Asuragen (United States)

AccuScan showed remarkable ultra-low limit of detection with a short turnaround time, low sample requirement and a simple workflow for MRD detection.

Female FMR1 premutation (FMR1 PM) carriers for fragile X syndrome (FXS) are at risk to have a child with FXS based on their CGG repeat size and AGG interruption number. Studies examining this risk in unselected populations of female PM carriers are lacking. This retrospective cohort study analyzed carrier status, CGG repeat length, AGG interruption result, and reproductive risk refinement in a population of female patients who underwent routine carrier screening for FXS. A total of 1536 PM carriers (0.43%) were identified, 95% of whom had between 55 and 90 CGG repeats. A number of 1334 carriers underwent AGG interruption testing. The majority had at least one AGG interruption and received a lower reproductive risk for FXS following AGG interruption testing (89% and 85%, respectively) as compared to their risk calculated based on CGG repeat size alone. The average change in risk across the population following AGG interruption testing was -3.4%, with a range from -50.8% to 48.9%. This article describes the range of CGG repeats and AGG interruptions in an unselected population of female PM carriers and suggests that most carriers would benefit from AGG interruption testing to refine their reproductive risk of having a child with FXS.

Abstract Introduction: Mutation analysis of circulating tumor DNA (ctDNA) in blood-based liquid biopsies provides a minimally invasive approach to detect and monitor disease. Existing next-generation sequencing (NGS) liquid biopsy techniques have laborious and/or inefficient workflows, heuristic error-correction algorithms, and variable performance with clinical tumor-plasma samples. We describe a method that combines a kittable and efficient wet-bench workflow with accurate dry-bench analytics to reduce costs and turnaround time, and is relevant to clinical research and patient testing. Methods: We developed a comprehensive, targeted NGS technology to enable the ultra-sensitive detection of variants from liquid biopsy samples. Input DNA molecules from Seraseq™ ctDNA v2 reference materials (SeraCare) and >50 matched FFPE and plasma samples were uniquely tagged with a random molecular barcode (MBC), amplified in an efficient PCR protocol, and sequenced using a targeted panel covering >500 somatic mutation hotspots. Sequence-ready libraries were prepared from input DNA within 9 hours. Data were analyzed with a robust bioinformatics pipeline tailored to the library chemistry to correct for multiple background errors. Variants were identified with a site-specific model, which effectively eliminated recurring non-biological aberrations that remained despite MBC-facilitated error-suppression. We verified variant calls in plasma by Droplet Digital™ PCR (Bio-Rad). To evaluate the limit of detection, healthy control and mutation-positive plasma admixtures were prepared and sequenced. Results: Input template molecules were efficiently recovered. The median number of unique MBCs across all amplicons was >90% of expectation based on input DNA quantities for both the tumor-associated plasma and Seraseq™ ctDNA v2 material. SNVs and indels were recognized with >90% sensitivity and PPV at 0.5% allele frequency (AF) in the Seraseq™ ctDNA mutation mixes. In both neat plasma samples and plasma admixtures, we correctly identified tumor mutations down to ~0.1% AF while maintaining a low false-positive rate (sensitivity and PPV remained high for AF ≥0.1%). Reference ctDNA material and tumor-associated plasma had analogous template amplification and variant frequency rates. Conclusions: A fast, efficient, and sensitive NGS panel approach was developed and evaluated, and it demonstrated the reliable and specific detection of rare variants in liquid biopsy specimens. The method is able to distinguish a low frequency ctDNA signal from the overwhelming background noise in plasma cell-free DNA using a streamlined workflow and purpose-built bioinformatics pipeline. This technology may provide an easy-to-use, high-performance, and adaptable NGS diagnostic framework for disease detection and therapeutic intervention monitoring. Citation Format: Jessica L. Larson, Liangjing Chen, Lando Ringel, Blake Printy, Farol L. Tomson, Yves Konigshofer, Sarah Statt, Joseph K. Kaplan, Shobha Gokul, Jeffrey Shelton, Gary J. Latham, Brian C. Haynes. A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5574.

Abstract 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

Abstract Introduction: Reliable assessment of cancer-associated RNA markers in lung cancer produced by gene-fusions or exon skipping events by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We evaluated a comprehensive system for targeted RNA-Seq that includes reagents for nucleic acid quantification, library prep, run controls, and companion bioinformatics software. The reproducibility of this system was evaluated in a multi-phase study design at 5 independent laboratories. Methods: Total nucleic acid (TNA) was isolated from formalin-fixed, paraffin-embedded (FFPE) residual non-small cell lung cancer (NSCLC) tumor biopsies and cell-lines (RT-112, H596, HCC78). These TNA isolates were used to prepare a set of 30 test samples, including a dilution series to assess assay sensitivity. A non-template control and kit positive control were also included. The sample set was evaluated using the QuantideX® NGS RNA Lung Cancer Kit RUO (Asuragen) and sequenced on the MiSeq® system (Illumina) at Asuragen and 4 independent laboratories. Analyses were conducted using QuantideX® NGS Reporter RUO (Asuragen), a software suite that includes a FASTQ processing pipeline and incorporates pre-analytical QC information into the fusion-caller algorithm and reporting tool. Results: Laboratories were trained on the assay workflow and companion bioinformatics software in less than two days followed by independent library preparation and sequencing workflows. A total of 266 sample libraries were evaluated from inputs down to <10 ng. A single library was excluded from sequencing due to a failed QC status. Specific targeted fusions (ALK, ROS1, FGFR3) and splice variants (MET exon 14 skipping) were detected in 132 libraries and were concordant at all sites. Designs to detect 3’/5’ expression imbalances reported the presence of gene fusions for ALK and ROS1 in a total of 96 libraries with one aberrant call and one missed call, which occurred in libraries flagged as “at risk” by the interpretive software. Conclusions: The accuracy of this novel targeted NGS assay for RNA fusions and splice variants in NSCLC was demonstrated in a multi-site laboratory evaluation using clinically-relevant specimens and low inputs of TNA. The ability of the panel to detect both common and rare gene fusion transcripts and exon skipping events within an integrated wet- and dry-bench workflow provides a foundation for the reliable detection of oncogenic RNA fusions and aberrant splicing events that can respond to current and emerging targeted therapies. This study highlighted the ease of implementation and consistent performance that can be achieved in different laboratories when the process from sample-to-report is highly integrated. Citation Format: Gary J. Latham, Richard Blidner, Brian C. Haynes, Shobha Gokul, Maria L. Aguirre, Stephen Hyter, Ziyan Y. Pessetto, Maria Curtis, Dan Su, Tom Halsey, Victor Weigman, Patrick Hurban, Andrew K. Godwin, Leon C. van Kempen. Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories [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 1764. doi:10.1158/1538-7445.AM2017-1764

OBJECTIVE: To present a case series of four sisters with similar premutation size Fragile X mental retardation 1 (FMR1) alleles and varying activation ratios for the normal allele (AR) to determine whether AR, in addition to CGG repeat size, could account for their discordant neurological phenotypes. BACKGROUND: Skewed X-inactivation may be responsible for mediating some of the phenotypic variability seen in FMR1 premutation carrier women. These discordant clinical phenotypes may be explained by variation in the combination of CGG repeat size and AR. DESIGN/METHODS: Four sisters with premutation size FMR1 repeats underwent detailed clinical characterization. CGG repeat length was determined by PCR and AR was determined using a newly developed commercial mPCR assay (Asuragen) and confirmed with results from Southern blot with densitometric image analysis. The mPCR assay was found to be simpler and faster, and results correlated well with AR determined based on Southern blot. RESULTS: Sister 1 had the largest CGG expansion (90) and the lowest AR (10[percnt]), with the most severe clinical presentation. Sister 2 had a lower CGG expansion (70) and an AR of 10[percnt], and had milder neurological symptoms. Sister 3 had a CGG expansion of 79, but a slightly higher AR of 15[percnt] and less neurological involvement. Sister 4 had a similar CGG expansion (80), but had the largest AR (40[percnt]) and was the only sister not to be affected by FXTAS or show any neurological signs. CONCLUSION: Women in the same family who have higher AR or lower CGG repeat sizes than their sisters may be less likely to show manifestations of FXTAS. Using AR data could be beneficial to supplement CGG repeat size when counseling premutation carrier women in the clinic if larger studies show similar patterns.

Molecular testing for oncogenic mutations or gene expression in fine-needle aspirations (FNAs) from thyroid nodules with indeterminate cytology identifies a subset of benign or malignant lesions with high predictive value. This study aimed to evaluate a novel diagnostic algorithm combining mutation detection and miRNA expression to improve the diagnostic yield of molecular cytology. Surgical specimens and preoperative FNAs (n = 638) were tested for 17 validated gene alterations using the miRInform Thyroid test and with a 10-miRNA gene expression classifier generating positive (malignant) or negative (benign) results. Cross-sectional sampling of thyroid nodules with atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) or follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) cytology (n = 109) was conducted at 12 endocrinology centers across the United States. Qualitative molecular results were compared with surgical histopathology to determine diagnostic performance and model clinical effect. Mutations were detected in 69% of nodules with malignant outcome. Among mutation-negative specimens, miRNA testing correctly identified 64% of malignant cases and 98% of benign cases. The diagnostic sensitivity and specificity of the combined algorithm was 89% (95% confidence interval [CI], 73-97%) and 85% (95% CI, 75-92%), respectively. At 32% cancer prevalence, 61% of the molecular results were benign with a negative predictive value of 94% (95% CI, 85-98%). Independently of variations in cancer prevalence, the test increased the yield of true benign results by 65% relative to mRNA-based gene expression classification and decreased the rate of avoidable diagnostic surgeries by 69%. Multiplatform testing for DNA, mRNA, and miRNA can accurately classify benign and malignant thyroid nodules, increase the diagnostic yield of molecular cytology, and further improve the preoperative risk-based management of benign nodules with AUS/FLUS or FN/SFN cytology.