustering analysis showed greater similarity of sequences shared vs. non-shared between subsets, consistent with an antigen driven differentiation process. Similar patterns were observed within the CD8 compartment. Identification of these subsets within single T cell RNA sequencing data revealed shared and distinct functional regulators, suggesting the enhanced effector capability of early compared to dysfunctionally differentiated populations. A validated transcriptional signature of TDS generated using TRACERx samples with paired flow cytometry and RNA sequencing data reflected loss of gene expression downstream of TCF7, and predicted worse survival within TRACERx and multiple TCGA cohorts including lung adenocarcinoma (LUAD). Conclusions Our finding support a model of neoantigen driven T cell differentiation within the tumour microenvironment that drives the depletion of progenitor-like cells and gain in abundance of dysfunctional subsets, resulting in a loss of immune fitness. Our analysis of transcriptomic data elucidates potential regulatory mechanisms and therapeutic targets within the subsets identified. Disclosure Information E. Ghorani: None. J. Reading: None. J. Henry: None. M. Robert de Massy: None. R. Rosenthal: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Achilles Therapeutics. V. Turati: None. A. Furness: None. A. Ben Aissa: None. S. Kumar Saini: None. S. Ramskov: None. A. Georgiou: None. M. Vila De Mucha: None. I. Uddin: None. T. Ronel: None. R. Salgado: None. T. Lund: None. J. Herrero: None. T. Enver: None. S. Hadrup: None. A. Hackshaw: None. K. Peggs: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. N. McGranahan,: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Achilles Therapeutics. B. Chain: None. C. Swanton: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Pfizer, AstraZeneca, BMS, Roche–Ventana and Boehringer Ingelheim. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; ApoGen Biotechnologies, Epic Bioscience and GRAIL, and has stock options in and is co-founder of Achilles Therapeutics. F. Consultant/Advisory Board; Modest; Pfizer, Novartis, GlaxoSmithKline, MSD, BMS, Celgene, AstraZeneca, Illumina, Genentech, Roche–Ventana, GRAIL, Medicxi and the Sarah Cannon Research Institute. S. Quezada: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Achilles Therapeutics.

Background There remains uncertainty about the prognostic significance of residual lung lesion on imaging after completion of treatment of low- or high-risk gestational trophoblastic neoplasia (GTN). Here, we determine if such residual lung lesions are associated with an increased risk of relapse. Methods We retrospectively screened our electronic database to identify patients with low- or high-risk GTN and lung metastases between 2004 and 18. Recurrences among patients with or without residual lung lesions on imaging were compared. Chi square analysis and Kaplan-Meier survival curves were constructed. As the numbers of cases were low, we combined this data with our previously published and non-overlapping patient cohort (1995–2004). Results Of 1304 GTN patients treated at our centre between 2004 and 18, 99 had lung metastases without other distant sites. There were 40 patients (40.4%) with residual lung lesions. Whilst an increased rate of relapse was observed among patients with residual lung lesions (4/40; 10.0%) compared to without such lesions (3/59; 5.1%), this difference was not statistically significant (p = .35). By combining the data with our previous cohort, there was an increase in relapse rate of patients with residual lung lesions (5/63; 7.9%) compared to those without such lesions (4/112; 3.6%). However, this difference was also not statistically significant (p = .21). Conclusion Residual lung lesions on imaging after completion of GTN treatment are common. However, this finding did not statistically increase relapse rate. Due to low number of recurrent events, a multi-centre, larger dataset would be needed to provide more definitive evidence.

Background Cancer mutations generate novel (neo-)peptides recognised by T cells, but the determinants of recognition are not well characterised. The difference in predicted class I major histocompatibility complex (MHC-I) binding affinity between wild-type and corresponding mutant peptides (differential agretopicity index; DAI) may reflect clinically relevant cancer peptide immunogenicity. Our aim was to explore the relationship between DAI, measures of immune infiltration and patient outcomes in advanced cancer. Patients and methods Cohorts of patients with advanced non-small-cell lung cancer (NSCLC; LUAD, n = 66) and melanoma (SKCM, n = 72) were obtained from The Cancer Genome Atlas. Three additional cohorts of immunotherapy treated patients with advanced melanoma (total n = 131) and NSCLC (n = 31) were analysed. Neopeptides and their clonal status were defined using genomic data. MHC-I binding affinity was predicted for each neopeptide and DAI values summarised as the sample mean DAI. Correlations between mean DAI and markers of immune activity were evaluated using measures of lymphocyte infiltration and immune gene expression. Results In univariate and multivariate analyses, mean DAI significantly correlated with overall survival in 3/5 cohorts, with evidence of superiority over nonsynonymous mutational and neoantigen burden. In these cohorts, the effect was seen for mean DAI of clonal but not subclonal peptides. In SKCM, the association between mean DAI and survival bordered significance (P = 0.068), reaching significance in an immunotherapy-treated melanoma cohort (P = 0.003). Mean DAI but not mutational nor neoantigen burden was positively correlated with independently derived markers of immune infiltration in both SKCM (P = 0.027) and LUAD (P = 0.024). Conclusions The association between mean DAI, survival and measures of immune activity support the hypothesis that DAI is a determinant of cancer peptide immunogenicity. Investigation of DAI as a marker of immunologically relevant peptides in further datasets and future clinical studies of neoantigen based immunotherapies is warranted.

Immune dysregulation is described in multiple myeloma. While preclinical models suggest a role for altered T-cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterize marrow-infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first-line therapy.We undertook detailed characterization of T cells from bone marrow (BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression-free survival.We found that patients with multiple myeloma had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared with healthy donors. Patients with higher frequencies of Tregs had shorter PFS and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared with patients with lower frequencies of Tregs. Analysis of CD4 and CD8 effectors revealed that low CD4effector (CD4BM-infiltrating T-cell subsets, specifically Tregs and PD-1-expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies.

Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8+ tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy.

Urine-derived lymphocytes (UDLs) may serve as a non-invasive dynamic biomarker, reflective of the immune checkpoint phenotype and T-cell receptor repertoire of tumor-infiltrating lymphocytes. Patients with UDLs expressing high levels of PD-1 had worse clinical outcomes in muscle invasive bladder cancer., Despite the advances in cancer immunotherapy, only a fraction of patients with bladder cancer exhibit responses to checkpoint blockade, highlighting a need to better understand drug resistance and identify rational immunotherapy combinations. However, accessibility to the tumor prior and during therapy is a major limitation in understanding the immune tumor microenvironment (TME). Herein, we identified urine-derived lymphocytes (UDLs) as a readily accessible source of T cells in 32 patients with muscle invasive bladder cancer (MIBC). We observed that effector CD8+ and CD4+ cells and regulatory T cells within the urine accurately map the immune checkpoint landscape and T cell receptor repertoire of the TME. Finally, an increased UDL count, specifically high expression of PD-1 (PD-1hi) on CD8+ at the time of cystectomy, was associated with a shorter recurrence-free survival. UDL analysis represents a dynamic liquid biopsy that is representative of the bladder immune TME that may be used to identify actionable immuno-oncology (IO) targets with potential prognostic value in MIBC., Graphical Abstract

Tumours have developed strategies to interfere with most steps required for anti-tumour immune responses. Although many populations contribute to anti-tumour responses, tumour-infiltrating cytotoxic T cells dominate, hence, many suppressive strategies act to inhibit these. Tumour-associated T cells are frequently restricted to stromal zones rather than tumour islands, raising the possibility that the tumour microenvironment, where crosstalk between malignant and “normal” stromal cells exists, may be critical for T cell suppression. We provide evidence of direct interactions between stroma and T cells driving suppression, showing that cancer-associated fibroblasts (CAFs) sample, process and cross-present antigen, killing CD8+ T cells in an antigen-specific, antigen-dependent manner via PD-L2 and FASL. Inhibitory ligand expression is observed in CAFs from human tumours, and neutralisation of PD-L2 or FASL reactivates T cell cytotoxic capacity in vitro and in vivo. Thus, CAFs support T cell suppression within the tumour microenvironment by a mechanism dependent on immune checkpoint activation.

In states of insulopaenic hyperglycaemia, counter-regulatory hormone (CRH) excess is a driver of ketogenesis and thus diabetic ketoacidosis. We present a case of hyperglycaemic hyperosmolar state in a patient with type 1 diabetes mellitus, associated with a mixed drug overdose including agents with documented CRH suppressive effects. We postulate that the drugs taken in overdose protected against diabetic ketoacidosis, and suggest further investigation of pharmacological therapies aimed at inhibiting CRH responses which may protect against this life-threatening complication in patients with type 1 diabetes. Copyright © 2016 John Wiley & Sons.

Objectives: The combination of 5-fluorouracil (5-FU), irinotecan and oxaliplatin (FOLFIRINOX) is considered the first-line chemotherapy for fit patients with advanced pancreatic ductal adenocarcinoma (PDAC) but carries an unfavourable adverse event (AE) profile. We retrospectively evaluated the tolerability and efficacy of a modified FOLFIRINOX (mFOLFIRINOX) regimen: intravenous oxaliplatin 85 mg/m2, irinotecan 135 mg/m2, folinic acid 400 mg/m2 and 5-FU infusion 2,400 mg/m2 over 46 h, with routine subcutaneous filgrastim on a 14-day cycle. Methods: Records of 18 patients with advanced PDAC who received treatment with mFOLFIRINOX were reviewed. Imaging of measurable disease was assessed for response, and survival was measured from the date of commencing chemotherapy to disease progression and/or death. Results: Grade 3 or 4 AEs (n; %) included vomiting (5; 28), nausea (4; 22), diarrhoea (3; 17) and non-neutropaenic fever (3; 17). For patients with stage IV disease, 12/15 (80%) achieved at least stable disease as the best radiological response, with 7/15 (47%) objective responses. In this subgroup, median overall and progression-free survival were 9.3 months (95% CI 8.3-10.4) and 7.2 months (95% CI 4.7-9.6), respectively. Conclusion: Compared to full-dose FOLFIRINOX, our modified regimen resulted in lower haematological but only marginally improved non-haematological toxicity rates, with comparable efficacy outcomes. Prospective studies are required to validate these findings.

Tumours have developed strategies to interfere with most steps required for anti-tumour immune responses. Although many populations contribute to anti-tumour responses, tumour-infiltrating cytotoxic T cells dominate, hence, many suppressive strategies act to inhibit these. Tumour-associated T cells are frequently restricted to stromal zones rather than tumour islands, raising the possibility that the tumour microenvironment, where crosstalk between malignant and “normal” stromal cells exists, may be critical for T cell suppression. We provide evidence of direct interactions between stroma and T cells driving suppression, showing that cancer-associated fibroblasts (CAFs) sample, process and cross-present antigen, killing CD8+ T cells in an antigen-specific, antigen-dependent manner via PD-L2 and FASL. Inhibitory ligand expression is observed in CAFs from human tumours, and neutralisation of PD-L2 or FASL reactivates T cell cytotoxic capacity in vitro and in vivo. Thus, CAFs support T cell suppression within the tumour microenvironment by a mechanism dependent on immune checkpoint activation., Tumours employ a variety of strategies to induce immune suppression. Here the authors show that cancer-associated fibroblasts process and cross-present tumour-antigens to T-cells resulting in antigen-specific T cell death and dysfunction via upregulation of both cell death and immune checkpoint signals.

Antigens expressed by cancer cells target them for elimination by tumor-infiltrating T cells ( 1 ). But, despite T cell recognition, advanced malignancies are often fatally progressive. T cell inhibitory (checkpoint) receptors, including programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte–associated protein 4 (CTLA-4), contribute to immune suppression and dysfunction in tumors. Checkpoint inhibitors (CPIs) developed to block these pathways and derepress T cell activity have considerably improved outcomes for various cancer types. However, beyond certain rare and highly sensitive tumors ( 2 ), responses remain limited to a fraction of patients, and both primary and acquired resistance are frequently observed. Although much work has focused on defining and overcoming T cell–intrinsic inhibitory mechanisms, such as checkpoint expression, less is known about what regulates tumor cell sensitivity to T cell attack. On pages 801 and 770 of this issue, Miao et al. ( 3 ) and Pan et al. ( 4 ), respectively, find that chromatin remodeling pathways contribute to cancer cell immune resistance through control of interferon-stimulated gene (ISG) expression. This has implications for our understanding of why CPIs fail and suggests that targeting these pathways may enhance tumor immunotherapy.

2590 Background: CD4 T helper cells are key orchestrators of immunity in states of persistent antigen exposure. In chronic viral infection, loss of immune control is associated with CD4 differentiation skewing (CD4ds) resulting from decline of early progenitors and gain in abundance of exhausted and terminally differentiated subsets. Here, we set out to identify whether a similar process occurs within the tumour microenvironment, contributing to immune dysfunction. Methods: Multiregional samples of tumour and non-tumour lung tissue from patients with untreated, surgically resected non-small cell lung cancer (NSCLC) within the first 100 recruited to the prospective lung TRACERx study were analysed by high dimensional flow cytometry of tumour infiltrating lymphocytes (TILs) and paired bulk tumour exome and RNA sequencing. We additionally reanalysed publically available single T cell and bulk tumour RNA sequencing from patients with NSCLC. Results: Unsupervised clustering and dimension reduction revealed a heterogenous landscape of CD4 TILs, with evidence of CD4ds in association with tumour mutational burden. Loss of PD1-CCR7+ T central memory enriched early differentiated cells was accompanied by gain in abundance of PD1+ populations with exhausted (CD57-ICOShiCTLA4hi) and terminally differentiated effector (CD57+Eomes+) phenotypes. Further characterisation of these populations by single cell RNA sequencing revealed differential expression of key genes involved in transcriptional regulation, co-inhibition and co-stimulatory pathways. A validated gene signature of CD4ds was associated with worse outcomes in TRACERx and TCGA cohorts. Conclusions: Our findings reveal remodelling of the CD4 differentiation landscape in association with tumour genomic characteristics, underscoring how mutational burden in the context of chronic stimulation may lead to loss of immune fitness and elucidating key regulatory pathways in potentially clinically relevant CD4 subsets.

Serum anti-Müllerian hormone (AMH) is an emerging indicator of ovarian reserve which may be predictive of reproductive capacity. Although AMH levels decline with chemotherapy, little is known about the relevance of this to subsequent fertility, and we set out to evaluate this association in patients with gestational trophoblastic neoplasia (GTN).The GTN database of our national referral center was screened from 2008-2012 for patients undergoing AMH testing, and subsequent fertility outcomes were reviewed.Of 470 treated patients, 3 underwent AMH testing for evaluation of potential subfertility 4-13 months following multiagent chemotherapy, with levels rangingfrom 0.07-4.62 pmol/L. All 3 were counseled by independent fertility specialists of the low probability of subsequent conception but went on to initiate spontaneously conceived pregnancies within 2-9 months, resulting in healthy infants.Low serum AMH is not a reliable predictor of reduced short-term fertility postchemotherapy for GTN and should be interpreted with caution when counseling patients in this setting.

A 35-year-old woman presented with rapidly progressive dyspnoea (WHO functional class III) two months after normal vaginal delivery at term, with no other symptoms. On examination, she was normotensive, with a raised jugular venous pressure, right ventricular (RV) heave, a prominent pulmonary second heart sound and mild pitting leg oedema. Oxygen saturations were 90% on air with a clear chest on auscultation. There was a single enlarged supraclavicular lymph node. ECG showed sinus tachycardia with right axis deviation. Renal and liver function were normal but brain natriuretic peptide and D-dimer were raised. Chest radiograph showed clear lungs and enlarged proximal pulmonary arteries, with no filling defects or parenchymal disease evident on CT pulmonary angiogram (CTPA). Transthoracic echocardiogram revealed signs of severe pulmonary hypertension (PH) with severely impaired RV function. Right heart catheterisation confirmed precapillary PH with a mean pulmonary artery pressure of 39 mm Hg, pulmonary wedge pressure of 13 mm Hg, cardiac output of 2.1 L/min and pulmonary vascular …

Background Intraplacental choriocarcinoma (IC) is a rare form of malignant gestational trophoblastic disease (GTD). We present a review of 62 cases, including four previously unreported, and a suggested management algorithm. Patients and methods IC cases and clinical data were identified within the Charing Cross Hospital (CXH) national GTD database (1986–2014) and by systematic literature search (1949–2014). Results 62 cases were identified including eight from CXH representing 0.03% of all GTD ( n =27,101) diagnosed between 1986 and 2014. Most cases were identified in the third trimester ( n =52; 84%) among asymptomatic women ( n =31; 50%) and with macroscopically normal placenta in 29% (18/62). In 29 non-metastatic cases with available data, 4 (14%) underwent adjuvant chemotherapy and 25 (86%) surveillance only, one of whom relapsed with metastatic disease cured with multi-agent chemotherapy. In 32 patients with metastatic disease (31 at presentation and one with relapse during surveillance), all 18 treated since 1990 achieved complete remission with multi-agent chemotherapy. Among 58 cases with available data, there were 20 fetal deaths and 38 live births with 2 neonatal deaths. Of the two (5%) cases of infantile choriocarcinoma, one was cured with intensive therapy and the other died shortly after commencing single agent treatment. A further neonatal death was due to fetomaternal haemorrhage. Conclusions IC usually occurs in the third trimester and is often asymptomatic with no macroscopic placental abnormalities. Prognosis with current therapy is generally excellent, even for patients presenting with metastatic disease. Around 60% of pregnancies affected by IC result in a live birth with a low neonatal mortality.

Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL.

12009Background: The lung TRACERx study is a prospective study exploring the cancer genome evolution of NSCLC. Data analysis from the first 100 patients enrolled into the study has shown an increas...

In Xenopus, the animal cap is very sensitive to BMP antagonists, which result in neuralization. In chick, however, only cells at the border of the neural plate can be neuralized by BMP inhibition. Here we compare the two systems. BMP antagonists can induce neural plate border markers in both ventral Xenopus epidermis and non-neural chick epiblast. However, BMP antagonism can only neuralize ectodermal cells when the BMP-inhibited cells form a continuous trail connecting them to the neural plate or its border, suggesting that homeogenetic neuralizing factors can only travel between BMP-inhibited cells. Xenopus animal cap explants contain cells fated to contribute to the neural plate border and even to the anterior neural plate, explaining why they are so easily neuralized by BMP-inhibition. Furthermore, chick explants isolated from embryonic epiblast behave like Xenopus animal caps and express border markers. We propose that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction.

We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV.

Abstract 3757 Replicating viruses that selectively lyse transformed cells are attractive agents for cancer therapy. The vaccine strain of measles virus has proven oncolytic activity in various murine models of malignancy including myeloma and lymphoma. These pre-clinical reports of MV efficacy have led to advanced phase clinical trial. In the study here we investigate the anti-tumour potential of MV in 2 novel disease targets:- adult B lineage acute lymphoblastic leukaemia (ALL) and Chronic lymphocytic leukaemia (CLL) using in-vitro and in-vivo models. MV derived from the Edmonston strain genetically engineered to express GFP was used to infect primary ALL (n = 6) and chronic lymphocytic leukaemia (CLL, n = 7) cells. All CLL and ALL cells expressed the MV receptor CD46 and were efficiently infected by MV-GFP as indicated by quantitation of viral nucleocapsid mRNA by RQ-PCR and immunoblotting of viral proteins N and H. Large multinucleated syncytia, characteristic of MV- induced cytopathology, were found in all infected ALL cultures, by contrast syncitium formation was much less prominent in the infected CLL specimens. Despite this, both CLL and ALL cells were efficiently killed by MV-GFP, as characterised by viability assays and immunoblotting for PARP cleavage. To further probe the contribution of cell to cell fusion in MV induced oncolysis we used a relatively non-fusogenic strain of MV:- MV-Moraten to infect CLL and ALL specimens. As expected ALL and CLL cells infected with MV-Moraten lacked the typical features of MV induced cytopathology. Despite this cell viability was markedly reduced in both ALL and CLL cultures infected with MV-Moraten compared to uninfected controls suggesting that intracellular fusion might be dispensable for MV-induced oncolysis in our two models. To test whether MV had therapeutic efficacy in-vivo we established subcutaneous xenografts of pre-B ALL in CB17/SCID mice using the Nalm-6 cell line and administered 1 × 107 pfu of MV (n==12) or UV inactivated MV (n=12) intratumorally on 10 occasions. In vivo MV administration had striking antitumour activity resulting in complete resolution of 11/12 or regression (1/12) of established subcutaneous pre-B ALL tumours by week 4. In contrast, all UV-MV treated tumours progressed. The differences in tumour growth between the MV treated and UV-MV control groups was significantly different (p < 0.0001, Figure 1). To test for MV-induced oncolysis in a model more closely related to ALL in humans we used a disseminated pre-B ALL model established by tail vein injection of 1 × 106 Nalm-6 cells. 1 × 106 pfu of MV or UV-MV was administered by the intravenous route six times. Eleven of twelve mice receiving replication competent MV remain disease free whereas 6/7 mice receiving tail vein administered UV MV had become moribund by 67 days (Figure 2). Bone marrow examination of moribund mice revealed 52 – 99% of CD19+/CD10+ Nalm-6 cells present. Overall, our data suggest that both ALL and CLL are targets for MV-mediated lysis in vitro. The significant antineoplastic activity of MV against both subcutaneous and disseminated ALL xenografts holds great promise towards developing vaccine MV as a therapeutic tool in adult ALL. Figure 1 Regression of Nalm-6 subcutaneous xenografts in SCID mice after intratumoral injection of MV. Figure 1. Regression of Nalm-6 subcutaneous xenografts in SCID mice after intratumoral injection of MV. Figure 2 Prolonged survival of disseminated Nalm-6 SCID xenografts after intravenous injection of MV. Figure 2. Prolonged survival of disseminated Nalm-6 SCID xenografts after intravenous injection of MV. Regression of Nalm-6 subcutaneous xenografts in SCID mice after intratumoral injection of MV. Prolonged survival of disseminated Nalm-6 SCID xenografts after intravenous injection of MV. Disclosures: No relevant conflicts of interest to declare.

Abstract 2460 Poster Board II-437 Replicating viruses that selectively lyse transformed cells are promising agents for cancer therapy. Attenuated measles virus (MV) has particular tropism for B cells and is oncolytic in murine models of myeloma and lymphoma. These results have led to phase 1 studies in myeloma. Here we investigated the anti-tumour potential of MV in adult B lineage acute lymphoblastic leukaemia (ALL) and Chronic lymphocytic leukaemia (CLL). GFP expressing attenuated MV derived from the Edmonston vaccine lineage (MV-Edm) was used to infect ALL (n = 6) or chronic lymphocytic leukaemia (CLL, n = 7) primary specimens. All CLL and ALL cells expressed the MV receptor CD46. The second MV receptor SLAM was consistently expressed in all the CLL cells but only in 5/6 of the ALL cells. Both ALL and CLL cells were efficiently infected by MV-Edm as indicated by quantitation of viral nucleocapsid mRNA by RQ-PCR and immunoblotting of viral proteins N,H and F. Large multinucleated syncytia, characteristic of MV- induced cytopathology, were found in all infected ALL cultures (Figure 1), by contrast syncitium formation was less prominent in the infected CLL specimens. Despite this, both CLL and ALL cells were efficiently killed by MV-Edm, as characterised by FACS and trypan blue based assays as well as immunoblotting for PARP cleavage. Specificity of lysis for tumor cells was confrmed by infection of normal mononuclear cell controls, which showed minimal syncitium formation and death even after prolonged infection. To investigate the contribution of cell-to-cell fusion in the pathogenesis of MV-induced oncolysis we investigated a relatively non-fusogenic MV:MV-Moraten in our models. Surprisingly, both CLL and ALL cultures infected with MV-Moraten demonstrated a reduction in cell viability compared to uninfected controls despite the absence of typical features of MV cytopathology. Our data suggest that both ALL and CLL are targets for MV-mediated lysis and that cell-cell- fusion might not be an important determinant of this effect in-vitro. Ongoing in vivo studies of MV oncolysis in B-cell malignancy should further inform the therapeutic potential of vaccine MV for these neoplasms. Figure 1 MV-Edm infection results in prominent syncytia formation in primary ALL cells (A and B). Figure 1. MV-Edm infection results in prominent syncytia formation in primary ALL cells (A and B). Disclosures: No relevant conflicts of interest to declare.

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.

Summary In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation., Graphical Abstract, Highlights • Tumor-infiltrating CD4+ T cells exhibit T helper and cytotoxic features • Treg cells limit IL-2 availability vital for the acquisition of cytotoxic features • T-bet is required for IFN-γ, but not GzmB, expression in CD4+ TILs • Blimp-1 is required for GzmB expression in response to IL-2 in CD4+ TILs, Śledzińska et al. show that upon depletion of regulatory T cells, a surplus of interleukin-2 in the tumor microenvironment supports acquisition of cytotoxic activity by T helper cells orchestrated by the transcription factor Blimp-1. These polyfunctional CD4+ T cells exhibit potent anti-tumor activity in an adoptive transfer setting with therapeutic implications.