Evaluation of the TruSight Oncology 500 Assay for Routine Clinical Testing of Tumor Mutational Burden and Clinical Utility for Predicting Response to Pembrolizumab.

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.

Development of a PCR Assay to Detect Low Level Trypanosoma cruzi in Blood Specimens Collected with PAXgene Blood DNA Tubes for Clinical Trials Treating Chagas Disease.

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.

Development and Validation of a Template-Independent Next-Generation Sequencing Assay for Detecting Low-Level Resistance-Associated Variants of Hepatitis C Virus.

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.

MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression.

microRNAs in the miR-106b family are overexpressed in multiple tumor types and are correlated with the expression of genes that regulate the cell cycle. Consistent with these observations, miR-106b family gain of function promotes cell cycle progression, whereas loss of function reverses this phenotype. Microarray profiling uncovers multiple targets of the family, including the cyclin-dependent kinase inhibitor p21/CDKN1A. We show that p21 is a direct target of miR-106b and that its silencing plays a key role in miR-106b-induced cell cycle phenotypes. We also show that miR-106b overrides a doxorubicin-induced DNA damage checkpoint. Thus, miR-106b family members contribute to tumor cell proliferation in part by regulating cell cycle progression and by modulating checkpoint functions.

Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression.

microRNAs (miRNAs) are abundant, approximately 21-nucleotide, noncoding regulatory RNAs. Each miRNA may regulate hundreds of mRNA targets, but the identities of these targets and the processes they regulate are poorly understood. Here we have explored the use of microarray profiling and functional screening to identify targets and biological processes triggered by the transfection of human cells with miRNAs. We demonstrate that a family of miRNAs sharing sequence identity with miRNA-16 (miR-16) negatively regulates cellular growth and cell cycle progression. miR-16-down-regulated transcripts were enriched with genes whose silencing by small interfering RNAs causes an accumulation of cells in G(0)/G(1). Simultaneous silencing of these genes was more effective at blocking cell cycle progression than disruption of the individual genes. Thus, miR-16 coordinately regulates targets that may act in concert to control cell cycle progression.

A population threshold for functional polymorphisms.

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".

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.

Genomic organization, transcript variants and comparative analysis of the human nucleoporin 155 (NUP155) gene.

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.