Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting.

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma.

PURPOSE: Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options. EXPERIMENTAL DESIGN: DNA was extracted from 40 µm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors. Sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries to high coverage for 3,230 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alteration. RESULTS: Mutations in the extracellular domain of ERBB2 were identified in 6 of 15 (40%) of MPUC: S310F (four cases), S310Y (one case), and R157W (one case). All six cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification and Erbb2 overexpression. In contrast, 6 of 64 (9.4%) non-MPUC harbored an ERBB2 alteration, including base substitution (three cases), amplification (two cases), and gene fusion (one case), which is higher than the 2 of 159 (1.3%) protein-changing ERBB2 mutations reported for urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared with non-MPUC is significant both between this series (P < 0.0084) and for all types of urinary tract cancer in COSMIC (P < 0.001). CONCLUSIONS: NGS of MPUC revealed a high incidence of mutation in the extracellular domain of ERBB2, a gene for which there are five approved targeted therapies. NGS can identify genomic alteration, which inform treatment options for the majority of MPUC patients.

Clinical next-generation sequencing successfully applied to fine-needle aspirations of pulmonary and pancreatic neoplasms.

BACKGROUND: Next-generation sequencing was performed on pulmonary and pancreatic fine-needle aspirations (FNAs) and on paired FNAs and resected primary tumors from the same patient. METHODS: DNA was isolated in formalin-fixed, paraffin-embedded cell blocks from 16 pulmonary FNAs, 23 pancreatic FNAs, and 5 resected pancreatic primary tumors. Next-generation sequencing was performed for 4561 exons of 287 cancer-related genes and for 47 introns of 19 genes on indexed, adaptor-ligated, hybridization-captured libraries using a proprietary sequencing system (the Illumina HiSeq 2000). RESULTS: Genomic profiles were generated successfully from 16 of 16 (100%) pulmonary FNAs, which included 14 nonsmall cell lung cancers (NSCLCs) and 2 small cell lung cancers (SCLCs). The NSCLC group included 6 adenocarcinomas, 5 squamous cell carcinomas, and 3 NSCLCs not otherwise specified. Genomic profiles were successfully obtained from 23 of 23 (100%) pancreatic FNAs and from 5 of 5 (100%) matched pancreatic primary tumors, which included 17 ductal adenocarcinomas, 3 mucinous adenocarcinomas, 2 adenocarcinomas NOS, and 1 neuroendocrine tumor. Eighty-one genomic alterations were identified in the 16 pulmonary FNAs (average, 5.1 genomic alterations per patient); and the most common genomic alterations were TP53, RB1, SOX2, PIK3CA, and KRAS. Eighty-seven genomic alterations were identified in the 23 pancreatic tumor FNAs (average, 3.8 genomic alterations per patient); and the most common genomic alterations were KRAS, TP53, CDKN2A/B, SMAD4, and PTEN. Among the pancreatic tumors, there was 100% concordance of 20 genomic alterations that were identified in 5 patient-matched FNA and surgical primary tumor pairs. CONCLUSIONS: The authors were able to perform next-generation sequencing reliably on FNAs of pulmonary and pancreatic tumors, and the genomic alterations discovered correlated well with those identified in matched resected pancreatic tumors.

lincRNAs act in the circuitry controlling pluripotency and differentiation.

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries.

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.

Targeted exon sequencing by in-solution hybrid selection.

This unit describes a protocol for the targeted enrichment of exons from randomly sheared genomic DNA libraries using an in-solution hybrid selection approach for sequencing on an Illumina Genome Analyzer II. The steps for designing and ordering a hybrid selection oligo pool are reviewed, as are critical steps for performing the preparation and hybrid selection of an Illumina paired-end library. Critical parameters, performance metrics, and analysis workflow are discussed.