Pembrolizumab is approved for treating patients with unresectable or metastatic solid tumors with high tumor mutational burden (TMB), as assessed by the Food and Drug Administration–approved ...companion diagnostic FoundationOneCDx, after progression on prior treatment. To expand TMB assessment for enriching response to pembrolizumab, TMB measurement from TruSight Oncology 500 (TSO500) was evaluated in archival pan-tumor samples from 294 patients enrolled in eight pembrolizumab monotherapy studies. TSO500 is a panel-based next-generation sequencing assay with broad availability, quick turnaround time, and a standardized bioinformatics pipeline. TSO500 TMB was evaluated for correlation and concordance with two reference methods: FoundationOneCDx and whole-exome sequencing. The TSO500 cut-off for TMB-high was selected based on the receiver-operating characteristic curve analysis against each reference method's cut-off for TMB-high. Clinical utility of the selected TSO500 cut-off (10 mutations/Mb) was assessed by calculating the sensitivity, specificity, positive and negative predictive values, and objective response rate enrichment. There was high correlation and concordance of TSO500 TMB with both reference methods. TSO500 TMB was associated significantly with the best overall response, and the selected cut-off had comparable clinical utility with respective cut-offs for the reference methods in predicting response to pembrolizumab. As a commercial assay with global kit distribution complete with an off-the-shelf software package, TSO500 may provide additional access to immunotherapy for patients with tumors with TMB ≥10 mutations/Mb.
Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with ...treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi's discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BackgroundVarious biomarkers have been investigated for their ability to identify patients more likely to respond to immunotherapy. Recently, the PD-1 inhibitor pembrolizumab was approved by the FDA ...for treating patients with unresectable or metastatic solid tumors with high TMB (TMB-H) who have no satisfactory alternative treatment options following progression on prior treatment. The FDA contemporaneously approved the FoundationOne®CDx (F1CDx; Foundation Medicine) as the companion diagnostic for TMB assessment for pembrolizumab. However, multiple comprehensive genomic profiling panels that can measure TMB are currently available or in development. We evaluated the performance of TruSight™ Oncology 500 (TSO500; Illumina) for assessing TMB and its clinical utility using F1CDx and whole exome sequencing (WES) as reference methods.MethodsPretreatment archival tumor samples from patients enrolled in 8 clinical trials of pembrolizumab monotherapy were evaluated for TMB by TSO500, F1CDx QSR pipeline v3.2.0, and WES. Correlation was assessed using Spearman’s rank correlation coefficient (ρ). The F1CDx and WES TMB cutpoints were 10 mut/Mb and 175 mut/exome, respectively. The TSO500 cutpoint was selected using the Youden index criterion. Concordance was assessed by calculating area under the receiver-operating curve (AUROC), positive percentage agreement (PPA), and negative percentage agreement (NPA). Statistical significance of the association of TMB measured by TSO500 with ORR was assessed using logistic regression adjusted for ECOG performance status and cancer type. Clinical utility of the selected TSO500 TMB cutpoint for discriminating responders and nonresponders was assessed by calculating sensitivity, specificity, positive predictive value, negative predictive value, ORR enrichment, and prevalence.ResultsTMB scores were valid for 294/294 patients assessed by TSO500, 269/270 assessed by F1CDx, and 293/294 assessed by WES. TMB assessed by TSO500 had good correlation with TMB assessed by F1CDx (ρ=0.76) and WES (ρ=0.74). Using Youden index criterion, 10 mut/Mb was the TSO500 cutpoint that corresponded with both the F1CDx and WES cutpoints. TSO500 reliably predicted TMB-H and non–TMB-H status as determined by the F1CDx (AUROC=0.99, PPA=97.4%, NPA=93.0%) and WES (AUROC=0.95, PPA=76.2%, NPA=96.1%) cutpoints. TMB measured by TSO500 was significantly associated with ORR (one-sided P<0.0001). Clinical utility metrics were generally similar for TSO500 and F1CDx (table 1) and TSO500 and WES (table 2).Abstract 80 Table 1Clinical Utility Metrics for the TSO500 TMB Cutpoint Compared with the F1CDx TMB Cutpoint (n=269)Abstract 80 Table 2Clinical Utility Metrics for the TSO500 TMB Cutpoint Compared with the WES TMB Cutpoint (n=293)ConclusionsTMB assessed by TSO500 is highly correlated and concordant with TMB assessed by F1CDx and WES. Similar to the validated and approved F1CDx TMB cutpoint of 10 mut/Mb, the TSO500 TMB cutpoint of 10 mut/Mb is predictive of response to pembrolizumab monotherapy.AcknowledgementsThis analysis and all included studies were sponsored by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.Trial RegistrationNAEthics ApprovalThe protocols and all amendments for the studies included in this analysis were approved by the appropriate ethics committee at each participating institution.ConsentNAReferenceNA
To develop hepatitis C virus (HCV) direct-acting antiviral (DAA) drugs that can treat most HCV genotypes and offer higher barriers for treatment-resistant mutations, it is important to study ...resistance-associated variants (RAVs). Current commercially available RAV detection assays rely on genotype- or subtype-specific template-dependent PCR amplification. These assays are limited to genotypes and subtypes that are often prevalent in developed countries because of availability of public sequence databases. To support global clinical trials of DAAs, we developed and validated a template-independent (TI) next-generation sequencing (NGS) assay for HCV whole genome sequencing that can perform HCV subtyping, detect HCV mixed genotype or subtype infection, and identify low-level RAVs at a 5% fraction of the viral population with sensitivity and positive predictive value ≥ 0.9. We compared TI-NGS with commercial genotype- or subtype-specific Sanger sequencing assays, and found that TI-NGS both confirmed most of variants called by Sanger sequencing and avoided biases likely caused by PCR primers used in Sanger sequencing. To confirm TI-NGS assay's variant calls at the discrepant positions with Sanger sequencing, we custom designed template-dependent NGS assays and obtained 100% concordance with the TI-NGS assay. The ability to reliably detect low-level RAVs in HCV samples of any subtype without PCR primer-related bias makes this TI-NGS assay an important tool in studying HCV DAA drug resistance.
Nucleoporin 155 (
Nup155) is a major component of the nuclear pore complex (NPC) involved in cellular nucleo-cytoplasmic transport. We have acquired the complete sequence and interpreted the genomic ...organization of the
Nup155 orthologos from human (
Homo sapiens) and pufferfish (
Fugu rubripes), which are approximately 80 and 8 kb in length, respectively. The human gene is ubiquitously expressed in many tissues analyzed and has two major transcript variants, resulted from an alternative usage of the 5′ cryptic or consensus splice donor in intron 1 and two polyadenylation signals. We have also cloned DNA complementary to RNAs of the
Nup155 orthologs from
Fugu and mouse. Comparative analysis of the
Nup155 orthologs in many species, including
H. sapiens, Mus musculus, Rattus norvegicus, F. rubripes, Arabidopsis thaliana, Drosophila melanogaster, and
Saccharomyces cerevisiae, has revealed two paralogs in
S. cerevisiae but only a single gene with increasing number of introns in more complex organisms. The amino acid sequences of the
Nup155 orthologos are highly conserved in the evolution of eukaryotes. Different gene orders in the human and
Fugu genomic regions harboring the
Nup155 orthologs advocate cautious interpretation of synteny in comparative genomic analysis even within the vertebrate lineage.
We sequenced 114 genes (for DNA repair, cell cycle arrest, apoptosis, and detoxification)in a mixed human population and observed a sudden increase in the number of functional polymorphisms below a ...minor allele frequency of approximately 6%. Functionality is assessed by considering the ratio in the number of nonsynonymous single nucletide polymorphisms (SNPs)to the number of synonymous or intron SNPs. This ratio is steady from below 1% in frequency-that regime traditionally associated with rare Mendelian diseases-all the way up to about 6% in frequency, after which it falls precipitously. We consider possible explanations for this threshold effect. There are four candidates as follows: (1). deleterious variants that have yet to be purified from the population, (2). balancing selection, in which a selective advantage accrues to the heterozygotes, (3). population-specific functional polymorphisms, and (4). adaptive variants that are accumulating in the population as a response to the dramatic environmental changes of the last 7000 approximately 17000 years.
Minimal introns are not "junk" Yu, Jun; Yang, Zhiyong; Kibukawa, Miho ...
Genome research
12, Številka:
8
Journal Article
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Odprti dostop
Intron-size distributions for most multicellular (and some unicellular) eukaryotes have a sharp peak at their "minimal intron" size. Across the human population, these minimal introns exhibit an ...abundance of insertion-deletion polymorphisms, the effect of which is to maintain their optimal size. We argue that minimal introns affect function by enhancing the rate at which mRNA is exported from the cell nucleus.
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