Analytical validation of NeXT Personal®, an ultra-sensitive personalized circulating tumor DNA assay.

We describe the analytical validation of NeXT Personal®, an ultra-sensitive, tumor-informed circulating tumor DNA (ctDNA) assay for detecting residual disease, monitoring therapy response, and detecting recurrence in patients diagnosed with solid tumor cancers. NeXT Personal uses whole genome sequencing of tumor and matched normal samples combined with advanced analytics to accurately identify up to ~1,800 somatic variants specific to the patient's tumor. A personalized panel is created, targeting these variants and then used to sequence cell-free DNA extracted from patient plasma samples for ultra-sensitive detection of ctDNA. The NeXT Personal analytical validation is based on panels designed from tumor and matched normal samples from two cell lines, and from 123 patients across nine cancer types. Analytical measurements demonstrated a detection threshold of 1.67 parts per million (PPM) with a limit of detection at 95% (LOD95) of 3.45 PPM. NeXT Personal showed linearity over a range of 0.8 to 300,000 PPM (Pearson correlation coefficient = 0.9998). Precision varied from a coefficient of variation of 12.8% to 3.6% over a range of 25 to 25,000 PPM. The assay targets 99.9% specificity, with this validation study measuring 100% specificity and in silico methods giving us a confidence interval of 99.92 to 100%. In summary, this study demonstrates NeXT Personal as an ultra-sensitive, highly quantitative and robust ctDNA assay that can be used to detect residual disease, monitor treatment response, and detect recurrence in patients.

Analytic validation of NeXT Dx™, a comprehensive genomic profiling assay.

We describe the analytic validation of NeXT Dx, a comprehensive genomic profiling assay to aid therapy and clinical trial selection for patients diagnosed with solid tumor cancers. Proprietary methods were utilized to perform whole exome and whole transcriptome sequencing for detection of single nucleotide variants (SNVs), insertions/deletions (indels), copy number alterations (CNAs), and gene fusions, and determination of tumor mutation burden and microsatellite instability. Variant calling is enhanced by sequencing a patient-specific normal sample from, for example, a blood specimen. This provides highly accurate somatic variant calls as well as the incidental reporting of pathogenic and likely pathogenic germline alterations. Fusion detection via RNA sequencing provides more extensive and accurate fusion calling compared to DNA-based tests. NeXT Dx features the proprietary Accuracy and Content Enhanced technology, developed to optimize sequencing and provide more uniform coverage across the exome. The exome was validated at a median sequencing depth of >500x. While variants from 401 cancer-associated genes are currently reported from the assay, the exome/transcriptome assay is broadly validated to enable reporting of additional variants as they become clinically relevant. NeXT Dx demonstrated analytic sensitivities as follows: SNVs (99.4%), indels (98.2%), CNAs (98.0%), and fusions (95.8%). The overall analytic specificity was >99.0%.

How the virus outsmarts the host: function and structure of cytomegalovirus MHC-I-like molecules in the evasion of natural killer cell surveillance.

Natural killer (NK) cells provide an initial host immune response to infection by many viral pathogens. Consequently, the viruses have evolved mechanisms to attenuate the host response, leading to improved viral fitness. One mechanism employed by members of the ß-herpesvirus family, which includes the cytomegaloviruses, is to modulate the expression of cell surface ligands recognized by NK cell activation molecules. A novel set of cytomegalovirus (CMV) genes, exemplified by the mouse m145 family, encode molecules that have structural and functional features similar to those of host major histocompatibility-encoded (MHC) class I molecules, some of which are known to contribute to immune evasion. In this review, we explore the function, structure, and evolution of MHC-I-like molecules of the CMVs and speculate on the dynamic development of novel immunoevasive functions based on the MHC-I protein fold.

Structural basis for dimerization in DNA recognition by Gal4.

Gal4 is a Zn2Cys6 binuclear cluster containing transcription factor that binds DNA as a homodimer and can activate transcription by interacting with the mutant Gal11P protein. Although structures have been reported of the Gal4 dimerization domain and the binuclear cluster domain bound to DNA as a dimer, the structure of the "complete" Gal4 dimer bound to DNA has not previously been described. Here we report the structure of a complete Gal4 dimer bound to DNA and additional biochemical studies to address the molecular basis for Gal4 dimerization in DNA binding. We find that Gal4 dimerization on DNA is mediated by an intertwined helical bundle that deviates significantly from the solution NMR structure of the free dimerization domain. Associated biochemical studies show that the dimerization domain of Gal4 is important for DNA binding and protein thermostability. We also map the interaction surface of the Gal4 dimerization domain with Gal11P.