Azeet Narayan

Ultrasensitive Measurement of Hotspot Mutations in Tumor DNA in Blood Using Error-Suppressed Multiplexed Deep Sequencing

Detection of cell-free tumor DNA in the blood has offered promise as a cancer biomarker, but practical clinical implementations have been impeded by the lack of a sensitive and accurate method for quantitation that is also simple, inexpensive, and readily scalable. Here we present an approach that uses next-generation sequencing to quantify the small fraction of DNA molecules that contain tumor-specific mutations within a background of normal DNA in plasma. Using layers of sequence redundancy designed to distinguish true mutations from sequencer misreads and PCR misincorporations, we achieved a detection sensitivity of approximately 1 variant in 5,000 molecules. In addition, the attachment of modular barcode tags to the DNA fragments to be sequenced facilitated the simultaneous analysis of more than 100 patient samples. As proof-of-principle, we showed the successful use of this method to follow treatment-associated changes in circulating tumor DNA levels in patients with non-small cell lung cancer. Our findings suggest that the deep sequencing approach described here may be applied to the development of a practical diagnostic test that measures tumor-derived DNA levels in blood.

Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis, and Concurrently Affect the Pathogen's Growth

Background The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung. Methodology/Principal Findings During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage. Conclusions/Significance While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptides binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.

High-throughput RNA profiling via up-front sample parallelization

We describe a method called modular, early-tagged amplification (META) RNA profiling that can quantify a broad panel of microRNAs or mRNAs simultaneously across many samples and requires far less sequence depth than existing digital profiling technologies. The method assigns quantitative tags during reverse transcription to permit up-front sample pooling before competitive amplification and deep sequencing. This simple, scalable and inexpensive approach improves the practicality of large-scale gene expression studies.

Loss of kinase activity in Mycobacterium tuberculosis multidomain protein Rv1364c

The alternative sigma factors are regulated by a phosphorylation‐mediated signal transduction cascade involving anti‐sigma factors and anti‐anti‐sigma factors. The proteins regulating Mycobacterium tuberculosis sigma factor F (SigF), anti‐SigF and anti‐anti‐SigF have been identified, but the factors catalyzing phosphorylation–dephosphorylation have not been well established. We identified a distinct pathogenic species‐specific multidomain protein, Rv1364c, in which the components of the entire signal transduction cascade for SigF regulation appear to be encoded in a single polypeptide. Sequence analysis of M. tuberculosis Rv1364c resulted in the prediction of various domains, namely a phosphatase (RsbU) domain, an anti‐SigF (RsbW) domain, and an anti‐anti‐SigF (RsbV) domain. We report that the RsbU domain of Rv1364c bears all the conserved features of the PP2C‐type serine/threonine phosphatase family, whereas its RsbW domain has certain substitutions and deletions in regions important for ATP binding. Another anti‐SigF protein in M. tuberculosis, UsfX (Rv3287c), shows even more unfavorable substitutions in the kinase domain. Biochemical assay with the purified RsbW domain of Rv1364c and UsfX showed the loss of ability of autophosphorylation and phosphotransfer to cognate anti‐anti‐SigF proteins or artificial substrates. Both the Rv1364c RsbW domain and UsfX protein display very weak binding with fluorescent ATP analogs, despite showing functional interactions characteristic of anti‐SigF proteins. In view of conservation of specific interactions with cognate sigma and anti‐anti‐sigma factor, the loss of kinase activity of Rv1364c and UsfX appears to form a missing link in the phosphorylation‐dependent interaction involved in SigF regulation in Mycobacterium.

Early Assessment of Lung Cancer Immunotherapy Response via Circulating Tumor DNA

Purpose: Decisions to continue or suspend therapy with immune checkpoint inhibitors are commonly guided by tumor dynamics seen on serial imaging. However, immunotherapy responses are uniquely challenging to interpret because tumors often shrink slowly or can appear transiently enlarged due to inflammation. We hypothesized that monitoring tumor cell death in real time by quantifying changes in circulating tumor DNA (ctDNA) levels could enable early assessment of immunotherapy efficacy. Experimental Design: We compared longitudinal changes in ctDNA levels with changes in radiographic tumor size and with survival outcomes in 28 patients with metastatic non–small cell lung cancer (NSCLC) receiving immune checkpoint inhibitor therapy. CtDNA was quantified by determining the allele fraction of cancer-associated somatic mutations in plasma using a multigene next-generation sequencing assay. We defined a ctDNA response as a >50% decrease in mutant allele fraction from baseline, with a second confirmatory measurement. Results: Strong agreement was observed between ctDNA response and radiographic response (Cohens kappa, 0.753). Median time to initial response among patients who achieved responses in both categories was 24.5 days by ctDNA versus 72.5 days by imaging. Time on treatment was significantly longer for ctDNA responders versus nonresponders (median, 205.5 vs. 69 days; P < 0.001). A ctDNA response was associated with superior progression-free survival [hazard ratio (HR), 0.29; 95% CI, 0.09–0.89; P = 0.03], and superior overall survival (HR, 0.17; 95% CI, 0.05–0.62; P = 0.007). Conclusions: A drop in ctDNA level is an early marker of therapeutic efficacy and predicts prolonged survival in patients treated with immune checkpoint inhibitors for NSCLC. Clin Cancer Res; 24(8); 1872–80. ©2018 AACR.

META RNA profiling: Multiplexed quantitation of targeted RNAs across large numbers of samples

META RNA profiling is a simple and inexpensive method to measure the expression of multiple targeted RNAs across many samples. By assigning sample-specific tags up-front during reverse-transcription, cDNAs from multiple samples can be pooled prior to amplification and deep sequencing. Such early parallelization of samples simplifies the workflow, minimizes cross-sample experimental variability, and reduces reagent and sequencing costs. Herein we describe the theoretical framework of the method and provide a detailed protocol to facilitate its implementation.

Measurement of circulating tumor DNA as a cancer biomarker in gastrointestinal malignancies using a novel next-generation sequencing method.

217 Background: Circulating tumor DNA (ctDNA) holds promise as a highly specific cancer biomarker. The presence of mutant tumor-derived DNA fragments in the blood provides an opportunity to non-invasively assess tumor mutation profiles and to quantify changes in tumor DNA levels over time. Methods: After obtaining informed consent, plasma samples were collected prospectively at multiple time points in a cohort of patients (pts) with various gastrointestinal (GI) malignancies in the locally advanced, metastatic and adjuvant settings. Hotspot regions of genes known to be commonly mutated in GI tumors were amplified by multiplexed PCR, and the resultant amplicons were subjected to next-generation ultra-deep sequencing. Suppression of sequencer and PCR errors allowed mutations to be identified and quantified with a sensitivity of approximately 1 variant in 5,000 molecules. Sample-specific barcoding allowed simultaneous analysis of up to 96 samples. Results: 29 out of 74 available samples from 17 pts were anal...