Exploring Gut Microbiota Alterations with Trimethoprim-Sulfamethoxazole and Dexamethasone in a Humanized Microbiome Mouse Model.

Along with the standard therapies for glioblastoma, patients are commonly prescribed trimethoprim-sulfamethoxazole (TMP-SMX) and dexamethasone for preventing infections and reducing cerebral edema, respectively. Because the gut microbiota impacts the efficacy of cancer therapies, it is important to understand how these medications impact the gut microbiota of patients. Using mice that have been colonized with human microbiota, this study sought to examine how TMP-SMX and dexamethasone affect the gut microbiome. Two lines of humanized microbiota (HuM) Rag1-/- mice, HuM1Rag and HuM2Rag, were treated with either TMP-SMX or dexamethasone via oral gavage once a day for a week. Fecal samples were collected pre-treatment (pre-txt), one week after treatment initiation (1 wk post txt), and three weeks post-treatment (3 wk post txt), and bacterial DNA was analyzed using 16S rRNA-sequencing. The HuM1Rag mice treated with TMP-SMX had significant shifts in alpha diversity, beta diversity, and functional pathways at all time points, whereas in the HuM2Rag mice, it resulted in minimal changes in the microbiome. Likewise, dexamethasone treatment resulted in significant changes in the microbiome of the HuM1Rag mice, whereas the microbiome of the HuM2Rag mice was mostly unaffected. The results of our study show that routine medications used during glioblastoma treatment can perturb gut microbiota, with some microbiome compositions being more sensitive than others, and these treatments could potentially affect the overall efficacy of standard-of-care therapy.

A Bacterial-Sourced Protein Diet Induces Beneficial Shifts in the Gut Microbiome of the Zebrafish, Danio rerio.

Background: Bacterial-sourced single-cell proteins (SCPs) offer an alternative protein source for diet formulation for Zebrafish (Danio rerio) and other aquaculture models. In addition, the use of a single-cell bacterial protein source derived from multiple species provides a unique insight into the interplay among nutrients in the diet, microbial populations in the diet, and the gut microbiome in D. rerio. Objective: Our objective in this study was to evaluate the impact of dietary replacement of fish protein hydrolysate in a standard reference (SR) with a single-cell bacterial protein source on D. rerio gut microbiome. Methods: We investigated gut microbial compositions of D. rerio fed an open-formulation standard reference (SR) diet or a bacterial-sourced protein (BP) diet, utilizing microbial taxonomic co-occurrence networks, and predicted functional profiles. Results: Microbial communities in the SR diet were primarily composed of Firmicutes. In contrast, the BP diet was mainly composed of Proteobacteria. Alpha diversity revealed significant differences in microbial communities between the 2 diets, and between the guts of D. rerio fed either of the 2 diets. D. rerio fed with the SR diet resulted in abundance of Aeromonas and Vibrio. In contrast, D. rerio fed with a BP diet displayed a large abundance of members from the Rhodobacteraceae family. Taxonomic co-occurrence networks display unique microbial interactions, and key taxons in D. rerio gut samples were dependent on diet and gender. Predicted functional profiling of the microbiome across D. rerio fed SR or BP diets revealed distinct metabolic pathway differences. Female D. rerio fed the BP diet displayed significant upregulation of pathways related to primary and secondary bile acid synthesis. Male D. rerio fed the BP diet revealed similar pathway shifts and, additionally, a significant upregulation of the polyketide sugar unit biosynthesis pathway. Conclusions: The use of a BP dramatically affects the composition and activity of the gut microbiome. Future investigations should further address the interplay among biological systems and diet and may offer insights into potential health benefits in preclinical and translational animal models.

Replacement of Dietary Fish Protein with Bacterial Protein Results in Decreased Adiposity Coupled with Liver Gene Expression Changes in Female Danio rerio.

Background: Effective use of Danio rerio as a preclinical model requires standardization of macronutrient sources to achieve scientific reproducibility across studies and labs. Objective: Our objective was to evaluate a bacterial-based single-cell protein (SCP) for the production of open-source standardized diets with defined health characteristics for the zebrafish research community. Methods: We completed a 16-wk feeding trial using juvenile D. rerio 31 d postfertilization (10 tanks per diet and 14 D. rerio per tank) with formulated diets containing either a typical fish protein ingredient [standard reference (SR) diet] or a novel bacterial SCP source [bacterial protein (BP) diet]. At the end of the feeding trial, growth metrics, body composition, reproductive success, and bulk transcriptomics of the liver (RNAseq on female D. rerio with confirmatory rtPCR) were performed for each diet treatment. Results: D. rerio fed the BP diet had body weight gains equivalent to the D. rerio fed fish protein, and females had significantly lower total carcass lipid, indicating reduced adiposity. Reproductive success was similar between treatments, suggesting normal physiological function. Genes differentially expressed in female D. rerio fed the BP diet compared with females fed the SR diet were overrepresented in the gene ontologies of metabolism, biosynthesis of cholesterol precursors and products, and protein unfolding responses. Conclusion: Protein source substantially affected body growth metrics and composition as well as gene expression. These data support the development of an open-source diet utilizing an ingredient that correlates with improved health profiles and reduced variability in notable outcomes.

Evaluation of tissue- and plasma-derived tumor mutational burden (TMB) and genomic alterations of interest in CheckMate 848, a study of nivolumab combined with ipilimumab and nivolumab alone in patients with advanced or metastatic solid tumors with high TMB.

BACKGROUND: An accumulation of somatic mutations in tumors leads to increased neoantigen levels and antitumor immune response. Tumor mutational burden (TMB) reflects the rate of somatic mutations in the tumor genome, as determined from tumor tissue (tTMB) or blood (bTMB). While high tTMB is a biomarker of immune checkpoint inhibitor (ICI) treatment efficacy, few studies have explored the clinical utility of bTMB, a less invasive alternative for TMB assessment. Establishing the correlation between tTMB and bTMB would provide insight into whether bTMB is a potential substitute for tTMB. We explored the tumor genomes of patients enrolled in CheckMate 848 with measurable TMB. The correlation between tTMB and bTMB, and the factors affecting it, were evaluated. METHODS: In the phase 2 CheckMate 848 (NCT03668119) study, immuno-oncology-naïve patients with advanced, metastatic, or unresectable solid tumors and tTMB-high or bTMB-high (≥10 mut/Mb) were prospectively randomized 2:1 to receive nivolumab plus ipilimumab or nivolumab monotherapy. Tissue and plasma DNA sequencing was performed using the Foundation Medicine FoundationOne CDx and bTMB Clinical Trial Assays, respectively. tTMB was quantified from coding variants, insertions, and deletions, and bTMB from somatic base substitutions. Correlations between tTMB and bTMB were determined across samples and with respect to maximum somatic allele frequency (MSAF). Assay agreement and variant composition were also evaluated. RESULTS: A total of 1,438 and 1,720 unique tissue and blood samples, respectively, were obtained from 1,954 patients and included >100 screened disease ontologies, with 1,017 unique pairs of tTMB and bTMB measurements available for assessment. Median tTMB and bTMB were 3.8 and 3.5 mut/Mb, respectively. A significant correlation between tTMB and bTMB (r=0.48, p<0.0001) was observed across all sample pairs, which increased to r=0.54 (p<0.0001) for samples with MSAF≥1%. Assay concordance was highest for samples with MSAF≥10% across multiple disease ontologies and observed for both responders and non-responders to ICI therapy. The variants contributing to tTMB and bTMB were similar. CONCLUSIONS: We observed that tTMB and bTMB had a statistically significant correlation, particularly for samples with high MSAF, and that this correlation applied across disease ontologies. Further investigation into the clinical utility of bTMB is warranted.

Replacement of Dietary Fish Protein With Bacterial Single Cell Protein Results in Decreased Adiposity Coupled With Liver Expression Changes in Female Danio Rerio.

Background: Effective use of Danio rerio as a preclinical model requires standardization of macronutrient sources to achieve scientific reproducibility across studies and labs. Our objective was to evaluate single cell protein (SCP) for production of open-source standardized diets with defined heath characteristics for the zebrafish research community. We completed a 16-week feeding trial using juvenile D. rerio 31 days post-fertilization (dpf) (10 tanks per diet, 14 D. rerio per tank) with formulated diets containing either a typical fish protein ingredient or a novel bacterial SCP source. At the end of the feeding trial, growth metrics, body composition, reproductive success, and bulk transcriptomics of the liver (RNAseq on female D. rerio only with confirmatory rtPCR) were performed for each diet treatment. Results: D. rerio fed the SCP containing diet had body weight gains equivalent to the D. rerio fed fish protein, and females had significantly lower total carcass lipid, indicating reduced adiposity. Reproductive success was similar between treatments. Genes differentially expressed in female D. rerio provided the bacterial SCP compared to females given fish protein were overrepresented in the gene ontologies of metabolism, biosynthesis of cholesterol precursors and products, and protein unfolding responses. Conclusion: These data support the development of an open-source diet utilizing an ingredient that correlates with improved health profiles and reduced variability in notable outcomes.

Body Metrics and the Gut Microbiome in Response to Macronutrient Limitation in the Zebrafish Danio rerio.

Background: Healthy and predictable physiologic homeostasis is paramount in animal models for biomedical research. Proper macronutrient intake is an essential and controllable environmental factor for maintaining animal health and promoting experimental reproducibility. Objective and Methods: Evaluate reductions in dietary macronutrient composition on body weight metrics, composition, and gut microbiome in Danio rerio. Methods: D. rerio were fed reference diets deficient in either protein or lipid content for 14 weeks. Results: Diets of reduced-protein or reduced-fat resulted in lower weight gain than the standard reference diet in male and female D. rerio. Females fed the reduced-protein diet had increased total body lipid, suggesting increased adiposity compared with females fed the standard reference diet. In contrast, females fed the reduced-fat diet had decreased total body lipid compared with females fed the standard reference diet. The microbial community in male and female D. rerio fed the standard reference diet displayed high abundances of Aeromonas, Rhodobacteraceae, and Vibrio. In contrast, Vibrio spp. were dominant in male and female D. rerio fed a reduced-protein diet, whereas Pseudomonas displayed heightened abundance when fed the reduced-fat diet. Predicted functional metagenomics of microbial communities (PICRUSt2) revealed a 3- to 4-fold increase in the KEGG (Kyoto Encyclopedia of Genes and Genomes) functional category of steroid hormone biosynthesis in both male and female D. rerio fed a reduced-protein diet. In contrast, an upregulation of secondary bile acid biosynthesis and synthesis and degradation of ketone bodies was concomitant with a downregulation in steroid hormone biosynthesis in females fed a reduced-fat diet. Conclusions: These study outcomes provide insight into future investigations to understand nutrient requirements to optimize growth, reproductive, and health demographics to microbial populations and metabolism in the D. rerio gut ecosystem. These evaluations are critical in understanding the maintenance of steady-state physiologic and metabolic homeostasis in D. rerio. Curr Dev Nutr 20xx;x:xx.

High-throughput amplicon sequencing datasets of coastal sediments from three locations of the Gulf of Mexico, USA.

We present high-throughput amplicon sequence (HTS) datasets of the purified microbial metacommunity DNA of coastal surface sediments from Portersville Bay (PVB) (n = 3), Bayou La Batre (BLB) (n = 3), and Mobile Bay (MOB) (n = 3) of the U.S. Gulf of Mexico (U.S. Gulf Coast). The PVB samples were collected from the oyster aquaculture Shellevator™ system; the BLB samples were from locations on the shoreline adjacent to wild oysters attached to rocks and likely polluted from sewage and possibly chemical contamination from boats, shipyards, and seafood processing facilities; and MOB samples were adjacent to aquaculture oysters in bottom cages. The amplicons of the V4 hypervariable segment of the 16S rRNA gene from each sample were sequenced on an Illumina MiSeq to generate these HTS datasets. The raw sequences were quality-checked, demultiplexed into FASTQ files, denoised using DADA2, and subsampled. Then, the FASTA formatted sequences were assigned the taxonomic ids to amplicon sequence variants (ASVs) against the silva-138-99-nb-classifier using the Quantitative Insights Into Microbial Ecology (QIIME2 v2022.2). The applicability of the HTS datasets was confirmed by microbial taxa analysis at the phylum level using the "qiime taxa collapse" command. All HTS datasets are available through the BioSample Submission Portal under the BioProject ID PRJNA876773 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA876773).

The hitchhikers' guide to RNA sequencing and functional analysis.

DNA and RNA sequencing technologies have revolutionized biology and biomedical sciences, sequencing full genomes and transcriptomes at very high speeds and reasonably low costs. RNA sequencing (RNA-Seq) enables transcript identification and quantification, but once sequencing has concluded researchers can be easily overwhelmed with questions such as how to go from raw data to differential expression (DE), pathway analysis and interpretation. Several pipelines and procedures have been developed to this effect. Even though there is no unique way to perform RNA-Seq analysis, it usually follows these steps: 1) raw reads quality check, 2) alignment of reads to a reference genome, 3) aligned reads' summarization according to an annotation file, 4) DE analysis and 5) gene set analysis and/or functional enrichment analysis. Each step requires researchers to make decisions, and the wide variety of options and resulting large volumes of data often lead to interpretation challenges. There also seems to be insufficient guidance on how best to obtain relevant information and derive actionable knowledge from transcription experiments. In this paper, we explain RNA-Seq steps in detail and outline differences and similarities of different popular options, as well as advantages and disadvantages. We also discuss non-coding RNA analysis, multi-omics, meta-transcriptomics and the use of artificial intelligence methods complementing the arsenal of tools available to researchers. Lastly, we perform a complete analysis from raw reads to DE and functional enrichment analysis, visually illustrating how results are not absolute truths and how algorithmic decisions can greatly impact results and interpretation.

A modified standard American diet induces physiological parameters associated with metabolic syndrome in C57BL/6J mice.

Investigations into the causative role that western dietary patterns have on obesity and disease pathogenesis have speculated that quality and quantity of dietary fats and/or carbohydrates have a predictive role in the development of these disorders. Standard reference diets such as the AIN-93 rodent diet have historically been used to promote animal health and reduce variation of results across experiments, rather than model modern human dietary habits or nutrition-related pathologies. In rodents high-fat diets (HFDs) became a classic tool to investigate diet-induced obesity (DIO). These murine diets often relied on a single fat source with the most DIO consistent HFDs containing levels of fat up to 45-60% (kcal), higher than the reported human intake of 33-35% (kcal). More recently, researchers are formulating experimental animal (pre-clinical) diets that reflect mean human macro- and micronutrient consumption levels described by the National Health and Nutrition Examination Survey (NHANES). These diets attempt to integrate relevant ingredient sources and levels of nutrients; however, they most often fail to include high-fructose corn syrup (HFCS) as a source of dietary carbohydrate. We have formulated a modified Standard American Diet (mSAD) that incorporates relevant levels and sources of nutrient classes, including dietary HFCS, to assess the basal physiologies associated with mSAD consumption. Mice proffered the mSAD for 15 weeks displayed a phenotype consistent with metabolic syndrome, exhibiting increased adiposity, fasting hyperglycemia with impaired glucose and insulin tolerance. Metabolic alterations were evidenced at the tissue level as crown-like structures (CLS) in adipose tissue and fatty acid deposition in the liver, and targeted 16S rRNA metagenomics revealed microbial compositional shifts between dietary groups. This study suggests diet quality significantly affects metabolic homeostasis, emphasizing the importance of developing relevant pre-clinical diets to investigate chronic diseases highly impacted by western dietary consumption patterns.

High-throughput amplicon sequencing datasets of the metacommunity DNA of the gut microbiota of Zebrafish Danio rerio fed diets with differential quantities of protein and fat contents.

In this paper, we present high-throughput amplicon sequence (HTS) datasets of the gut microbiota of male and female Zebrafish Danio rerio fed diets consisting of sub-optimal and above-optimal quantities of proteins and fats. The HTS datasets were generated using an Illumina MiSeq targeting the V4 hypervariable segment of the 16S rRNA gene. The raw sequence reads were quality checked, demultiplexed into FASTQ files, denoised using DADA2 (q2-dada2 denoise-paired), and subsampled. Taxonomic ids were then assigned to amplicon sequence variants (ASVs) against the silva-138-99-nb-classifier for taxonomic output using the Quantitative Insights Into Microbial Ecology (QIIME2 v2021.4). The resultant taxa list was generated at the phylum level to confirm the applicability of the HTS dataset using the "qiime taxa collapse" command. These HTS datasets of the metagenome can be accessed through the BioSample Submission Portal (https://www.ncbi.nlm.nih.gov/bioproject/) under the BioProject IDs PRJNA772302 and PRJNA772305.

Fibrotic activity quantified in serum by measurements of type III collagen pro-peptides can be used for prognosis across different solid tumor types.

Due to activation of fibroblast into cancer-associated fibroblasts, there is often an increased deposition of extracellular matrix and fibrillar collagens, e.g. type III collagen, in the tumor microenvironment (TME) that leads to tumor fibrosis (desmoplasia). Tumor fibrosis is closely associated with treatment response and poor prognosis for patients with solid tumors. To assure that the best possible treatment option is provided for patients, there is medical need for identifying patients with high (or low) fibrotic activity in the TME. Measuring unique collagen fragments such as the pro-peptides released into the bloodstream during fibrillar collagen deposition in the TME can provide a non-invasive measure of the fibrotic activity. Based on data from 8 previously published cohorts, this review provides insight into the prognostic value of quantifying tumor fibrosis by measuring the pro-peptide of type III collagen in serum of a total of 1692 patients with different solid tumor types and discusses the importance of tumor fibrosis for understanding prognosis and for potentially guiding future drug development efforts that aim at overcoming the poor outcome associated with a fibrotic TME.

Microbial Composition and Genes for Key Metabolic Attributes in the Gut Digesta of Sea Urchins Lytechinus variegatus and Strongylocentrotus purpuratus Using Shotgun Metagenomics.

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.

Development and Performance of a CD8 Gene Signature for Characterizing Inflammation in the Tumor Microenvironment across Multiple Tumor Types.

Across multiple tumor types, immune checkpoint inhibitors (ICIs) have demonstrated clinical benefit to patients with cancer, yet there is a need to identify predictive biomarkers of response to these therapies. A multiparameter gene expression profiling-based tumor inflammation assay may offer robust characterization of the tumor microenvironment, thereby extending the utility of single-gene analysis or immunohistochemistry (IHC) in predicting response to ICIs. The authors interrogated 1778 commercially procured, formalin-fixed, paraffin-embedded samples using gene expression profiling and pathology-assisted digital CD8 IHC. A machine-learning approach was used to develop gene expression signatures that predicted CD8+ immune cell abundance as surrogates for tumor inflammation in melanoma and squamous cell carcinoma of the head and neck samples. An assay for a 16-gene CD8 signature was developed and analytically validated across 12 tumor types. CD8 signature scores correlated with CD8 IHC in a platform-independent manner, and inflammation prevalence was similar between assay methods for all tumor types except prostate cancer and small cell lung cancer. In retrospective analyses, CD8 signature scores were associated with progression-free survival and overall survival with nivolumab in patients with urothelial carcinoma from CheckMate 275. This study demonstrated that the CD8 signature assay can be used to accurately quantify CD8+ immune cell abundance in the tumor microenvironment and has potential clinical utility for determining patients with cancer likely to respond to ICIs.

Bioinformatic Methods and Bridging of Assay Results for Reliable Tumor Mutational Burden Assessment in Non-Small-Cell Lung Cancer.

INTRODUCTION: Tumor mutational burden (TMB) has emerged as a clinically relevant biomarker that may be associated with immune checkpoint inhibitor efficacy. Standardization of TMB measurement is essential for implementing diagnostic tools to guide treatment. OBJECTIVE: Here we describe the in-depth evaluation of bioinformatic TMB analysis by whole exome sequencing (WES) in formalin-fixed, paraffin-embedded samples from a phase III clinical trial. METHODS: In the CheckMate 026 clinical trial, TMB was retrospectively assessed in 312 patients with non-small-cell lung cancer (58% of the intent-to-treat population) who received first-line nivolumab treatment or standard-of-care chemotherapy. We examined the sensitivity of TMB assessment to bioinformatic filtering methods and assessed concordance between TMB data derived by WES and the FoundationOne® CDx assay. RESULTS: TMB scores comprising synonymous, indel, frameshift, and nonsense mutations (all mutations) were 3.1-fold higher than data including missense mutations only, but values were highly correlated (Spearman's r = 0.99). Scores from CheckMate 026 samples including missense mutations only were similar to those generated from data in The Cancer Genome Atlas, but those including all mutations were generally higher. Using databases for germline subtraction (instead of matched controls) showed a trend for race-dependent increases in TMB scores. WES and FoundationOne CDx outputs were highly correlated (Spearman's r = 0.90). CONCLUSIONS: Parameter variation can impact TMB calculations, highlighting the need for standardization. Encouragingly, differences between assays could be accounted for by empirical calibration, suggesting that reliable TMB assessment across assays, platforms, and centers is achievable.

First-Line Nivolumab Plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer (CheckMate 568): Outcomes by Programmed Death Ligand 1 and Tumor Mutational Burden as Biomarkers.

PURPOSE: CheckMate 568 is an open-label phase II trial that evaluated the efficacy and safety of nivolumab plus low-dose ipilimumab as first-line treatment of advanced/metastatic non-small-cell lung cancer (NSCLC). We assessed the association of efficacy with programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB). PATIENTS AND METHODS: Two hundred eighty-eight patients with previously untreated, recurrent stage IIIB/IV NSCLC received nivolumab 3 mg/kg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks. The primary end point was objective response rate (ORR) in patients with 1% or more and less than 1% tumor PD-L1 expression. Efficacy on the basis of TMB (FoundationOne CDx assay) was a secondary end point. RESULTS: Of treated patients with tumor available for testing, 252 patients (88%) of 288 were evaluable for PD-L1 expression and 98 patients (82%) of 120 for TMB. ORR was 30% overall and 41% and 15% in patients with 1% or greater and less than 1% tumor PD-L1 expression, respectively. ORR increased with higher TMB, plateauing at 10 or more mutations/megabase (mut/Mb). Regardless of PD-L1 expression, ORRs were higher in patients with TMB of 10 or more mut/Mb (n = 48: PD-L1, ≥ 1%, 48%; PD-L1, < 1%, 47%) versus TMB of fewer than 10 mut/Mb (n = 50: PD-L1, ≥ 1%, 18%; PD-L1, < 1%, 5%), and progression-free survival was longer in patients with TMB of 10 or more mut/Mb versus TMB of fewer than 10 mut/Mb (median, 7.1 v 2.6 months). Grade 3 to 4 treatment-related adverse events occurred in 29% of patients. CONCLUSION: Nivolumab plus low-dose ipilimumab was effective and tolerable as a first-line treatment of advanced/metastatic NSCLC. TMB of 10 or more mut/Mb was associated with improved response and prolonged progression-free survival in both tumor PD-L1 expression 1% or greater and less than 1% subgroups and was thus identified as a potentially relevant cutoff in the assessment of TMB as a biomarker for first-line nivolumab plus ipilimumab.

After 50 years, a personal reflection on the development of internal thoracic artery (ITA) grafting.

It has been 50 years since the first publications of evidence that internal thoracic artery (ITA) grafts successfully bypassed obstructions of coronary arteries in dogs and in humans. The evidence consisted of in vivo measurements of blood flow through the grafts and selective angiograms of the grafts months following operation. It may be of interest to recount how that success came about.

Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden.

BACKGROUND: Nivolumab plus ipilimumab showed promising efficacy for the treatment of non-small-cell lung cancer (NSCLC) in a phase 1 trial, and tumor mutational burden has emerged as a potential biomarker of benefit. In this part of an open-label, multipart, phase 3 trial, we examined progression-free survival with nivolumab plus ipilimumab versus chemotherapy among patients with a high tumor mutational burden (≥10 mutations per megabase). METHODS: We enrolled patients with stage IV or recurrent NSCLC that was not previously treated with chemotherapy. Those with a level of tumor programmed death ligand 1 (PD-L1) expression of at least 1% were randomly assigned, in a 1:1:1 ratio, to receive nivolumab plus ipilimumab, nivolumab monotherapy, or chemotherapy; those with a tumor PD-L1 expression level of less than 1% were randomly assigned, in a 1:1:1 ratio, to receive nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy. Tumor mutational burden was determined by the FoundationOne CDx assay. RESULTS: Progression-free survival among patients with a high tumor mutational burden was significantly longer with nivolumab plus ipilimumab than with chemotherapy. The 1-year progression-free survival rate was 42.6% with nivolumab plus ipilimumab versus 13.2% with chemotherapy, and the median progression-free survival was 7.2 months (95% confidence interval [CI], 5.5 to 13.2) versus 5.5 months (95% CI, 4.4 to 5.8) (hazard ratio for disease progression or death, 0.58; 97.5% CI, 0.41 to 0.81; P<0.001). The objective response rate was 45.3% with nivolumab plus ipilimumab and 26.9% with chemotherapy. The benefit of nivolumab plus ipilimumab over chemotherapy was broadly consistent within subgroups, including patients with a PD-L1 expression level of at least 1% and those with a level of less than 1%. The rate of grade 3 or 4 treatment-related adverse events was 31.2% with nivolumab plus ipilimumab and 36.1% with chemotherapy. ical; CheckMate 227 ClinicalTrials.gov number, NCT02477826 .). CONCLUSIONS: Progression-free survival was significantly longer with first-line nivolumab plus ipilimumab than with chemotherapy among patients with NSCLC and a high tumor mutational burden, irrespective of PD-L1 expression level. The results validate the benefit of nivolumab plus ipilimumab in NSCLC and the role of tumor mutational burden as a biomarker for patient selection. (Funded by Bristol-Myers Squibb and Ono Pharmaceut

Comparative assessment of clinical response in patients with rheumatoid arthritis between PF-05280586, a proposed rituximab biosimilar, and rituximab.

AIMS: To evaluate potential differences between PF-05280586 and rituximab sourced from the European Union (rituximab-EU) and USA (rituximab-US) in clinical response (Disease Activity Score in 28 Joints [DAS28] and American College of Rheumatology [ACR] criteria), as part of the overall biosimilarity assessment of PF-05280586. METHODS: A randomised, double-blind, pharmacokinetic similarity trial was conducted in patients with active rheumatoid arthritis refractory to anti-tumour necrosis factor therapy on a background of methotrexate. Patients were treated with 1000 mg of PF-05280586, rituximab-EU or rituximab-US on days 1 and 15 and followed over 24 weeks for pharmacokinetic, clinical response and safety assessments. Key secondary end points were the areas under effect curves for DAS28 and ACR responses. Mean differences in areas under effect curves were compared against respective reference ranges established by observed rituximab-EU and rituximab-US responses using longitudinal nonlinear mixed effects models. RESULTS: The analysis included 214 patients. Demographics were similar across groups with exceptions in some baseline disease characteristics. Baseline imbalances and group-to-group variation were accounted for by covariate effects in each model. Predictions from the DAS28 and ACR models tracked the central tendency and distribution of observations well. No point estimates of mean differences were outside the reference range for DAS28 or ACR scores. The probabilities that the predicted differences between PF-05280586 vs. rituximab-EU or rituximab-US lie outside the reference ranges were low. CONCLUSIONS: No clinically meaningful differences were detected in DAS28 or ACR response between PF-05280586 and rituximab-EU or rituximab-US as the differences were within the pre-specified reference ranges. TRIAL REGISTRATION NUMBER: NCT01526057.

A phase I pharmacokinetics trial comparing PF-05280586 (a potential biosimilar) and rituximab in patients with active rheumatoid arthritis.

AIMS: Pharmacokinetic (PK) similarity was assessed among PF-05280586 (a proposed biosimilar) vs. rituximab sourced from the European Union (rituximab-EU) and the United States (rituximab-US). Pharmacodynamics (PD), overall safety and immunogenicity were also evaluated. METHODS: Patients with active rheumatoid arthritis on a background of methotrexate and inadequate response to one or more tumour necrosis factor antagonist therapies were randomized to intravenous PF-05280586, rituximab-EU or rituximab-US 1000 mg doses on study days 1 and 15. RESULTS: A total of 220 patients were randomized to receive study treatment as assigned. Of these, 198 met per-protocol population criteria for inclusion in the PK data analysis. PF-05280586, rituximab-EU and rituximab-US exhibited similar PK profiles following administration of assigned study drug on days 1 and 15. The 90% confidence intervals of test-to-reference ratios for Cmax , AUCT , AUC0-∞ and AUC2-week were within the bioequivalence margin of 80.00-125.00% for comparisons of PF-05280586 with rituximab-EU, PF-05280586 with rituximab-US, and rituximab-EU with rituximab-US. All treatments resulted in a rapid and profound reduction in CD19+ B cells and sustained profound B cell suppression up to week 25. The incidence of antidrug antibody (ADA) response (n = 7, 10 and 9 for PF-05280586, rituximab-EU and rituximab-US, respectively), time to ADA emergence and ADA titres were similar across treatments. None of the ADA-positive samples was positive for neutralizing activity. No clinically meaningful differences in adverse events were identified. CONCLUSIONS: The study demonstrated PK similarity among PF-05280586, rituximab-EU and rituximab-US. In addition, all treatments showed comparable CD19+ B cell depletion PD responses, as well as safety and immunogenicity profiles.

Amidation inhibitors 4-phenyl-3-butenoic acid and 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester are novel HDAC inhibitors with anti-tumorigenic properties.

4-Phenyl-3-butenoic acid (PBA) is an inhibitor of peptidylglycine alpha-amidating monooxygenase with anti-inflammatory properties that has been shown to inhibit the growth of ras-mutated epithelial and human lung carcinoma cells. In this report, we show that PBA also increases the acetylation levels of selected histone subtypes in a dose and time dependent manner, an effect that is attributable to the inhibition of histone deacetylase (HDAC) enzymes. Comparison studies with the known HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) using high resolution two-dimensional polyacrylamide gels and Western analysis provide evidence that PBA acts as an HDAC inhibitor within cells. PBA and a more potent amidation inhibitor, 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me), inhibit HDAC enzymes in vitro at micromolar concentrations, with IC50 values approximately 30 fold lower for AOPHA-Me than PBA for selected HDAC isoforms. Overall, these results indicate that PBA and AOPHA-Me are novel anti-tumorigenic HDAC inhibitors.