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We developed and characterized a next-generation sequencing (NGS) technology for streamlined analysis of DNA and RNA using low-input, low-quality cancer specimens. A single-workflow, targeted NGS panel for non–small cell lung cancer (NSCLC) was designed covering 135 RNA and 55 DNA disease-relevant targets. This multiomic panel was used to assess 219 formalin-fixed paraffin-embedded NSCLC surgical resections and core needle biopsies. Mutations and expression phenotypes were identified consistent with previous large-scale genomic studies, including mutually exclusive DNA and RNA oncogenic driver events. Evaluation of a second cohort of low cell count fine-needle aspirate smears from the BATTLE-2 trial yielded 97% agreement with an independent, validated NGS panel that was used with matched surgical specimens. Collectively, our data indicate that broad, clinically actionable insights that previously required independent assays, workflows, and analyses to assess both DNA and RNA can be conjoined in a first-tier, highly multiplexed NGS test, thereby providing faster, simpler, and more economical results.

Newborn screening is designed for presymptomatic identification of serious conditions with effective early interventions. Clinical laboratories must perform prospective pilot studies to ensure that the analytical performance and workflow for a given screening test are appropriate. We assessed the potential to screen newborns for fragile X syndrome, a monogenic neurodevelopmental disorder, by establishing a customized, high-throughput PCR and analysis software system designed to detect fragile X mental retardation 1 gene repeat expansions from dried blood spots (DBSs). Assay precision, accuracy, sensitivity, and specificity were characterized across the categorical range of repeat expansions. The assay consistently resolved genotypes within three CGG repeats of reference values up to at least 137 repeats and within six repeats for larger expansions up to 200 repeats. Accuracy testing results were concordant with reference results. Full and premutation alleles were detected from subnanogram DNA inputs eluted from DBSs and from mixtures with down to 1% relative abundance of the respective expansion. Analysis of 963 deidentified newborn DBS samples identified 957 normal and 6 premutation specimens, consistent with previously published prevalence estimates. These studies demonstrate that the assay system can support high-throughput newborn screening programs.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench ("wet bench") and data analyses conducted using bioinformatics pipelines ("dry bench"). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible "tag" PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills over 600,000 people annually. Here, we report integrated computational and experimental investigations of the role and mechanisms of transcriptional regulation in cryptococcal infection. Major cryptococcal virulence traits include melanin production and the development of a large polysaccharide capsule upon host entry; shed capsule polysaccharides also impair host defenses. We found that both transcription and translation are required for capsule growth and that Usv101 is a master regulator of pathogenesis, regulating melanin production, capsule growth, and capsule shedding. It does this by directly regulating genes encoding glycoactive enzymes and genes encoding three other transcription factors that are essential for capsule growth: GAT201, RIM101, and SP1. Murine infection with cryptococci lacking Usv101 significantly alters the kinetics and pathogenesis of disease, with extended survival and, unexpectedly, death by pneumonia rather than meningitis. Our approaches and findings will inform studies of other pathogenic microbes., IMPORTANCE Cryptococcus neoformans causes fatal meningitis in immunocompromised individuals, mainly HIV positive, killing over 600,000 each year. A unique feature of this yeast, which makes it particularly virulent, is its polysaccharide capsule; this structure impedes host efforts to combat infection. Capsule size and structure respond to environmental conditions, such as those encountered in an infected host. We have combined computational and experimental tools to elucidate capsule regulation, which we show primarily occurs at the transcriptional level. We also demonstrate that loss of a novel transcription factor alters virulence factor expression and host cell interactions, changing the lethal condition from meningitis to pneumonia with an exacerbated host response. We further demonstrate the relevant targets of regulation and kinetically map key regulatory and host interactions. Our work elucidates mechanisms of capsule regulation, provides methods and resources to the research community, and demonstrates an altered pathogenic outcome that resembles some human conditions.

DICER1 is essential for the generation of mature miRNAs and other short noncoding RNAs. Several lines of investigation implicate DICER1 as a tumor suppressor. Reduced DICER1 levels and changes in miRNA abundance have been associated with aggressive tumor phenotypes. The global effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. We used short hairpin RNA to stably knock down DICER1 in endometrial cancer cell lines to begin to determine how reduced DICER1 activity contributes to tumor phenotypes. DICER1 knockdown did not affect cell proliferation but caused enhanced cell migration and growth in soft agar. miRNA and mRNA profiling in KLE cells revealed overall decreases in miRNA levels and changes in the relative abundance of many mRNAs. One of the most striking changes in mRNA levels was the upregulation of IFN-stimulated genes (ISG), the majority of which lack known miRNA target sequences. IFNβ, a key upstream regulator of the IFN response, was significantly increased in DICER1 knockdowns in the AN3CA, Ishikawa, and KLE endometrial cancer cell lines and in the normal endometrial cell line EM-E6/E7/TERT. IFNβ secreted in media from KLE and EM-E6/E7/TERT shDcr cells was sufficient to activate an IFN response in HT29 cells. The reduced miRNA processing in DICER1 knockdowns was associated with increases in pre-miRNAs in the cytoplasm. Our findings suggest that elevated pre-miRNA levels trigger the IFN response to double-stranded RNA. We thus report a novel effect of reduced DICER1 function in cancer cells. Mol Cancer Res; 10(3); 316–25. ©2012 AACR.

Introduction: HER2-positive breast cancers comprise 20-30% by subtype and represent the second poorest prognosis. In addition to HER2 signaling activation via protein overexpression or gene amplification (measured by IHC or ISH), somatic mutations in the ERBB2 (HER2) gene have been identified in 2-4% of breast cancers and reported to drive HER2 pathway signaling and tumorigenesis. Irreversible HER2 tyrosine kinase inhibitors such as neratinib have been shown to potently inhibit breast tumors harboring somatic HER2 activating mutations and are under active clinical investigation. A targeted molecular characterization strategy is thus needed to support clinical trial research. Here, we present an integrated NGS system that identifies multiple classes of ERBB2aberrations, including single nucleotide variants, insertion/deletion mutations, and copy number variations (CNVs). Methods: Genomic DNA was isolated from FFPE human tumor tissues or FFPE-embedded engineered cell lines expressing ERBB2 mutations. Pre-analytical sample QC was performed by a novel qPCR assay that quantifies amplifiable DNA templates suitable for library preparation. NGS libraries were prepared using the QuantideX® NGS ERBB2 Mutations assay, with two-pool PCR-based enrichment to detect multiple types of ERBB2 genomic alterations. Libraries were sequenced (Illumina MiSeq®) and analyzed using QuantideX® Reporter software, directly incorporating the pre-analytical QC data from each DNA sample to improve variant call accuracy. Results: The QuantideX® NGS ERBB2 Mutations system targets 28% of ERBB2 coding bases covering exons 8 and 17 - 24, including codons 713-989 of the tyrosine kinase domain. The assay also detects focal, whole-arm and polysomy-induced HER2 amplification using sentinel flanking amplicons, and amplicons covering CEN17 and copy number-neutral chromosomal regions identified in the TCGA breast cancer cohort. The assay was analytically validated in a CLIA-laboratory which included an analysis of 6 mixtures and titrations of synthesized DNA bearing 31 ERBB2 mutations, 17 engineered cell lines and FFPE cell-line blocks, and 12 surgically-resected FFPE carcinomas. A multi-day/operator study revealed 100% sensitivity and PPV for detection of ERBB2 SNVs, indels and complex variants down to 5% mutation positive in wild-type DNA. The NGS system was also used to profile ERBB2 variants in 473 FFPE human tumor tissues, including 280 breast carcinomas. Mutation-positive samples were confirmed by an orthogonal NGS method. A subset of 46 FFPE tissue samples were assessed for CNVs by NGS and compared with matched droplet digital PCR (ddPCR) analysis. A correlation of R2 > 0.91 between NGS and ddPCR underscores the potential of the assay to reveal both ERBB2 mutations and copy number changes. Conclusions: The QuantideX® NGS ERBB2system integrates optimized wet- and dry-bench elements to achieve broad and accurate detection of ERBB2 mutations and copy number variants from challenging FFPE specimens. The technology has the potential to advance breast cancer clinical research and aid in the selection of patients for clinical trials, particularly those that assess HER2-directed therapies such as afatinib, lapatinib, and neratinib. Citation Format: Zhu H, Zeigler R, Bridger S, Larson JL, Kincaid A, Cardwell R, Haynes BC, Andruss BF, Latham GJ. A fit-for-purpose NGS system that reports ERBB2 (HER2) mutations and copy number variants for clinical trials research and drug development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-10.

Introduction: Ovarian cancer is a leading cause of cancer related death in women. A comprehensive genomic characterization of platinum-sensitive tumors is required to further refine the definition of molecular subtypes and identify targeted therapies for this patient population. To this end we have performed a large-scale genomic and transcriptomic characterization of 348 primary FFPE tissues from a cohort of platinum-sensitive HGSOC patients collected from multiple clinical sites. Methods: Macrodissection of FFPE resected tumor slides or sectioned blocks was performed to enrich for tumor content. RNA and DNA were each isolated from 2x 5µm sections of FFPE material. RNA expression and gene fusions were profiled by whole transcriptome RNA-Seq. DNA variants were analyzed by the AmpliSeq™ Cancer Hotspot Panel (Thermo Fisher). A subset of tumor and matched germline (PBL) specimens (N=181) were assessed for TP53 mutations by the QuantideX® NGS TP53 Assay (Asuragen, Inc.). CNV analysis of FFPE tumor DNA was performed using the OncoScan® FFPE Assay Kit (Affymetrix) and microsatellite instability was characterized by comparing matched tumor and PBL specimens with capillary electrophoresis using the Bethesda panel. Germline BRCA1/2 mutation status was determined by profiling PBL specimens with a custom AmpliSeq™ NGS panel. Results: The spectrum of DNA mutations and CNVs was consistent with other HGSOC cohorts with mutations in TP53 present in the majority of specimens (87% for specimens with full exon coverage of TP53). Germline mutations in BRCA1 and BRCA2 were identified at 10.3% and 6.8% respectively. Analysis of recurrent whole chromosomal arm gain and loss displayed a striking agreement with the TCGA HGSOC cohort. Unsupervised analysis of the RNA-Seq expression data through non-negative matrix factorization revealed 4 distinct transcriptional subtypes, corresponding to the 4 established CLOVAR subtypes: differentiated, immunoreactive, mesenchymal and proliferative. Patients classified as mesenchymal had the poorest prognosis. Further integrative analysis identified additional associations between the CLOVAR subtypes and other molecular indications. Conclusions: Through integrative genomic analyses on this challenging set of clinical specimens we have reproduced and refined the molecular subtypes of platinum-sensitive HGSOC and highlighted alterations that may lead to improved diagnostic and precision medicine strategies. In contrast to previous large-scale molecular characterization efforts such as TCGA where fresh-frozen tissues were collected under highly controlled settings, this study is based on molecular profiling of FFPE tissues collected at dozens of clinical sites. Thus, our study serves as a model for future molecular characterization efforts of FFPE specimens collected in real-world clinical settings. Citation Format: Brian C. Haynes, Marie E. Fahey, Darcy Myers, Diane Ilsley, Gary J. Latham, Elizabeth B. Somers, Nicholas C. Nicolaides, Charles Schweizer, Daniel J. O’Shannessy. Comprehensive genomic characterization of a large cohort of platinum-sensitive, high-grade serous ovarian cancer (HGSOC) FFPE specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1743. doi:10.1158/1538-7445.AM2017-1743

Introduction: The promise of precision medicine relies on the identification of DNA and RNA markers that can individualize patient management. Methods such as next-generation sequencing (NGS) can deduce DNA or RNA sequences, but both types of nucleic acid have not been efficiently and effectively combined into a single NGS workflow. We describe a comprehensive methodology for targeted clinical NGS that reports DNA and RNA variants, provides a streamlined workflow, and accommodates low-input total nucleic acid (TNA) from challenging clinical specimens. Methods: Sample QC was performed using a novel qPCR assay that quantifies discrete populations of amplifiable DNA and RNA from TNA material. PCR-based target enrichment was performed using QuantideX® NGS reagents (Asuragen) and sequenced on the MiSeq® System (Illumina). Bioinformatic analyses were conducted using QuantideX® Reporter (Asuragen), a software suite that directly incorporates pre-analytical QC information into the variant calling. Results: Targeted DNA- and RNA-seq panels were developed that query 54 lung cancer DNA targets and 135 RNA targets, including >100 gene fusions and mRNA expression markers associated with clinical actionability. Gene-specific primers were formulated as multiplex PCR or RT-PCR reactions to independently interrogate DNA or RNA variants, respectively, from TNA or combined into a 189-plex RT-PCR to report multi-categorical nucleic acid variants. Integration of the bioinformatics pipeline and variant caller with wet-lab QC results enhanced mutation call sensitivity at The NGS panels were assessed with cell-line and synthetic controls and a residual clinical cohort of 97 NSCLC FFPE specimens. Mutations were accurately detected from as few as 5-10 DNA templates and down to Conclusions: QuantideX targeted NGS chemistries can unify the analysis of DNA and RNA markers associated with lung cancer and report SNVs, indels, fusions, and aberrantly expressed mRNA transcripts from a single NGS run. This novel technology offers reliable, accurate and comprehensive molecular characterizations of challenging tumor specimens by integrating wet-bench and dry-bench methods and improving the efficacy of routine laboratory testing. Citation Format: Gary J. Latham, Julie Krosting, Michael Dodge, Robert Zeigler, Liangjing Chen, Jason Plyler, Shobha Gokul, Junya Fujimoto, Vassiliki Papadimitrakopoulou, Ignacio Wistuba, Richard Blidner, Brian Haynes. A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1389.

DICER1 is required for generation of mature microRNAs (miRNAs) and other short noncoding RNAs. Reduced DICER1 levels are associated with adverse outcomes in a variety of cancers and multiple lines of evidence suggest DICER1 is a tumor suppressor. The effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. To determine how reduced DICER1 levels contribute to tumor phenotypes we used shRNA to stably knock down DICER1 in endometrial cancer cell lines for extended periods (up to 25 passages). Reduced DICER1 levels had no effect on cell proliferation but were associated with enhanced cell migration and increased growth in soft agar. MiRNA profiling for the KLE cancer cell line DICER1 knockdowns (two different DICER1 shRNA constructs) revealed that miRNA levels were decreased overall. Not surprisingly, many mRNA transcripts were upregulated in the DICER1 knockdowns. A striking number of interferon stimulated genes (ISGs) showed upregulation. Upregulation of six ISG transcripts was validated by qRT-PCR in three independent knockdowns of DICER1 in KLE cells. IFNα, a key upstream regulator of the interferon response, was significantly increased in DICER1 knockdowns (KLE and additional endometrial cancer cell lines). IFNα protein secreted in media of DICER1 knockdown cells activated an interferon response in HT29 colon carcinoma cells. The majority of ISGs with increased transcript levels lacked miRNA target sequences and as such it seemed unlikely that the increase in expression levels was secondary to reduced miRNA levels. We tested the hypothesis that defects in miRNA processing could trigger an interferon response. We showed that DICER1 knockdowns in which miRNA processing is reduced have increased levels of pre-microRNAs in the cytoplasm. Pre-microRNAs are double-stranded molecules and long enough (60-100 nt) to activate the dsRNA sensors in the cytoplasm, such as Mda5/IFIH1, that typically sense viral dsRNA (> 30 nt) and stimulate the interferon response. Knockdown of DROSHA, necessary for processing of pri-miRNAs to pre-miRNAs, did not result in accumulation of pre-miRNAs and did not cause an interferon response. Our findings suggest elevated pre-microRNA levels trigger the interferon response to double stranded RNA (incompletely processed miRNA and other species). Rescue experiments (restoring DICER1 function) are currently underway to shed additional light on how reduced double-stranded RNA processing might elicit an interferon response. Work is ongoing to determine the relationship between the observed interferon response and the migration and soft agar phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2312. doi:1538-7445.AM2012-2312

DICER1 is required for the generation of mature microRNAs (22-24 nt miRNAs) and other short noncoding RNAs. Several lines of evidence point to DICER1's role as a tumor suppressor. In particular, reduced DICER1 levels are associated with adverse outcomes in a variety of human and mouse cancers. To date the effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. Furthermore, DICER1 may have non-canonical (unrelated to miRNA activity) functions in tumorigenesis. In model organisms, small RNAs initiate transcriptional silencing. DICER1 may have a similar role in transcriptional silencing and DNA methylation in mammalian cells. To begin to determine how reduced DICER1 levels contribute to tumor phenotypes we used shRNA to stably knock down DICER1 in endometrial cancer cell lines for extended periods (up to 25 passages). Reduced DICER1 levels (measured by Western blot) had no effect on cell proliferation but were associated with enhanced cell migration. We performed microRNA, mRNA, and DNA methylation profiling experiments in the KLE endometrial cancer cell line. MiRNA levels were decreased overall and the fold reductions for individual miRNAs were highly correlated in two different DICER1 shRNA knockdowns. RNAseq analysis (Illumina) revealed a 4-fold increase in the TARBP2 (TRBP) and MOV-10 transcripts. TARBP2 and MOV-10 are integral components of the DICER1 complex and the observed upregulation of their transcripts could reflect selection to stabilize DICER1 in the knockdowns. Furthermore, miR-103 and miR-107, which target DICER1 mRNA, were downregulated in the knockdown cells, possibility reflecting selection to maintain DICER1 activity. Consistent with decreases in miRNA levels, many mRNA transcripts were upregulated with DICER1 knockdown (average 1.1 fold increase across 8077 informative transcripts). Most striking was the number of interferon responsive genes that showed upregulation. Among the 8077 informative transcripts, 28 were classified as interferon responsive, and for those the average and median fold increase was 3.1 and 2.1 respectively (range 0.9 – 12.9). The majority of those interferon responsive genes with increased transcript levels lack miRNA target sequences. Previous work has shown that shRNAs do not induce a general antiviral interferon response. We are currently performing studies to confirm that the effect observed is DICER1 dependent. Technical and biologic validations for those interferon responsive genes that show upregulation based on RNAseq are underway. Increases in IFI6 and IFI44L transcript levels have been confirmed by qPCR. We hypothesize a transcription-based mechanism of DICER1 control of interferon responsive transcripts and are attempting to determine if there is such an effect and whether it is a direct or indirect effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1173. doi:10.1158/1538-7445.AM2011-1173