The transcription factor c-Myb regulates CD8+ T cell stemness and antitumor immunity.

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8+ T cell memory compartment. Following viral infection, CD8+ T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8+ T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8+ T cell stemness and highlight its therapeutic potential.

Type I interferon blockade with anifrolumab in patients with systemic lupus erythematosus modulates key immunopathological pathways in a gene expression and proteomic analysis of two phase 3 trials.

INTRODUCTION: Anifrolumab is a type I interferon (IFN) receptor 1 (IFNAR1) blocking antibody approved for treating patients with systemic lupus erythematosus (SLE). Here, we investigated the immunomodulatory mechanisms of anifrolumab using longitudinal transcriptomic and proteomic analyses of the 52-week, randomised, phase 3 TULIP-1 and TULIP-2 trials. METHODS: Patients with moderate to severe SLE were enrolled in TULIP-1 and TULIP-2 and received intravenous anifrolumab or placebo alongside standard therapy. Whole-blood expression of 18 017 genes using genome-wide RNA sequencing (RNA-seq) (pooled TULIP; anifrolumab, n=244; placebo, n=258) and 184 plasma proteins using Olink and Simoa panels (TULIP-1; anifrolumab, n=124; placebo, n=132) were analysed. We compared treatment groups via gene set enrichment analysis using MetaBase pathway analysis, blood transcriptome modules, in silico deconvolution of RNA-seq and longitudinal linear mixed effect models for gene counts and protein levels. RESULTS: Compared with placebo, anifrolumab modulated >2000 genes by week 24, with overlapping results at week 52, and 41 proteins by week 52. IFNAR1 blockade with anifrolumab downregulated multiple type I and II IFN-induced gene modules/pathways and type III IFN-λ protein levels, and impacted apoptosis-associated and neutrophil extracellular traps-(NET)osis-associated transcriptional pathways, innate cell activating chemokines and receptors, proinflammatory cytokines and B-cell activating cytokines. In silico deconvolution of RNA-seq data indicated an increase from baseline of mucosal-associated invariant and γδT cells and a decrease of monocytes following anifrolumab treatment. DISCUSSION: Type I IFN blockade with anifrolumab modulated multiple inflammatory pathways downstream of type I IFN signalling, including apoptotic, innate and adaptive mechanisms that play key roles in SLE immunopathogenesis.

Trial of Anifrolumab in Active Systemic Lupus Erythematosus.

BACKGROUND: Anifrolumab, a human monoclonal antibody to type I interferon receptor subunit 1 investigated for the treatment of systemic lupus erythematosus (SLE), did not have a significant effect on the primary end point in a previous phase 3 trial. The current phase 3 trial used a secondary end point from that trial as the primary end point. METHODS: We randomly assigned patients in a 1:1 ratio to receive intravenous anifrolumab (300 mg) or placebo every 4 weeks for 48 weeks. The primary end point of this trial was a response at week 52 defined with the use of the British Isles Lupus Assessment Group (BILAG)-based Composite Lupus Assessment (BICLA). A BICLA response requires reduction in any moderate-to-severe baseline disease activity and no worsening in any of nine organ systems in the BILAG index, no worsening on the Systemic Lupus Erythematosus Disease Activity Index, no increase of 0.3 points or more in the score on the Physician Global Assessment of disease activity (on a scale from 0 [no disease activity] to 3 [severe disease]), no discontinuation of the trial intervention, and no use of medications restricted by the protocol. Secondary end points included a BICLA response in patients with a high interferon gene signature at baseline; reductions in the glucocorticoid dose, in the severity of skin disease, and in counts of swollen and tender joints; and the annualized flare rate. RESULTS: A total of 362 patients received the randomized intervention: 180 received anifrolumab and 182 received placebo. The percentage of patients who had a BICLA response was 47.8% in the anifrolumab group and 31.5% in the placebo group (difference, 16.3 percentage points; 95% confidence interval, 6.3 to 26.3; P = 0.001). Among patients with a high interferon gene signature, the percentage with a response was 48.0% in the anifrolumab group and 30.7% in the placebo group; among patients with a low interferon gene signature, the percentage was 46.7% and 35.5%, respectively. Secondary end points with respect to the glucocorticoid dose and the severity of skin disease, but not counts of swollen and tender joints and the annualized flare rate, also showed a significant benefit with anifrolumab. Herpes zoster and bronchitis occurred in 7.2% and 12.2% of the patients, respectively, who received anifrolumab. There was one death from pneumonia in the anifrolumab group. CONCLUSIONS: Monthly administration of anifrolumab resulted in a higher percentage of patients with a response (as defined by a composite end point) at week 52 than did placebo, in contrast to the findings of a similar phase 3 trial involving patients with SLE that had a different primary end point. The frequency of herpes zoster was higher with anifrolumab than with placebo. (Funded by AstraZeneca; ClinicalTrials.gov number, NCT02446899.).

The Pathogenesis, Molecular Mechanisms, and Therapeutic Potential of the Interferon Pathway in Systemic Lupus Erythematosus and Other Autoimmune Diseases.

Therapeutic success in treating patients with systemic lupus erythematosus (SLE) is limited by the multivariate disease etiology, multi-organ presentation, systemic involvement, and complex immunopathogenesis. Agents targeting B-cell differentiation and survival are not efficacious for all patients, indicating a need to target other inflammatory mediators. One such target is the type I interferon pathway. Type I interferons upregulate interferon gene signatures and mediate critical antiviral responses. Dysregulated type I interferon signaling is detectable in many patients with SLE and other autoimmune diseases, and the extent of this dysregulation is associated with disease severity, making type I interferons therapeutically tangible targets. The recent approval of the type I interferon-blocking antibody, anifrolumab, by the US Food and Drug Administration for the treatment of patients with SLE demonstrates the value of targeting this pathway. Nevertheless, the interferon pathway has pleiotropic biology, with multiple cellular targets and signaling components that are incompletely understood. Deconvoluting the complexity of the type I interferon pathway and its intersection with lupus disease pathology will be valuable for further development of targeted SLE therapeutics. This review summarizes the immune mediators of the interferon pathway, its association with disease pathogenesis, and therapeutic modalities targeting the dysregulated interferon pathway.

Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

Generation of a functional humanized Delta-like ligand 4 transgenic mouse model.

Humanized mouse models are important tools in many areas of biological drug development including, within oncology research, the development of antagonistic antibodies that have the potential to block tumor growth by controlling vascularization and are key to the generation of in vivo proof-of-concept efficacy data. However, due to cross reactivity between human antibodies and mouse target such studies regularly require mouse models expressing only the human version of the target molecule. Such humanized knock-in/knock-out, KIKO, models are dependent upon the generation of homozygous mice expressing only the human molecule, compensating for loss of the mouse form. However, KIKO strategies can fail to generate homozygous mice, even though the human form is expressed and the endogenous mouse locus is correctly targeted. A typical strategy for generating KIKO mice is by ATG fusion where the human cDNA is inserted downstream of the endogenous mouse promoter elements. However, when adopting this strategy it is possible that the mouse promoter fails to express the human form in a manner compensating for loss of the mouse form or alternatively the human protein is incompatible in the context of the mouse pathway being investigated. So to understand more around the biology of KIKO models, and to overcome our failure with a number of ATG fusion strategies, we developed a range of humanized models focused on Delta-like 4 (Dll4), a target where we initially failed to generate a humanized model. By adopting a broader biologic strategy, we successfully generated a humanized DLL4 KIKO which led to a greater understanding of critical biological aspects for consideration when developing humanized models.

Synergistic actions of blocking angiopoietin-2 and tumor necrosis factor-α in suppressing remodeling of blood vessels and lymphatics in airway inflammation.

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.

HERC5 is a prognostic biomarker for post-liver transplant recurrent human hepatocellular carcinoma.

BACKGROUND AND AIMS: Orthotopic liver transplantation (OLT) can be an effective treatment option for certain patients with early stage hepatocellular carcinoma (HCC) meeting Milan, UCSF, or Hangzhou criteria. However, HCC recurrence rates post-OLT range from 20 to 40 %, with limited follow-up options. Elucidating genetic drivers common to primary and post-OLT recurrent tumors may further our understanding and help identify predictive biomarkers of recurrence-both to ultimately help manage clinical decisions for patients undergoing OLT. METHODS: Whole exome and RNA sequencing in matched primary and recurrent tumors, normal adjacent tissues, and blood from four Chinese HCC patients was conducted. SiRNA knockdown and both qRT-PCR and Western assays were performed on PLCPRF5, SNU449 and HEPG2 cell lines; immunohistochemistry and RNA Sequencing were conducted on the primary tumors of Chinese HCC patients who experienced tumor recurrence post-OLT (n = 9) or did not experience tumor recurrence (n = 12). RESULTS: In three independent HCC studies of patients undergoing transplantation (n = 21) or surgical resection (n = 242, n = 44) of primary tumors (total n = 307), HERC5 mRNA under-expression correlated with shorter: time to tumor recurrence (p = 0.007 and 0.02) and overall survival (p = 0.0063 and 0.023), even after adjustment for relevant clinical variables. HERC5 loss drives CCL20 mRNA and protein over-expression and associates with regulatory T cell infiltration as measured by FOXP3 expression. Further, matched primary and recurrent tumors from the 4 HCC patients indicated clonal selection advantage of Wnt signaling activation and CDKN2A inactivation. CONCLUSIONS: HERC5 plays a crucial role in HCC immune evasion and has clinical relevance as a reproducible prognostic marker for risk of tumor recurrence and survival in patients.

A phase 1b clinical trial evaluating sifalimumab, an anti-IFN-α monoclonal antibody, shows target neutralisation of a type I IFN signature in blood of dermatomyositis and polymyositis patients.

OBJECTIVE: To assess the pharmacodynamic effects of sifalimumab, an investigational anti-IFN-α monoclonal antibody, in the blood and muscle of adult dermatomyositis and polymyositis patients by measuring neutralisation of a type I IFN gene signature (IFNGS) following drug exposure. METHODS: A phase 1b randomised, double-blinded, placebo controlled, dose-escalation, multicentre clinical trial was conducted to evaluate sifalimumab in dermatomyositis or polymyositis patients. Blood and muscle biopsies were procured before and after sifalimumab administration. Selected proteins were measured in patient serum with a multiplex assay, in the muscle using immunohistochemistry, and transcripts were profiled with microarray and quantitative reverse transcriptase PCR assays. A 13-gene IFNGS was used to measure the pharmacological effect of sifalimumab. RESULTS: The IFNGS was suppressed by a median of 53-66% across three time points (days 28, 56 and 98) in blood (p=0.019) and 47% at day 98 in muscle specimens post-sifalimumab administration. Both IFN-inducible transcripts and proteins were prevalently suppressed following sifalimumab administration. Patients with 15% or greater improvement from baseline manual muscle testing scores showed greater neutralisation of the IFNGS than patients with less than 15% improvement in both blood and muscle. Pathway/functional analysis of transcripts suppressed by sifalimumab showed that leucocyte infiltration, antigen presentation and immunoglobulin categories were most suppressed by sifalimumab and highly correlated with IFNGS neutralisation in muscle. CONCLUSIONS: Sifalimumab suppressed the IFNGS in blood and muscle tissue in myositis patients, consistent with this molecule's mechanism of action with a positive correlative trend between target neutralisation and clinical improvement. These observations will require confirmation in a larger trial powered to evaluate efficacy.

Increased IR-A/IR-B ratio in non-small cell lung cancers associates with lower epithelial-mesenchymal transition signature and longer survival in squamous cell lung carcinoma.

BACKGROUND: To evaluate the insulin receptor isoform mRNA expression status in non-small cell lung cancer (NSCLC) patients. METHODS: RNA-seq data from 614 NSCLC [355 adenocarcinomas (LUAD) and 259 squamous cell carcinomas (LUSC)] and 92 normal lung specimens were obtained from The Cancer Genome Atlas (TCGA) to evaluate the mRNA expression of insulin receptor isoform A (IR-A) and insulin receptor isoform B (IR-B). The differential expression status of the insulin receptor isoforms in NSCLC patients was confirmed using qRT-PCR assays with lung cancer cDNA arrays and primary tumor samples. RESULTS: The mRNA expression levels of IR-B were significantly lower in some NSCLC samples compared to normal lung specimens, including both LUAD and LUSC. Notably, no IR-B transcripts were detected - only the IR-A isoform was expressed in 11% of NSCLC patients. This decrease in IR-B expression contributed to an elevated IR-A/IR-B ratio, which was also associated with lower epithelial-mesenchymal transition gene signatures in NSCLC and longer patient survival under standard of care in LUSC. In addition to NSCLC, RNA-seq data from TCGA revealed a similar increase in IR-A/IR-B ratio in many other cancer types, with high prevalence in acute myeloid leukemia, glioblastoma multiforme, and brain lower grade glioma. CONCLUSIONS: Our results indicate a common reduction of the mRNA expression level of IR-B and an increased IR-A/IR-B mRNA ratio in NSCLC and other tumor types. The relationship of altered IR-A/IR-B ratios with cancer progression and patient survival should be prospectively explored in future studies.

Opposing roles of membrane and soluble forms of the receptor for advanced glycation end products in primary respiratory syncytial virus infection.

Respiratory syncytial virus (RSV), a common respiratory pathogen in infants and the older population, causes pulmonary inflammation and airway occlusion that leads to impairment of lung function. Here, we have established a role for receptor for advanced glycation end products (RAGE) in RSV infection. RAGE-deficient (ager(-/-)) mice were protected from RSV-induced weight loss and inflammation. This protection correlated with an early increase in type I interferons, later decreases in proinflammatory cytokines, and a reduction in viral load. To assess the contribution of soluble RAGE (sRAGE) to RSV-induced disease, wild-type and ager(-/-) mice were given doses of sRAGE following RSV infection. Of interest, sRAGE treatment prevented RSV-induced weight loss and neutrophilic inflammation to a degree similar to that observed in ager(-/-) mice. Our work further elucidates the roles of RAGE in the pathogenesis of respiratory infections and highlights the opposing roles of membrane and sRAGE in modulating the host response to RSV infection.

Combining pharmacometric models with predictive and prognostic biomarkers for precision therapy in Crohn's disease: A case study of brazikumab.

Pharmacometric models were used to investigate the utility of biomarkers in predicting the efficacy (Crohn's Disease Activity Index [CDAI]) of brazikumab and provide a data-driven framework for precision therapy for Crohn's disease (CD). In a phase IIa trial in patients with moderate to severe CD, treatment with brazikumab, an anti-interleukin 23 monoclonal antibody, was associated with clinical improvement. Brazikumab treatment effect was determined to be dependent on the baseline IL-22 (BIL22) or baseline C-reactive protein (BCRP; predictive biomarkers), and placebo effect was found to be correlated with the baseline CDAI (a prognostic biomarker). A maximal total inhibition on CDAI input function of 50.6% and 42.4% was predicted for patients with extremely high BIL22 or BCRP, compared to a maximal total inhibition of 20.9% and 17.8% for patients with extremely low BIL22 or BCRP, respectively, which were mainly due to the placebo effect. We demonstrated that model-derived baseline biomarker levels that achieve 50% of maximum unbound systemic concentration of 22.8 pg/mL and 8.03 mg/L for BIL22 and BCRP as the cutoffs to select subpopulations can effectively identify high-response subgroup patients with improved separation of responders when compared to using the median values as the cutoff. This work exemplifies the utility of pharmacometrics to quantify biomarker-driven responses in biologic therapies and distinguish between predictive and prognostic biomarkers, complementing clinical efforts of identifying subpopulations with higher likelihood of response to brazikumab.

Interferon gamma expression and mortality in unselected cohorts of urothelial bladder cancer patients.

BACKGROUND: The role of interferon gamma (IFN-γ) expression in long-term survival has not been studied in patients with urinary bladder cancer (UBC). IFN-γ expression was characterized among various UBC patient cohorts to assess if IFN-γ status is associated with overall survival (OS). METHODS: A tumor-based IFN-γ gene signature was evaluated among adult UBC patients newly diagnosed between 2004 and 2017 from two hospital systems in New York. Patient cohorts included metastatic (stage IV or progressing to stage IV [MBC]), muscle-invasive (stages T2a to T4a [MIBC]), and non-muscle-invasive (carcinoma in situ or stages 0a, 0is, and I [NMIBC]) disease. Descriptive analyses were conducted comparing IFN-γ signature in the highest tertile to those in the lowest two tertiles. RESULTS: 234 patients with bladder cancer were evaluated (56 MBC, 38 MIBC, and 140 NMIBC). Median OS was only reached in the MIBC cohort for those with an IFN-γ signature in the lowest two tertiles (15.03 months [95% CI, 8.50-50.60]). Those with an IFN-γ signature in the highest tertile had a decreased risk of mortality in all cohorts indicating better survival, but this was statistically significant in only the MIBC cohort (adjusted HR = 0.09 [95% CI, 0.01-0.73]). CONCLUSION: IFN-γ signature status was associated with a decreased mortality risk in all cohorts, particularly MIBC, indicating that it may be a prognostic marker of survival in patients with UBC.

Patients with systemic lupus erythematosus, myositis, rheumatoid arthritis and scleroderma share activation of a common type I interferon pathway.

OBJECTIVE: To characterise activation of the type I interferon (IFN) pathway in patients with systemic lupus erythematosus (SLE), dermatomyositis (DM), polymyositis (PM), rheumatoid arthritis (RA) and systemic scleroderma (SSc) and to evaluate the potential to develop a molecular diagnostic tool from the peripheral blood that reflects this activation in disease-affected tissues. METHODS: Overexpressed transcripts were identified in the whole blood (WB) of 262 patients with SLE, 44 with DM, 33 with PM, 28 with SSc and 89 with RA and compared with 24 healthy subjects using Affymetrix microarrays. A five gene type I IFN signature was assessed in these subjects to identify subpopulations showing both activation and concordance of the type I IFN pathway in the peripheral blood and disease-affected tissues of each disease and to correlate activation of this pathway in the WB with clinical measurements. RESULTS: A common set of 36 type I IFN inducible transcripts were identified among the most overexpressed in the WB of all subjects. Significant activation of the type I IFN pathway in subgroups of each of the five diseases studied was observed. Baseline disease activity measurements correlated with a type I IFN gene signature in the WB of subjects with SLE, PM and SSc, as did various serum autoantibody levels in subjects with SLE and DM. This signature was also well correlated between disease-affected tissue and WB in subjects with SLE, DM, PM and SSc. CONCLUSIONS: The results indicate that the type I IFN pathway is activated in patient subsets of five rheumatic diseases and suggest that these subsets may benefit from anti-IFN therapy.

Long-Term Safety and Efficacy of Anifrolumab in Adults With Systemic Lupus Erythematosus: Results of a Phase II Open-Label Extension Study.

OBJECTIVE: To investigate long-term safety and tolerability of anifrolumab, a human monoclonal antibody to the type I interferon (IFN) receptor subunit 1, in patients with moderate-to-severe systemic lupus erythematosus (SLE). METHODS: This 3-year, multinational, open-label extension study included adult patients who completed treatment (48 weeks of anifrolumab or placebo; 12-week follow-up) in the MUSE phase IIb randomized controlled trial (RCT). Patients initially received 1,000 mg of anifrolumab intravenously every 4 weeks, which was reduced to 300 mg every 4 weeks based on the benefit/risk profile established in the MUSE trial. Adverse events (AEs) were assessed monthly. Exploratory end points included the SLE Disease Activity Index 2000 (SLEDAI-2K), Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI), pharmacodynamics, and health-related quality of life (HRQoL). RESULTS: Of the 246 patients who completed the RCT, 218 (88.6%) enrolled in the open-label extension study, of which 139 (63.8%) completed 3 years of treatment. Approximately 69.7% of patients reported ≥1 AE during the first year of open-label extension treatment. Frequency and patterns of serious AEs and AEs of special interest over 3 years were consistent with those reported for 1 year of treatment in the RCT. Few patients (6.9%) discontinued treatment due to AEs. No new safety signals were identified. Improvement in the SLEDAI-2K was sustained over 3 years. SDI and Short Form 36 health survey scores remained stable. Neutralization of type I IFN gene signatures was maintained in the IFN-high population, and C3, C4, and anti-double-stranded DNA showed trends toward sustained improvement. CONCLUSION: Long-term anifrolumab treatment demonstrates an acceptable safety profile with sustained improvement in SLE disease activity, HRQoL, and serologic measures.

A Blood-based Assay for Assessment of Tumor Mutational Burden in First-line Metastatic NSCLC Treatment: Results from the MYSTIC Study.

PURPOSE: Tumor mutational burden (TMB) has been shown to be predictive of survival benefit in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors. Measuring TMB in the blood (bTMB) using circulating cell-free tumor DNA (ctDNA) offers practical advantages compared with TMB measurement in tissue (tTMB); however, there is a need for validated assays and identification of optimal cutoffs. We describe the analytic validation of a new bTMB algorithm and its clinical utility using data from the phase III MYSTIC trial. PATIENTS AND METHODS: The dataset used for the clinical validation was from MYSTIC, which evaluated first-line durvalumab (anti-PD-L1 antibody) ± tremelimumab (anticytotoxic T-lymphocyte-associated antigen-4 antibody) or chemotherapy for metastatic NSCLC. bTMB and tTMB were evaluated using the GuardantOMNI and FoundationOne CDx assays, respectively. A Cox proportional hazards model and minimal P value cross-validation approach were used to identify the optimal bTMB cutoff. RESULTS: In MYSTIC, somatic mutations could be detected in ctDNA extracted from plasma samples in a majority of patients, allowing subsequent calculation of bTMB. The success rate for obtaining valid TMB scores was higher for bTMB (809/1,001; 81%) than for tTMB (460/735; 63%). Minimal P value cross-validation analysis confirmed the selection of bTMB ≥20 mutations per megabase (mut/Mb) as the optimal cutoff for clinical benefit with durvalumab + tremelimumab. CONCLUSIONS: Our study demonstrates the feasibility, accuracy, and reproducibility of the GuardantOMNI ctDNA platform for quantifying bTMB from plasma samples. Using the new bTMB algorithm and an optimal bTMB cutoff of ≥20 mut/Mb, high bTMB was predictive of clinical benefit with durvalumab + tremelimumab versus chemotherapy.

Safety and Efficacy of Durvalumab and Tremelimumab Alone or in Combination in Patients with Advanced Gastric and Gastroesophageal Junction Adenocarcinoma.

PURPOSE: This randomized, multicenter, open-label, phase Ib/II study assessed durvalumab and tremelimumab in combination or as monotherapy for chemotherapy-refractory gastric cancer or gastroesophageal junction (GEJ) cancer. PATIENTS AND METHODS: Second-line patients were randomized 2:2:1 to receive durvalumab plus tremelimumab (arm A), or durvalumab (arm B) or tremelimumab monotherapy (arm C), and third-line patients received durvalumab plus tremelimumab (arm D). A tumor-based IFNγ gene signature was prospectively evaluated as a potential predictive biomarker in second- and third-line patients receiving the combination (arm E). The coprimary endpoints were objective response rate and progression-free survival (PFS) rate at 6 months. RESULTS: A total of 113 patients were treated: 6 in phase Ib and 107 (arm A, 27; arm B, 24; arm C, 12; arm D, 25; arm E, 19) in phase II. Overall response rates were 7.4%, 0%, 8.3%, 4.0%, and 15.8% in the five arms, respectively. PFS rates at 6 months were 6.1%, 0%, 20%, 15%, and 0%, and 12-month overall survival rates were 37.0%, 4.6%, 22.9%, 38.8%, and NA, respectively. Treatment-related grade 3/4 adverse events were reported in 17%, 4%, 42%, 16%, and 11% of patients, respectively. CONCLUSIONS: Response rates were low regardless of monotherapy or combination strategies. No new safety signals were identified. Including use of a tumor-based IFNγ signature and change in baseline and on-treatment circulating tumor DNA are clinically feasible and may be novel strategies to improve treatment response in this difficult-to-treat population.

Type I interferon inhibitor anifrolumab in active systemic lupus erythematosus (TULIP-1): a randomised, controlled, phase 3 trial.

BACKGROUND: Type I interferons are involved in systemic lupus erythematosus (SLE) pathogenesis. In a phase 2 trial, anifrolumab, a human monoclonal antibody to type I interferon receptor subunit 1, suppressed interferon gene signatures and substantially reduced SLE disease activity. Here, we sought to confirm the efficacy of anifrolumab versus placebo in a phase 3 trial of adult patients with SLE and moderate-to-severe disease activity despite standard-of-care treatment. METHODS: TULIP-1 was a double-blind, randomised, controlled, phase 3 trial done at 123 sites in 18 countries. Included patients were aged 18-70 years, with moderate-to-severe SLE, and ongoing stable treatment with either prednisone or equivalent, an antimalarial, azathioprine, mizoribine, mycophenolate mofetil or mycophenolic acid, or methotrexate. Patients were randomly assigned (2:1:2) to receive placebo, anifrolumab 150 mg, or anifrolumab 300 mg intravenously every 4 weeks for 48 weeks. Stable standard-of-care treatment continued except for mandatory attempts at oral corticosteroid tapering for patients receiving prednisone or equivalent of 10 mg/day or more at baseline. The primary outcome was the difference between the proportion of patients who achieved an SLE responder index-4 (SRI-4) response at week 52 with anifrolumab 300 mg versus with placebo. Key secondary outcomes were the difference between the anifrolumab 300 mg group and the placebo group in: proportion of patients in the interferon gene signature test-high subgroup who achieved SRI-4 at week 52; proportion of patients on 10 mg/day or more corticosteroids at baseline who achieved a sustained dose reduction to 7·5 mg/day or less from week 40 to 52; proportion of patients with a cutaneous lupus erythematosus disease area and severity index (CLASI) activity score of 10 or higher at baseline who achieved a 50% or more reduction in CLASI score by week 12; proportion of patients who achieved SRI-4 at week 24; and annualised flare rate through week 52. Other measures of disease activity were also assessed at week 52, including the British Isles Lupus Assessment Group-based composite lupus assessment (BICLA). Safety was also assessed. Efficacy and safety analyses were done in the population of patients who received at least one dose of study drug. This trial was registered at ClinicalTrials.gov (NCT02446912). FINDINGS: Between June 9, 2015, and June 16, 2017, 457 patients were randomly assigned to the anifrolumab 300 mg group (n=180), the anifrolumab 150 mg group (n=93), or the placebo group (n=184). The proportion of patients at week 52 with an SRI-4 response was similar between anifrolumab 300 mg (65 [36%] of 180) and placebo (74 [40%] of 184; difference -4·2 [95% CI -14·2 to 5·8], p=0·41). Similarly, proportions of patients with an SRI-4 response at week 24, and at week 52 in patients in the interferon gene signature test-high subgroup, did not differ between the anifrolumab and placebo groups. In patients with baseline oral corticosteroids of at least 10 mg/day, sustained dose reduction to 7·5 mg/day or less was achieved by 42 (41%) of 103 patients in the anifrolumab 300 mg group and 33 (32%) of 102 patients in the placebo group (difference 8·9 [95% CI -4·1 to 21·9]). In patients with CLASI activity score of at least 10 at baseline, at least 50% reduction by week 12 was achieved by 24 (42%) of 58 patients in the anifrolumab 300 mg group and 14 (25%) of 54 in the placebo group (difference 17·0 [95% CI -0·3 to 34·3]). Annualised flare rates were 0·60 for anifrolumab and 0·72 for placebo (rate ratio 0·83 [95% CI 0·60 to 1·14]). BICLA response was achieved by 67 (37%) of 180 patients receiving anifrolumab 300 mg versus 49 (27%) of 184 receiving placebo (difference 10·1 [95% CI 0·6 to 19·7]). Anifrolumab's safety profile was similar to that observed in phase 2, with similar proportions of patients having a serious adverse event between groups (25 [14%] of 180 for anifrolumab 300 mg, ten [11%] of 93 for anifrolumab 150 mg, and 30 [16%] of 184 for placebo). INTERPRETATION: The primary endpoint was not reached. However, several secondary endpoints, including reduction in oral corticosteroid dose, CLASI responses, and BICLA responses, suggest clinical benefit of anifrolumab compared with placebo. Conclusive evidence for the efficacy of anifrolumab awaits further phase 3 trial data. Despite the inherent limitations of a 1-year phase 3 study, such as incomplete knowledge of applicability to the general population and scarce detection of rare safety signals, in addition to complications from prespecified restricted medication rules, our results suggest that anifrolumab might have the potential to provide a treatment option for patients who have active SLE while receiving standard therapy. FUNDING: AstraZeneca.

Early Reduction in ctDNA Predicts Survival in Patients with Lung and Bladder Cancer Treated with Durvalumab.

PURPOSE: Immunotherapy has transformed the treatment of many solid tumors, with some patients deriving long-term benefit, but how to identify such patients remains unclear. Somatic mutations detected in circulating tumor DNA (ctDNA) from plasma can be an indicator of disease progression, response to therapy, and clonality of primary and metastatic lesions. Hence, ctDNA analysis can provide a valuable noninvasive and tumor-specific marker for longitudinal monitoring of tumor burden. We explored the use of ctDNA to predict survival on durvalumab, an anti-PD-L1 therapy. EXPERIMENTAL DESIGN: Variant allele frequencies (VAF) of somatic mutations in 73 genes were assessed in ctDNA using targeted sequencing in a discovery cohort consisting of 28 patients with non-small cell lung cancer (NSCLC) and two validation NSCLC and urothelial cancer (UC) cohorts of 72 and 29 patients, respectively, to correlate ctDNA changes with clinical outcomes. RESULTS: Somatic variants were detected in 96% of patients. Changes in VAF preceded radiographic responses, and patients with reduction in VAF at 6 weeks had significantly greater reduction in tumor volume, with longer progression-free and overall survival. CONCLUSIONS: ctDNA VAF changes are strongly correlated with duration of treatment, antitumor activity, and clinical outcomes in NSCLC and UC. Early on-treatment reduction in ctDNA VAF may be a useful predictor of long-term benefit from immunotherapy. Prospective studies should validate these findings and the value of utilizing early changes in ctDNA for therapeutic decision making by identifying nonresponders to checkpoint inhibitor monotherapies and guiding combination therapies.

Interferon Gamma Messenger RNA Signature in Tumor Biopsies Predicts Outcomes in Patients with Non-Small Cell Lung Carcinoma or Urothelial Cancer Treated with Durvalumab.

Purpose: To identify a predictive biomarker for durvalumab, an anti-programmed death ligand 1 (PD-L1) mAb.Experimental Design: RNA sequencing of 97 advanced-stage non-small cell lung carcinoma (NSCLC) biopsies from a nonrandomized phase Ib/II clinical trial (1108/NCT01693562) were profiled to identify a predictive signature; 62 locally advanced or metastatic urothelial cancer tumors from the same study were profiled to confirm predictive utility of the signature. Thirty NSCLC patients provided pre- and posttreatment tumors for messenger RNA (mRNA) analysis. NSCLC with ≥25% tumor cells and urothelial cancer with ≥25% tumor or immune cells stained for PD-L1 at any intensity were scored PD-L1 positive (PD-L1+). Kaplan-Meier and Cox proportional hazards analyses were used to adjust for gender, age, prior therapies, histology, ECOG status, liver metastasis, and smoking. Tumor mutation burden (TMB) was calculated using data from The Cancer Genome Atlas (TCGA).Results: In the NSCLC discovery set, a four-gene IFNγ-positive (IFNγ+) signature comprising IFNγ, CD274, LAG3, and CXCL9 was associated with higher overall response rates, longer median progression-free survival, and overall survival compared with signature-low patients. IFNγ+-signature NSCLC patients had improved survival regardless of IHC PD-L1 status. These associations were replicated in a urothelial cancer cohort. The IFNγ+ signature was induced 2-fold (P = 0.003) by durvalumab after 8 weeks of therapy in patients with NSCLC, and baseline signature was associated with TMB but not survival in TCGA data.Conclusions: The IFNγ+ mRNA signature may assist in identifying patients with improved outcomes with durvalumab, independent of PD-L1 assessed by IHC. Clin Cancer Res; 24(16); 3857-66. ©2018 AACR.