Associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 with molecular subtypes, PD-L1 expression, and FGFR3 mutational status in two advanced urothelial bladder cancer cohorts.

AIMS: Treatment options for advanced urothelial carcinoma (aUC) rapidly evolved: besides immunomodulative therapeutic options and inhibitors targeting Fibroblast growth factor receptor (FGFR) alterations, two new antibody-drug conjugates (ADC), sacituzumab govitecan (SG) and enfortumab vedotin (EV), have been approved. However, little is known about the associations of specific aUC properties and the surface target expression of TROP2 and NECTIN-4. Our aim was to characterize associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 protein and gene expression with morphomolecular and clinicopathological characteristics of aUC in two large independent cohorts. METHODS AND RESULTS: The TCGA BLCA (n = 405) and the CCC-EMN (n = 247) cohorts were retrospectively analysed. TROP2/TACSTD2 and NECTIN-4/NECTIN-4 are highly expressed at the protein and transcript level in aUC, and their expression status did not correlate with patient survival in both cohorts. NECTIN-4/NECTIN-4 expression was higher in luminal tumours and reduced in squamous aUCs. NECTIN-4 was negative in 10.6% of samples, and 18.4% of samples had low expression (H-score <15). The TROP2 negativity rate amounted to 6.5%. TACSTD2 and NECTIN-4 expression was reduced in neuroendocrine-like and/or protein-based double-negative tumours. TROP2- and NECTIN-4-negative tumours included one sarcomatoid and four neuroendocrine aUC. FGFR3 alterations and PD-L1 expression on tumour and immune cells did not associate with TROP2 or NECTIN-4 expression. CONCLUSIONS: TACSTD2/TROP2 and NECTIN-4/NECTIN-4 are widely expressed in aUC, independent of FGFR3 alterations or PD-L1 expression, thus representing a suitable target for ADC treatment in the majority of aUC. The expression loss was associated with aggressive morphomolecular aUC subtypes, i.e. neuroendocrine(-like) and sarcomatoid aUC.

RNA-seq RNAaccess identified as the preferred method for gene expression analysis of low quality FFPE samples.

Clinical tumor tissues that are preserved as formalin-fixed paraffin-embedded (FFPE) samples result in extensive cross-linking, fragmentation, and chemical modification of RNA, posing significant challenges for RNA-seq-based gene expression profiling. This study sought to define an optimal RNA-seq protocol for FFPE samples. We employed a common RNA extraction method and then compared RNA-seq library preparation protocols including RNAaccess, RiboZero and PolyA in terms of sequencing quality and concordance of gene expression using FFPE and case-matched fresh-frozen (FF) triple-negative breast cancer (TNBC) tissues. We found that RNAaccess, a method based on exome capture, produced the most concordant results. Applying RNAaccess to FFPE gastric cancer tissues, we established a minimum RNA DV200 requirement of 10% and a RNA input amount of 10ng that generated highly reproducible gene expression data. Lastly, we demonstrated that RNAaccess and NanoString platforms produced highly concordant expression profiles from FFPE samples for shared genes; however, RNA-seq may be preferred for clinical biomarker discovery work because of the broader coverage of the transcriptome. Taken together, these results support the selection of RNA-seq RNAaccess method for gene expression profiling of FFPE samples. The minimum requirements for RNA quality and input established here may allow for inclusion of clinical FFPE samples of sub-optimal quality in gene expression analyses and ultimately increasing the statistical power of such analyses.

Filgotinib Modulates Inflammation-Associated Peripheral Blood Protein Biomarkers in Adults with Active Rheumatoid Arthritis and Prior Inadequate Response to Methotrexate.

INTRODUCTION: Our aim was to evaluate protein biomarker changes related to the administration of filgotinib, a Janus kinase (JAK) 1 preferential inhibitor, in patients with moderately to severely active rheumatoid arthritis (RA) with inadequate response to methotrexate. METHODS: Plasma and serum samples were collected from patients enrolled in FINCH 1 (NCT02889796), a Phase 3 trial. Patients with stable backgrounds of methotrexate were randomly assigned once-daily oral filgotinib 200 or 100 mg, subcutaneous adalimumab 40 mg every 2 weeks (W), or placebo. Up to 35 biomarkers were analyzed at baseline, W4, and W12 with enzyme-linked immunosorbent assays and chemiluminescence and electrochemiluminescence assays. RESULTS: At baseline, four distinct biomarker clusters were identified. The strongest intragroup correlations were in bone-cartilage resorption/inflammation and JAK/signal transducer and activator of transcription (STAT) signaling activity. At baseline, significant positive correlations were identified for cytokines with patient-reported pain and with patient measures of fatigue. Filgotinib reduced levels of cytokines associated with inflammation and cell migration as early as W4 and through W12. Compared to adalimumab, filgotinib induced significant reductions in bone-related turnover biomarkers, N-telopeptide of type 1 collagen and C-telopeptide 1, as well as biomarkers associated with baseline disease activity. No baseline predictors of therapeutic response to filgotinib were identified. CONCLUSIONS: Filgotinib reduced peripheral protein biomarkers associated with JAK/STAT signaling, inflammatory signaling, immune cell migration, and bone resorption as soon as W4 in FINCH 1. Effects were dose-dependent and consistent with the clinical efficacy of filgotinib observed in FINCH 1. The changes in peripheral biomarkers associated with filgotinib treatment in methotrexate-experienced patients are consistent with changes observed in both methotrexate-naïve and biologic disease-modifying antirheumatic drug-experienced RA populations. These data demonstrate dose-dependent effects of preferential JAK1 inhibition by filgotinib on peripheral blood protein biomarkers in methotrexate-experienced patients with RA. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02889796.

Consistent Effects of Early Remdesivir on Symptoms and Disease Progression Across At-Risk Outpatient Subgroups: Treatment Effect Heterogeneity in PINETREE Study.

INTRODUCTION: In the PINETREE study, early remdesivir treatment reduced risk of coronavirus disease 2019 (COVID-19)-related hospitalizations or all-cause death versus placebo by 87% by day 28 in high-risk, non-hospitalized patients. Here we report results of assessment of heterogeneity of treatment effect (HTE) of early outpatient remdesivir, focusing on time from symptom onset and number of baseline risk factors (RFs). METHODS: PINETREE was a double-blind, placebo-controlled trial of non-hospitalized patients with COVID-19 who were randomized within 7 days of symptom onset and had ≥ 1 RF for disease progression (age ≥ 60 years, obesity [body mass index ≥ 30], or certain coexisting medical conditions). Patients received remdesivir intravenously (200 mg on day 1 and 100 mg on days 2 and 3) or placebo. RESULTS: In this subgroup analysis, HTE of remdesivir by time from symptom onset at treatment initiation and number of baseline RFs was not detected. Treatment with remdesivir reduced COVID-19-related hospitalizations independent of stratification by time from symptom onset to randomization. Of patients enrolled ≤ 5 days from symptom onset, 1/201 (0.5%) receiving remdesivir and 9/194 (4.6%) receiving placebo were hospitalized (hazard ratio [HR] 0.10; 95% confidence interval [CI] 0.01-0.82). Of those enrolled at > 5 days from symptom onset, 1/78 (1.3%) receiving remdesivir and 6/89 (6.7%) receiving placebo were hospitalized (HR 0.19; 95% CI 0.02-1.61). Remdesivir was also effective in reducing COVID-19-related hospitalizations when stratified by number of baseline RFs for severe disease. Of patients with ≤ 2 RFs, 0/159 (0.0%) receiving remdesivir and 4/164 (2.4%) receiving placebo were hospitalized; of those with ≥ 3 RFs, 2/120 (1.7%) receiving remdesivir and 11/119 (9.2%) receiving placebo were hospitalized (HR 0.16; 95% CI 0.04-0.73). CONCLUSIONS: In the outpatient setting, benefit of remdesivir initiated within 7 days of symptoms appeared to be consistent across patients with RFs. Therefore, it may be reasonable to broadly treat patients with remdesivir regardless of comorbidities. TRIAL REGISTRATION: ClinicalTrials.gov number NCT04501952.

Randomized, open-label, phase 2 study of andecaliximab plus nivolumab versus nivolumab alone in advanced gastric cancer identifies biomarkers associated with survival.

BACKGROUND: Matrix metalloproteinase-9 (MMP9) selectively cleaves extracellular matrix proteins contributing to tumor growth and an immunosuppressive microenvironment. This study evaluated andecaliximab (ADX), an inhibitor of MMP9, in combination with nivolumab (NIVO), for the treatment of advanced gastric cancer. METHODS: Phase 2, open-label, randomized multicenter study evaluating the efficacy, safety, and pharmacodynamics of ADX+NIVO versus NIVO in patients with pretreated metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and adverse events (AEs). We explored the correlation of efficacy outcomes with biomarkers. RESULTS: 144 patients were randomized; 141 were treated: 81% white, 69% male, median age was 61 years in the ADX+NIVO group and 62 years in the NIVO-alone group. The ORR was 10% (95% CI 4 to 19) in the ADX+NIVO group and 7% (95% CI 2 to 16) in the NIVO-alone group (OR: 1.5 (95% CI 0.4 to 6.1; p=0.8)). There was no response or survival benefit associated with adding ADX. AE rates were comparable in both treatment groups; the most common AEs were fatigue, decreased appetite, nausea, and vomiting. Programmed cell death ligand 1, interferon-γ (IFN), and intratumoral CD8+ cell density were not associated with treatment response or survival. The gene signature most correlated with shorter survival was the epithelial-to-mesenchymal gene signature; high transforming growth factor (TGF)-ß fibrosis score was negatively associated with OS (p=0.036). Gene expression analysis of baseline tumors comparing long-(1+ years) and short-term (<1 year) survivors showed that GRB7 was associated with survival beyond 1 year. Human epidermal growth factor receptor 2 (HER2)-positive disease was associated with significantly longer survival (p=0.0077). Median tumor mutation burden (TMB) was 2.01; patients with TMB ≥median had longer survival (p=0.0025) and improved PFS (p=0.016). Based on a model accounting for TMB, TGF-ß fibrosis, and HER2, TMB was the main driver of survival in this patient population. CONCLUSION: Combination of ADX+NIVO had a favorable safety profile but did not improve efficacy compared with NIVO alone in patients with pretreated metastatic gastric or GEJ adenocarcinoma. HER2 positivity, higher TMB or GRB7, and lower TGF-ß were associated with improved outcomes. TRIAL REGISTRATION NUMBER: NCT02864381 or GS-US-296--2013.

Compassionate Use of Remdesivir for Patients with Severe Covid-19.

BACKGROUND: Remdesivir, a nucleotide analogue prodrug that inhibits viral RNA polymerases, has shown in vitro activity against SARS-CoV-2. METHODS: We provided remdesivir on a compassionate-use basis to patients hospitalized with Covid-19, the illness caused by infection with SARS-CoV-2. Patients were those with confirmed SARS-CoV-2 infection who had an oxygen saturation of 94% or less while they were breathing ambient air or who were receiving oxygen support. Patients received a 10-day course of remdesivir, consisting of 200 mg administered intravenously on day 1, followed by 100 mg daily for the remaining 9 days of treatment. This report is based on data from patients who received remdesivir during the period from January 25, 2020, through March 7, 2020, and have clinical data for at least 1 subsequent day. RESULTS: Of the 61 patients who received at least one dose of remdesivir, data from 8 could not be analyzed (including 7 patients with no post-treatment data and 1 with a dosing error). Of the 53 patients whose data were analyzed, 22 were in the United States, 22 in Europe or Canada, and 9 in Japan. At baseline, 30 patients (57%) were receiving mechanical ventilation and 4 (8%) were receiving extracorporeal membrane oxygenation. During a median follow-up of 18 days, 36 patients (68%) had an improvement in oxygen-support class, including 17 of 30 patients (57%) receiving mechanical ventilation who were extubated. A total of 25 patients (47%) were discharged, and 7 patients (13%) died; mortality was 18% (6 of 34) among patients receiving invasive ventilation and 5% (1 of 19) among those not receiving invasive ventilation. CONCLUSIONS: In this cohort of patients hospitalized for severe Covid-19 who were treated with compassionate-use remdesivir, clinical improvement was observed in 36 of 53 patients (68%). Measurement of efficacy will require ongoing randomized, placebo-controlled trials of remdesivir therapy. (Funded by Gilead Sciences.).

Hypomethylation within gene promoter regions and type 1 diabetes in discordant monozygotic twins.

Genetic susceptibility to type 1 diabetes (T1D) is well supported by epidemiologic evidence; however, disease risk cannot be entirely explained by established genetic variants identified so far. This study addresses the question of whether epigenetic modification of the inherited DNA sequence may contribute to T1D susceptibility. Using the Infinium HumanMethylation450 BeadChip array (450k), a total of seven long-term disease-discordant monozygotic (MZ) twin pairs and five pairs of HLA-identical, disease-discordant non-twin siblings (NTS) were examined for associations between DNA methylation (DNAm) and T1D. Strong evidence for global hypomethylation of CpG sites within promoter regions in MZ twins with TID compared to twins without T1D was observed. DNA methylation data were then grouped into three categories of CpG sites for further analysis, including those within: 1) the major histocompatibility complex (MHC) region, 2) non-MHC genes with reported T1D association through genome wide association studies (GWAS), and 3) the epigenome, or remainder of sites that did not include MHC and T1D associated genes. Initial results showed modest methylation differences between discordant MZ twins for the MHC region and T1D-associated CpG sites, BACH2, INS-IGF2, and CLEC16A (DNAm difference range: 2.2%-5.0%). In the epigenome CpG set, the greatest methylation differences were observed in MAGI2, FANCC, and PCDHB16, (DNAm difference range: 6.9%-16.1%). These findings were not observed in the HLA-identical NTS pairs. Targeted pyrosequencing of five candidate CpG loci identified using the 450k array in the original discordant MZ twins produced similar results using control DNA samples, indicating strong agreement between the two DNA methylation profiling platforms. However, findings for the top five candidate CpG loci were not replicated in six additional T1D-discordant MZ twin pairs. Our results indicate global DNA hypomethylation within gene promoter regions may contribute to T1D; however, findings do not support the involvement of large DNAm differences at single CpG sites alone in T1D.

Genome-Wide Assessment of Differential DNA Methylation Associated with Autoantibody Production in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies associated with specific clinical manifestations. Previous studies have shown an association between differential DNA methylation and SLE susceptibility, but have not investigated SLE-related autoantibodies. Our goal was to determine whether DNA methylation is associated with production of clinically relevant SLE-related autoantibodies, with an emphasis on the anti-dsDNA autoantibody. In this study, we characterized the methylation status of 467,314 CpG sites in 326 women with SLE. Using a discovery and replication study design, we identified and replicated significant associations between anti-dsDNA autoantibody production and the methylation status of 16 CpG sites (pdiscovery<1.07E-07 and preplication<0.0029) in 11 genes. Associations were further investigated using multivariable regression to adjust for estimated leukocyte cell proportions and population substructure. The adjusted mean DNA methylation difference between anti-dsDNA positive and negative cases ranged from 1.2% to 19%, and the adjusted odds ratio for anti-dsDNA autoantibody production comparing the lowest and highest methylation tertiles ranged from 6.8 to 18.2. Differential methylation for these CpG sites was also associated with anti-SSA, anti-Sm, and anti-RNP autoantibody production. Overall, associated CpG sites were hypomethylated in autoantibody positive compared to autoantibody negative cases. Differential methylation of CpG sites within the major histocompatibility region was not strongly associated with autoantibody production. Genes with differentially methylated CpG sites represent multiple biologic pathways, and have not been associated with autoantibody production in genetic association studies. In conclusion, hypomethylation of CpG sites within genes from different pathways is associated with anti-dsDNA, anti-SSA, anti-Sm, and anti-RNP production in SLE, and these associations are not explained by genetic variation. Thus, studies of epigenetic mechanisms such as DNA methylation represent a complementary method to genetic association studies to identify biologic pathways that may contribute to the clinical heterogeneity of autoimmune diseases.

Genome-wide DNA methylation profiles indicate CD8+ T cell hypermethylation in multiple sclerosis.

OBJECTIVE: Determine whether MS-specific DNA methylation profiles can be identified in whole blood or purified immune cells from untreated MS patients. METHODS: Whole blood, CD4+ and CD8+ T cell DNA from 16 female, treatment naïve MS patients and 14 matched controls was profiled using the HumanMethylation450K BeadChip. Genotype data were used to assess genetic homogeneity of our sample and to exclude potential SNP-induced DNA methylation measurement errors. RESULTS: As expected, significant differences between CD4+ T cells, CD8+ T cells and whole blood DNA methylation profiles were observed, regardless of disease status. Strong evidence for hypermethylation of CD8+ T cell, but not CD4+ T cell or whole blood DNA in MS patients compared to controls was observed. Genome-wide significant individual CpG-site DNA methylation differences were not identified. Furthermore, significant differences in gene DNA methylation of 148 established MS-associated risk genes were not observed. CONCLUSION: While genome-wide significant DNA methylation differences were not detected for individual CpG-sites, strong evidence for DNA hypermethylation of CD8+ T cells for MS patients was observed, indicating a role for DNA methylation in MS. Further, our results suggest that large DNA methylation differences for CpG-sites tested here do not contribute to MS susceptibility. In particular, large DNA methylation differences for CpG-sites within 148 established MS candidate genes tested in our study cannot explain missing heritability. Larger studies of homogenous MS patients and matched controls are warranted to further elucidate the impact of CD8+ T cell and more subtle DNA methylation changes in MS development and pathogenesis.

Considerations for normalization of DNA methylation data by Illumina 450K BeadChip assay in population studies.

Analysis of epigenetic mechanisms, particularly DNA methylation, is of increasing interest for epidemiologic studies examining disease etiology and impacts of environmental exposures. The Infinium HumanMethylation450 BeadChip(®) (450K), which interrogates over 480,000 CpG sites and is relatively cost effective, has become a popular tool to characterize the DNA methylome. For large-scale studies, minimizing technical variability and potential bias is paramount. The goal of this paper was to evaluate the performance of several existing and novel color channel normalizations designed to reduce technical variability and batch effects in 450K analysis from a large population study. Comparative assessment of 10 normalization procedures included the GenomeStudio(®) Illumina procedure, the lumi smooth quantile approach, and the newly proposed All Sample Mean Normalization (ASMN). We also examined the performance of normalizations in combination with correction for the two types of Infinium chemistry utilized on the 450K array. We observed that the performance of the GenomeStudio(®) normalization procedure was highly variable and dependent on the quality of the first sample analyzed in an experiment, which is used as a reference in this procedure. While the lumi normalization was able to decrease batch variability, it increased variation among technical replicates, potentially reducing biologically meaningful findings. The proposed ASMN procedure performed consistently well, both at reducing batch effects and improving replicate comparability. In summary, the ASMN procedure can improve existing color channel normalization, especially for large epidemiologic studies, and can be successfully implemented to enhance a 450K DNA methylation data pipeline.