Abhijit A. Patel

Splicing double: insights from the second spliceosome

Almost 20 years after the discovery of introns and RNA splicing, a second spliceosome was uncovered. Although this new spliceosome is structurally and functionally analogous to the well-characterized major-class splicing apparatus, it mediates the excision of a minor class of evolutionarily conserved introns that have non-canonical consensus sequences. This unanticipated diversity in the splicing machinery is refining both the mechanistic understanding and evolutionary models of RNA splicing.

The splicing of U12‐type introns can be a rate‐limiting step in gene expression

Some protein‐coding genes in metazoan genomes contain a minor class of introns that are excised by a distinct, low‐abundance spliceosome. We have developed a quantitative RT–PCR assay that allows comparison of the relative rates of intron removal from the transcripts present in a pre‐mRNA population. We show that the U12‐type introns are more slowly spliced than the major‐class (U2‐type) introns from three endogenous pre‐mRNAs in human tissue culture cells. In Drosophila melanogaster S2 cells, using minigene constructs designed to produce nearly identical mRNAs, we observe increased expression of fluorescent protein and mature mRNA upon mutation of a U12‐type to a U2‐type intron. These results provide evidence that the level of gene expression in vivo is lowered by the presence of a U12‐type intron and implicate the U12‐type spliceosome as a target in the post‐transcriptional regulation of gene expression.

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.

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.

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.

A novel multiple biomarker panel for the early detection of high-grade serous ovarian carcinoma

INTRODUCTION Since the majority of patients are diagnosed at an advanced stage, ovarian cancer remains the most lethal gynecologic malignancy. There is no single biomarker with the sensitivity and specificity required for effective cancer screening; therefore, we investigated a panel of novel biomarkers for the early detection of high-grade serous ovarian carcinoma. METHODS Twelve serum biomarkers with high differential gene expression and validated antibodies were selected: IL-1Ra, IL-6, Dkk-1, uPA, E-CAD, ErbB2, SLPI, HE4, CA125, LCN2, MSLN, and OPN. They were tested using Simple Plex™, a multi-analyte immunoassay platform, in samples collected from 172 patients who were either healthy, had benign gynecologic pathologies, or had high-grade serous ovarian adenocarcinomas. The receiver operating characteristic (ROC) curve, ROC area under the curve (AUC), and standard error (SE) of the AUC were obtained. Univariate ROC analyses and multivariate ROC analyses with the combination of multiple biomarkers were performed. RESULTS The 4-marker panel consisting of CA125, HE4, E-CAD, and IL-6 had the highest ROC AUC. When evaluated for the ability to distinguish early stage ovarian cancer from a non-cancer control, not only did this 4-marker panel (AUC=0.961) performed better than CA 125 alone (AUC=0.851; P=0.0150) and HE4 alone (AUC=0.870; P=0.0220), but also performed significantly better than the 2- marker combination of CA125+HE4 (AUC=0.922; P=0.0278). The 4-marker panel had the highest average sensitivity under the region of its ROC curve corresponding to specificity ranging from 100% down to ~95%. CONCLUSION The four-marker panel, CA125, HE4, E-CAD, and IL-6, shows potential in detecting serous ovarian cancer at earlier stages. Additional validation studies using the biomarker combination in ovarian cancer patients are warranted.

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.

Publisher Correction: Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

In the version of this Article originally published, owing to a technical error, the Life Sciences Reporting Summary was not included; this summary is now available.

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...