Nucleic Acid Input Research Articles

Abstract 1330: Scalable single-day workflows for variant calling with the Genexus Integrated Sequencer

Abstract The purpose of this study is to test the speed, scalability, and flexibility of variant detection with the Genexus Integrated Sequencer. This research use only instrument uses nucleic acid input and executes the steps of library preparation, sequencing, analysis, and variant calling. Taking advantage of the instrument's flexibility in sample plexy, the authors used the Oncomine™ Comprehensive Assay v3 and commercially available control samples to detect SNPs, InDels, and fusions reliably at up to 6-plex paired DNA and RNA samples. The instrument can accommodate 1, 2, 4, or 6 paired DNA and RNA samples when using 1, 2, 3, or 4 lanes of the GX5 chip. Regardless of the number of samples analyzed, the system achieved at least 98% sensitivity, 98% positive predictive value, 95% agreement to known truth for de novo variants, and 98% agreement to known truth for hotspot variants in the Acrometrix™ Oncomine Hotspot Control. Also regardless of sample plexy, the system achieved at least 97% agreement to known truth for Fusions within the SeraSeq® Fusion RNA Mix v3. The flexible workflows allow for fast turn-around times of as little as 18 hours for a single sample pair and less than 30 hours for 6 paired DNA and RNA samples. Stability of system initialization is an important feature for flexible use of an NGS system, and read length is a limiting performance metric for evaluating stability. In order to test the Genexus system's read length and stability features the Oncomine™ TCR Beta-LR Assay GX was used. Instruments were initialized with sequencing consumables and tested for performance on day 1 and day 15. There was no statistically significant difference in total reads (3.5 ± 1.0 M at day 1 vs. 3.1 ± 1.0 M at day 15), the fraction of reads that are low quality reads (13% ± 2% vs. 14% ± 2%), or mean read length (301 ± 6 bases vs. 296 ± 6 bases) between the two time points. In conclusion, the Genexus integrated sequencer is a robust NGS system that facilitates rapid turn-around time, flexibility in sample plexy, and stability in system consumables use. Citation Format: Jason Gioia, Iris R. Casuga, Frances Chan, James Commins, David Conners, Marla Kratzer, Collyn Seeger, David Yaccarino. Scalable single-day workflows for variant calling with the Genexus Integrated Sequencer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1330.

Abstract 1335: Analytical evaluation of a targeted solid tumor next-generation sequencing assay

Abstract With the growing interest in personalized medicine for cancer patients, next generation sequencing (NGS) variant screening assays are becoming a powerful clinical tool for tumor classification. The AmpliSeqTM for Illumina Focus Panel is a targeted sequencing approach that interrogate 52 of the most common solid tumor genes for single nucleotide variants (SNVs), insertion/deletions (indels), and copy number variants (CNVs) in DNA and gene fusions in RNA concurrently. To evaluate the performance of the Focus assay, an analytical performance study was conducted. Using an automated workflow with high quality DNA reference samples, both intra-assay and inter-assay experiments resulted in high reproducibility (R2 >0.95) for detecting expected variants at various frequencies. Similarly, using high quality RNA reference samples, 100% of all expected fusions were detected in all experiments, demonstrating accuracy and precision of the Focus panel assay with high quality samples. To evaluate assay sensitivity, limit of detection (LOD) studies were carried out by titrating the nucleic acid input into the assay. Using a 5% variant allele frequency threshold (empirically established in the analysis pipeline), the assay detected all expected SNVs and indels across sample across sample input amounts. Using extracted DNA from a human cell line model (HL-60) with a confirmed MYC gene gain, results showed reliable MYC gain detection down to 4 copies in a diploid background. Similarly, extracted RNA from a confirmed EML4-ALK fusion positive cell line (H2228) diluted into background RNA showed 100% positive fusion calling down to 250pg or 133 copies. Finally, to understand the assay performance on clinically-relevant FFPE samples, somatic variant detection in 8 human cell lines and 68 total FFPE samples was evaluated. For FFPE DNA samples, the libraries averaged >90% on-target alignment and coverage uniformity with >3000x mean coverage across all samples. While FFPE RNA showed lower alignment for poor quality samples, the assay showed overall high positive and negative predictive value for variant and fusion calling. In summary, the AmpliSeqTM for Illumina Focus panel is an automation-compatible small panel NGS assay with robust performance to assess somatic variants in solid tumors. Citation Format: Jeff H. Tsai, Valerie Montel, Nathalie Mouttham. Analytical evaluation of a targeted solid tumor next-generation sequencing assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1335.

Analytical validation of a novel targeted next-generation sequencing assay for mutation detection in thyroid nodule aspirates and tissue.

The identification of somatic mutations in cancer specimens enables detection of molecular markers for personalized treatment. We recently developed a novel molecular assay and evaluated its clinical performance as an ancillary molecular method for indeterminate thyroid nodule cytology. Herein we describe the analytical validation of the novel targeted next-generation sequencing (NGS) assay in thyroid samples from different sources. We present validation data of a novel NGS-based panel on 463 thyroid samples, including 310 fine-needle aspiration (FNA) specimens from different sources (FNA collected in preservative solution, liquid-based, and stained smears), 10 fresh frozen, and 143 formalin-fixed paraffin-embedded (FFPE) thyroid tissue specimens. Sequencing performance in the different samples was evaluated along with reproducibility, repeatability, minimum nucleic acid input to detect variants, and analytical sensitivity of the assay. All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228 × (in liquid-based FNA) to 3661 × (in FNA stained smears), and coverage uniformity ranging from 86% (in FFPE) to 95% (in FNA collected in preservative solution), with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2-5% mutant allele frequency. This validation study of a novel NGS-based assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types, including FNA from different sources and FF and FFPE thyroid samples, thus providing a robust and reliable assay to genotype thyroid nodules, which may improve thyroid cancer diagnosis and care.

Abstract B46: Development of a fully integrated sample-to-report system for a pan-cancer application

Abstract Introduction: We have developed a new sample-to-report solution for testing solid tissue cancers using the Ion Torrent System and Software. The assay is designed for research applications from either formalin-fixed, paraffin-embedded (FFPE) solid tumor samples or cell-free total nucleic acid (cfTNA) from liquid biopsy. The new sequencer is a fully automated system requiring minimal hands-on time and touch points for a user to go from nucleic acid to variant calls for somatic variant testing across multiple cancer types. Methods: The assay is a new amplicon-based assay targeting specific somatic variants in 50 genes with coverage for multiple cancer types. The assay uses AmpliSeq HD chemistry capable of distinguishing true sample biologic variants from errors generated during library preparation, templating, and sequencing through incorporation of molecular tags during target amplification. The assay is optimized for use with the Ion Torrent System. With minimal hands-on time (~15 minutes), the user prepares the system with prefilled reagents strips to start a fully automated run that includes library prep, templating, sequencing, variant calling, and a final report if desired. Results: The assay is designed to detect somatic variants in 50 unique genes, where 45 of these genes include substitutions, deletions, and insertions. 14 genes are targeted for copy number variation, and 19 genes are drivers of gene fusions and alternate splice forms. The content has been selected based on published accounts of target actionability and prevalence across multiple cancer types. An example of run setup is shared along with timing (~30min) to input sample information using the Torrent Suite Software. Data are shown from a multiplexed sequencing run using FFPE DNA (10 ng) and RNA (10ng) as input. The automated FFPE run on the Ion Torrent System completed in about 30 hours. Data are also presented from a sequencing run that included multiple liquid biopsy libraries utilizing cell-free total nucleic acid (cfTNA) extracted from plasma. In this run mode, variant calling is optimized for highly sensitive and specific variant calling below 1% allelic frequency for all targeted variant types assay including CNVs, fusions, and alternative splice forms and was accomplished in less than 24 hours. Conclusion: The assay and system enable detection of key oncology variants in 50 genes using both solid tissue and liquid biopsy samples as input. This fully automated solution for oncology research generates variant calls from nucleic acid input in a single 24-hour period with minimal hands-on time and touch-points from the user. This system is not yet released but is in development. Citation Format: Kelli Bramlett, Ru Cao, Jeff Schageman, Yanchun Li, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Rasika Sunnadeniya, Oana Lungu, Varun Bagai, Desiree Martin, Khalid Hanif, Jian Gu. Development of a fully integrated sample-to-report system for a pan-cancer application [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B46.

NTRK testing: First results of the QuiP-EQA scheme and a comprehensive map of NTRK fusion variants and their diagnostic coverage by targeted RNA-based NGS assays.

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA-/DNA-based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH-based and 18 in the NGS-based round robin test, the latter additionally subdivided into low-input and high-input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin-fixed and paraffin-embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS-based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH- and tNGS-based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA-based tNGS panels.

Preoperative detection of malignancy in fine-needle aspiration cytology (FNAC) smears with indeterminate cytology (Bethesda III, IV) by a combined molecular classifier

AimsAnalysis of molecular markers in addition to cytological analysis of fine-needle aspiration (FNA) samples is a promising way to improve the preoperative diagnosis of thyroid nodules. Previously, we have developed...

Detection of Low Frequency T-Cell Clones Via a Next Generation Sequencing (NGS) Based Immune-Oncology Research Assay

Introduction: The ability to detect and quantitate low frequency T-cell clones enables numerous hematology/oncology research applications, including identification and assessment of biomarkers associated minimal residual disease (MRD). Rare clone detection via NGS requires highly efficient library preparation and accurate sequencing methodologies, because single nucleotide substitution sequencing errors mimic the natural variation in the T-cell repertoire, resulting in detection of artifactual low frequency clones. Here we present an experimental framework and corresponding performance of rare clone detection utilizing the OncomineTM TCR Beta short read (TCRb-SR) assay, using Ampliseq-based library preparation targeting the highly variable CDR3 region of TCRb using either DNA or RNA as input, with sample-to-result in 2 days. Methods: To evaluate detection sensitivity of the TCRB-SR assay, we utilized Jurkat cell line DNA and RNA because the presence of a single T cell clone enables precise control of dilution studies. Commercially procured Jurkat gDNA or RNA was spiked into peripheral blood leukocyte gDNA or RNA from 10-1 to 10-6 absolute clone frequency to create specimens with a known T-cell clone at frequencies commonly observed in MRD research applications. Peripheral blood leukocyte gDNA or RNA was used as the background for spike in studies due to its high T-Cell diversity. Six to twenty technical replicates were analyzed per dilution point, with DNA inputs ranging from 100ng to 1ug and RNA inputs ranging from 25ng to 100ng to evaluate the minimum detectable clone frequency as a function of nucleic acid input. Libraries were prepared following the TCRB-SR manufacturer's instructions for both DNA and RNA, followed by templating and sequencing using Ion Chef and S5 systems. Data processing was performed in Torrent Suite software (v5.10) followed by read alignment to the IMGT database of variable, diversity, and joining genes using Ion Reporter software (v5.10). For the 10-6 target frequency with gDNA as the input, four 1ug libraries were combined for analysis in Ion Reporter. Analytical sensitivity was calculated at each target clone frequency by detection of the Jurkat clone as defined by V-gene, Joining gene, and CDR3 nucleotide sequence. Results: Detection sensitivity was dependent on the amount gDNA or RNA input. For gDNA inputs, we observed 100% sensitivity at 10-3 with 100ng input, 100% sensitivity at 10-4 with 250ng input, 95% sensitivity at 10-5 with 1ug input, and 100% sensitivity at 10-6 with 4ug input. For RNA inputs, we observed 100% sensitivity at 10-5 with 25ng input and 100% sensitivity at 10-6 with 100ng input. In addition, we observe a strong linearity of observed clone frequencies at each dilution level, with an r-squared of 0.97. Conclusions: Here we demonstrate the ability to detect T-cell clones down to 10-6 from gDNA or RNA inputs with high sensitivity and linearity utilizing the OncomineTM TCR Beta short read assay. We present data demonstrating detection of clones with absolute frequencies of 10-6 utilizing 4ug gDNA input or 100ng RNA input, highlighting strong performance at nucleic acid input levels typically seen in clinical research samples. Taken together, we show feasibility for rare clone detection in either gDNA or RNA enabling research and development for T-cell minimal residual disease applications. For research use only, not for use in diagnostic procedures. Disclosures Williamson: Thermo Fisher Scientific: Employment. Looney:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Sidhu:Thermo Fisher Scientific: Employment. Solano:Thermo Fisher Scientific: Employment. Salazar:Thermo Fisher Scientific: Employment. Tian:Thermo Fisher Scientific: Employment. Ramsamooj:Thermo Fisher Scientific: Consultancy.

P1.03-11 Molecular Testing of Small Bronchoscopy Specimens Using NanoString Technology

Molecular testing for driver variants in oncogenes is crucial for NSCLC management to predict response to targeted therapy. In the majority of cases, NSCLC is diagnosed by trans-thoracic needle aspiration or more commonly bronchoscopy techniques resulting in small diagnostic tissue biopsies or cytological samples. As such specimens may be inadequate for molecular testing, we tested the accuracy of a novel digital molecular barcoding assay to detect actionable mutations in a single-centre cohort. A consecutive cohort of 46 specimens (19 endobronchial biopsy, EnBx; 18 transbronchial biopsy, TBBx; seven bronchoalveolar lavage, BAL; and two transbronchial needle aspirate, TBNA) were obtained ancillary to primary diagnostic specimens from 36 patients undergoing EBUS-guided bronchoscopy at The Prince Charles Hospital. Specimens containing at least 5ng DNA after standard column-based extraction methods were analysed using the NanoString SNV Solid Tumour Panel for testing of 104 somatic variants across 25 genes of clinical significance. NanoString variants calls were compared with routine clinical testing results from the primary diagnostic sample. Agreement analyses for variants common to both methods revealed the positive, negative and overall percentage agreement (PPA, NPA, OPA). One discordant case was validated using droplet digital PCR. Using NanoString, molecular analysis was feasible for 60.1% (28/46) of specimens. At least one variant was identified in 8/28 (28.6%) cases (Table 1). Two (7.1%) cases harboured dual mutations. KRAS mutations were detected in six (21.4%) cases, and EGFR in two (7.1%). Two patients would be eligible for targeted therapy. Agreement analysis for the two methods revealed PPA, NPA and OPA of 100%, 88.9% and 92.3%. In one discordant case, NanoString identified a KRAS G12C mutation and was confirmed by ddPCR with a mutant allele frequency of 5.5%. The mean time for reporting clinical mutation test results was 19.6 days. Of the 18 excluded cases with insufficient DNA, five had routine testing results for comparison however 3/5 cases cited insufficient DNA for reliable EGFR testing.Table 1Clinical and molecular characteristics of bronchoscopy samples used for molecular testingClinical molecular testingNanoStringConcordanceCase no.Sample typeHistological classificationDNA yield (μg)Mutation testing result (MAF)Mutation testing methodTAT to result (days)SNV panel resultAgreement (Yes/No)1BALNo evidence of malignancy2.81Not performedN/AN/AWTNCA2TBNAAC4.39Not performedN/AN/AWTNCA3TBBxSCC0.39Not performedN/AN/AKRAS G12RNCA4TBBxAC1.82EGFR exon 19 (L747_P753>S) del (24%)NGS TruSight21EGFR exon 19 (L747_P753>S) delYes5TBBxNo evidence of malignancy1.24Not performedN/AN/AWTNCA6TBBxNo evidence of malignancy0.62Not performedN/AN/AWTNCA7EnBxAC1.41WT for EGFR, KRAS, NRAS, BRAFNGS TruSight13WTYes8EnBxSCC0.57Not performedN/AN/AWTNCA9TBBxSCC0.53Not performedN/AN/AWTNCA10TBBxAC0.51WT for EGFR, KRAS, NRAS, BRAFNGS TruSight20KRAS G12CNo*11TBBxAC0.31EGFR L858R (12%), EGFR T790M (6%)NGS TruSight13EGFR L858R, EGFR T790MYes12TBBxNo evidence of malignancy0.69Not performedN/AN/AWTNCA13TBBxNSCLC4.26WT for EGFRcastPCR48WTYes14EnBxAC1.37KRAS G12C (10%)NGS TruSight23KRAS G12CYes15EnBxNSCLC19.6KRAS G12A (25%)NGS TruSight20KRAS G12AYes16EnBxCarcinoid1.16Not performedN/AN/AWTNCA17TBBxSCC5.4Not performedN/AN/AWTNCA18EnBxAC4.03WT for EGFR, KRAS, NRAS, BRAFNGS TruSight19WTYes19EnBxSCC2.12Not performedN/AN/AWTNCA20TBBxAC3.85WT for EGFRcastPCR14KRAS G12C, NRAS Q61KNCA21EnBxSCC6.4Not performedN/AN/AWTNCA22TBBxAC1.63WT for EGFRcastPCR20KRAS G12CNCA23EnBxAC0.234WT for EGFR, KRAS, NRAS, BRAFNGS TruSight16WTYes24BALAC0.871BRAF G466V (12%)NGS TruSight24WTYes25TBBxAC0.0918BRAF G466V (12%)NGS TruSightN/AWTYes26TBBxAC0.893WT for EGFRcastPCR13WTYes27TBBxSCC0.66Not performedN/AN/AWTNCA28EnBxAC4.4WT for EGFRcastPCR10WTYesMean2.5819.6 Open table in a new tab The performance of the NanoString platform for SNV characterisation was highly concordant with alternate clinical testing methods for those with sufficient DNA. Advantages of NanoString include its multiplex capacity, high sensitivity, low nucleic acid input, reduced turn-around time (<24hr) compared to alternate testing methods. The NanoString platform is a robust method for identification of actionable variants in NSCLC where at least 5ng of DNA is available.

Coupling of DNA Circuit and Templated Reactions for Quadratic Amplification and Release of Functional Molecules.

DNA-based circuitry empowers logic gated operations and amplifications but is restricted to nucleic acid output. Templated reactions enable the translation of nucleic acid cues into diverse small-molecule outputs but are more limited in their amplification. Herein, we demonstrate the coupling of a DNA circuit to templated reactions in order to achieve high levels of amplification in the output of small molecules, in response to nucleic acid input. We demonstrate that the coupling of the DNA circuit to templated reactions allows for the detection of the fM concentration of analyte and can respond with the release of a cytotoxic drug.

PATH-26. NEURO-ONCOLOGY NEXT-GENERATION SEQUENCING 219-GENE PANEL FOR COMPREHENSIVE CLINICAL TESTING

Molecular parameters were incorporated in the WHO classification of central nervous system (CNS) tumors. Despite advances in understanding the molecular biology of CNS tumors, targetable genomic abnormalities are lacking. To identify diagnostically, prognostically and potential therapeutically relevant abnormalities, we developed a next generation sequencing (NGS) assay for formalin-fixed paraffin-embedded (FFPE) tissue that evaluates 219 genes associated with adult and pediatric CNS tumors. This test consists of a DNA and an RNA subpanel for the detection of sequence alterations and gene rearrangements (known gene fusions and abnormal transcript variants, and novel fusion transcripts that contain any of the interrogated genes as a partner). The assay utilizes an amplicon-based approach with molecular barcode chemistry (to allow traceability of PCR artifacts/duplicates), Illumina sequencing and custom bioinformatics pipelines. Analytical validation included 175 samples. Overall concordance with alternative methods were 99% and 96% and success rates were 97% and 95% for the DNA and RNA subpanels, respectively. Inter and intra-assay reproducibility was 100% for both subpanels. The limit of detection (analytical sensitivity) for nucleic acid input and tumor content were 8.5 ng and 30%, and 10 ng and 10% for the DNA and RNA subpanels, respectively. The analytical specificity was high, with per base DNA sequencing false positive rate <0.4% and absent fusion transcript detection in non-neoplastic samples. We developed a robust 219-gene neuro-oncology NGS assay suitable for clinical testing of FFPE specimens, including small biopsies with low tumor content. This test, combined with chromosomal microarray analysis, detects nearly all single nucleotide variants, fusion rearrangements and copy number changes associated with CNS tumors. These tests are intended to assist in the diagnosis, prognosis and therapeutic management of adult and pediatric patients with CNS tumors, and have the potential to unravel novel genomic abnormalities and expand the understanding of the molecular biology of such tumors.

Mitigation of the effect of variability in digital PCR assays through use of duplexed reference assays for normalization.

Digital PCR has been promoted as a technique for obtaining absolute measures of the amount of nucleic acid target sequence in a sample, but still lacks standardization in data reporting. The initial method of representing data as copies per microliter produced inconsistent results and made inter-assay comparisons difficult. Normalizing copies to amount of nucleic acid gives more uniform results, but factors influencing the effective concentration of nucleic acid in the final digital PCR assay must be considered. Using droplet digital PCR and previously validated reference genes duplexed with target genes, a method of normalization was developed to estimate the amount of input nucleic acid in individual assays, subsequently reporting the number of copies of target gene relative to this amount. Correcting for the actual amount of amplifiable nucleic acid present demonstrated a higher correlation between various dilutions of sample mRNA and allowed more accurate comparisons of digital PCR results.

Analytical performance of the ThyroSeq v3 genomic classifier for cancer diagnosis in thyroid nodules.

Molecular tests have clinical utility for thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology, although their performance requires further improvement. This study evaluated the analytical performance of the newly created ThyroSeq v3 test. ThyroSeq v3 is a DNA- and RNA-based next-generation sequencing assay that analyzes 112 genes for a variety of genetic alterations, including point mutations, insertions/deletions, gene fusions, copy number alterations, and abnormal gene expression, and it uses a genomic classifier (GC) to separate malignant lesions from benign lesions. It was validated in 238 tissue samples and 175 FNA samples with known surgical follow-up. Analytical performance studies were conducted. In the training tissue set of samples, ThyroSeq GC detected more than 100 genetic alterations, including BRAF, RAS, TERT, and DICER1 mutations, NTRK1/3, BRAF, and RET fusions, 22q loss, and gene expression alterations. GC cutoffs were established to distinguish cancer from benign nodules with 93.9% sensitivity, 89.4% specificity, and 92.1% accuracy. This correctly classified most papillary, follicular, and Hurthle cell lesions, medullary thyroid carcinomas, and parathyroid lesions. In the FNA validation set, the GC sensitivity was 98.0%, the specificity was 81.8%, and the accuracy was 90.9%. Analytical accuracy studies demonstrated a minimal required nucleic acid input of 2.5 ng, a 12% minimal acceptable tumor content, and reproducible test results under variable stress conditions. The ThyroSeq v3 GC analyzes 5 different classes of molecular alterations and provides high accuracy for detecting all common types of thyroid cancer and parathyroid lesions. The analytical sensitivity, specificity, and robustness of the test have been successfully validated and indicate its suitability for clinical use. Cancer 2018;124:1682-90. © 2018 American Cancer Society.

Abstract 3732: Analytical performance of TruSight® Tumor 170 on small nucleotide variations and gene amplifications using DNA from formalin-fixed, paraffin-embedded (FFPE) solid tumor samples

Abstract Expanding the paradigm of solid tumor profiling from single-gene testing to comprehensive panels presents many challenges. One such challenges is the ability of these panels to detect genetic alterations from FFPE samples, where the DNA is of low abundance and often heavily compromised. Despite these challenges, next-generation sequencing (NGS) offers the ability to assess multiple variants simultaneously in an ever-expanding list of relevant tumor genes. To that end, Illumina developed a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes that is FFPE optimized. The assay consists of a DNA workflow for the identification of single and multiple nucleotide variants (SNVs, MNVs), small insertions and deletions (indels), gene amplifications, as well as a RNA workflow for the identification of splice variants and gene fusions. Following sequencing on the NextSeq® or HiSeq® instruments, the analytical pipeline initiates variant calling. The DNA aligner and variant callers were first optimized against the simulated read data from &amp;gt;40,000 COSMIC[1] mutations reported in the exons of the 170 genes. To reduce false positive variant calling due to systematic errors, each variant call was evaluated against its locus specific background error distribution. This distribution was compiled from a panel of FFPE normal samples and was also used to normalize against systematic bias in read coverage to increase the accuracy of amplification calling. Furthermore, gene amplification calling was improved by the addition of enhancer probes to the hybrid capture pool. The analytical sensitivity and specificity of TruSight® Tumor 170* was assessed on a large collection of FFPE samples and reference material. A panel of 72 cancer samples, including multiple tissue types, reference standards, and cell line and FFPE mixes were used to evaluate the limit of detection. The samples contained 533 SNVs, 80 indels including deletions up to 30 base pairs and insertions up to 31 base pairs, 4 MNVs, and 31 gene amplifications, characterized by orthogonal testing methods. Using 40 ng DNA input, detection sensitivity of the &amp;gt;1000 variants (including replicates) tested at variant allele frequencies down to ~5% was at 99.6%, while detection sensitivity of gene amplifications as low as 1.45x to 2.2x was at 98%. For limit of blank samples, a panel of 24 normal samples was used. Again using 40 ng DNA input, we show &amp;gt;99% specificity for small variant calling and &amp;gt;95% specificity for gene amplification calling. These data demonstrates the TruSight® Tumor 170 is able to detect multiple variant types within a single sample at low nucleic acid input, while exhibiting high analytical sensitivity and specificity for low allele fraction detection. [1] Forbes, et al. (2015) *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Danny Chou, Xiao Chen, Austin Purdy, Li Teng, Byron Luo, Chen Zhao, Laurel Ball, Allan Castaneda, Katie Clark, Brian Crain, Anthony Daulo, Manh Do, Tingting Du, Sarah Dumm, Yonmee Han, Michael Havern, Chia-Ling Hsieh, Tingting Jiang, Suzanne Johansen, Scott Lang, Rachel Liang, Jennifer S. LoCoco, Jaime McLean, Yousef Nassiri, Jason Rostron, Jennifer Silhavy, June Snedecor, Natasha Talago, Kevin Wu, Clare Zlatkov, Ali Kuraishy, Karen Gutekunst, Sohela De Rozieres, Matthew Friedenberg, Han-Yu Chuang, Anne C. Jager. Analytical performance of TruSight® Tumor 170 on small nucleotide variations and gene amplifications using DNA from formalin-fixed, paraffin-embedded (FFPE) solid tumor samples [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 3732. doi:10.1158/1538-7445.AM2017-3732

Defining RNA-Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA.

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif–small molecule interactions identified via selection. Named High Throughput Structure–Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif–small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule–RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.

Identification of Multiple, Patient-Specific MLL Fusion Transcript Isoforms in Childhood Leukemia Using Anchored Multiplex PCR-Based Enrichment (AMP-E)

Chromosomal translocations involving 11q23, resulting in rearrangements of the mixed lineage leukemia gene (MLL, re-named KMT2A) are frequent events in childhood leukemia. MLL is highly promiscuous, with approximately 80 fusions now characterized. Although fluorescence in situ hybridization (FISH) has high specificity for detecting MLL-rearrangements (MLL-r), sensitivity is limited and the translocation partner gene (TPG) cannot always be identified. In contrast, long-distance inverse-PCR (LDI-PCR) permits sequence-specific characterization of MLL breakpoints and the resultant fusion gene, which can then be used for monitoring minimal residual disease (MRD). A limitation of LDI-PCR is the relatively large input of DNA (≈ 1μg) required, with a blast cell percentage of > 20-30% to achieve sufficient sensitivity. Next-generation sequencing (NGS) approaches such as RNAseq and whole-genome sequencing (WGS) have the potential to identify multiple gene fusions, however their ability to detect the full spectrum of MLL fusions is limited by coverage, read depth and thereby cost. Such limitations can potentially be overcome with targeted sequencing panels, although their performance against "gold standard" assays, such as LDI-PCR, is unknown. We therefore aimed to assess the ability of a novel, targeted NGS approach for characterizing patient-specific MLLgene rearrangements from low inputs of RNA.The Archer™ FusionPlex™ Heme and Myeloid panels utilize anchored multiplex PCR-based enrichment (AMP-E) to rapidly enrich a number of targets, including MLL, creating libraries for NGS. The NGS libraries are generated using rapid workflows and are compatible with nucleic acid inputs of ≈ 20-200ng. Briefly, double stranded cDNA is generated from patient RNA and subjected to end repair, adenylation and ligation with unique, half-functional adaptors. Following two rounds of nested PCR with primers attached to common sequencing adaptors, the resulting target amplicons become functional and ready for clonal amplification and sequencing. Using AMP-E, we tested 23 paediatric MLL-r samples (15 ALL, 8 AML) that had previously been analyzed by LDI-PCR and were known to harbor 8 different MLL fusions, including MLL-AFF1 (n = 8), -MLLT3 (5), -MLLT10 (3), -ELL (2), -DCP1A (1), -MLLT1 (1), - AFF3 (1), and -TNRC18 (1). A patient sample known to express BCR-ABL1 was used as a positive control and a cytogenetically normal AML sample in remission was used as a negative control in each panel. The median blast count for samples analyzed was 86.1% (range 25%-97%). On average, 100ng of RNA was used per sample, with RIN values ranging from 2.7 to 9.1. Libraries generated using either the Archer™ FusionPlex™ Heme or Myeloid kit were sequenced to sufficient read depths by Illumina MiSeq® and NextSeq®, respectively. Bioinformatic analyses were performed with the Archer™ Analysis 4.1 software. Results were then compared with fusions identified by LDI-PCR.There was high concordance between AMP-E and LDI-PCR, with all MLL fusion genes identified by LDI-PCR also detected by AMP-E. Of note, an ALL sample with t(11;19), unable to be characterized by LDI-PCR, was identified by AMP-E to express MLL-MLLT1. The control BCR-ABL1 fusion was identified in every run and there were no false-negative results. Furthermore, AMP-E identified multiple MLL-fusion transcripts in 56.5% of patients. Analysis of paired diagnosis-relapse samples from an AML patient with MLL-MLLT3demonstrated that the two discrete transcripts present at diagnosis persisted at relapse, with emergence of a third transcript.In summary, detection of MLL gene fusions in acute leukemia using AMP-E is both sensitive and specific. The low RNA requirement, rapid workflow, compatibility with Illumina MiSeq® and cloud-based proprietary analysis software, together with the array of additional fusions and mutations detected by the Archer™ panels, show promise for translation into clinical diagnostic settings. The persistence of discrete transcript isoforms at relapse also highlights the potential for AMP-E to identify multiple, patient-specific MLL fusion transcripts which may have utility in refining prognostication, MRD monitoring and informing future functional studies of MLL-driven leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Towards a DNA Nanoprocessor: Reusable Tile‐Integrated DNA Circuits

AbstractModern electronic microprocessors use semiconductor logic gates organized on a silicon chip to enable efficient inter‐gate communication. Here, arrays of communicating DNA logic gates integrated on a single DNA tile were designed and used to process nucleic acid inputs in a reusable format. Our results lay the foundation for the development of a DNA nanoprocessor, a small and biocompatible device capable of performing complex analyses of DNA and RNA inputs.

Towards a DNA Nanoprocessor: Reusable Tile-Integrated DNA Circuits.

Modern electronic microprocessors use semiconductor logic gates organized on a silicon chip to enable efficient inter-gate communication. Here, arrays of communicating DNA logic gates integrated on a single DNA tile were designed and used to process nucleic acid inputs in a reusable format. Our results lay the foundation for the development of a DNA nanoprocessor, a small and biocompatible device capable of performing complex analyses of DNA and RNA inputs.

Abstract 1381: NGS-based CNV detection sensitivity is dependent upon nucleic acid input quality

Abstract Copy number variations (CNV) impact more of the cancer genome than all other mutation types combined. Recent advances in next-generation sequencing (NGS) have enabled simultaneous detection of CNVs and other somatic mutations from FFPE-derived samples, but NGS-based detection of low level CNVs (ie 2-3x) remains challenging. Nucleic acid from FFPE is a common starting material for NGS-based cancer genotyping; however, this material is often of low complexity due to a variety of factors including limited mass amount, excessive fragmentation, or chemical crosslinking. Current practices often measure input mass, or the nanograms of DNA that are added to a reaction, yet it is input complexity, or the amount of nucleic acid available for NGS library generation, that truly dictates the amount of information that can be recovered from a given sample. Archer™ VariantPlex™ assays are targeted NGS panels that permit simultaneous detection of SNVs, in/dels, and CNVs using Anchored Multiplex PCR (AMP™). Molecular barcoded adapters are ligated to each input molecule prior to any amplification. This permits the unique identification of individual input molecules thus facilitating precise copy number measurements. In addition, AMP enables amplification of highly fragmented FFPE inputs as short fragments are captured between the ligated adapter and the enrichment probe. To determine the effect of input quality on sensitivity of CNV calling we characterized over 150 tumor sample input qualities and their resulting library metrics. In addition we modeled the effect of low tumor cellularity on CNV sensitivity by carrying out dilution experiments of CNV-positive samples into samples of normal copy number. Using Archer VariantPlex assays in conjunction with Archer Analysis, we have successfully detected CNVs as small as 2X in both FFPE and cell line DNA. We found that input nucleic acid quality, as measured by a qPCR-based assay called Archer PreSeq™ DNA QC, strongly impacted the sensitivity of CNV calling. Assessment of input complexity using the PreSeq DNA QC Assay is predictive of limit of detection for CNVs and identifies an input quantity that will result in high quality NGS libraries. Our dilution experiments confirmed the expected relationship between actual and measured copy number in our population-averaging assay. Nucleic acid damage typical of FFPE samples reduces CNV calling sensitivity; however, this loss of sensitivity can be partially mitigated by increasing the input quantity. This corroborates the notion that input complexity is the major driver of information-capture from NGS based assays. Finally, tumor cellularity displays a predictable effect on the measured CNV value. Citation Format: Josh Haimes, James Covino, Namitha Namoj, Elina Baravik, Laura Johnson, Joshua Stahl, Brady P. Culver, Brian Kudlow. NGS-based CNV detection sensitivity is dependent upon nucleic acid input quality. [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 1381.

Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing.

Targeted therapy combined with companion diagnostics has led to the advancement of next-generation sequencing (NGS) for detection of molecular alterations. However, using a diagnostic test to identify patient populations with low prevalence molecular alterations, such as gene rearrangements, poses efficiency, and cost challenges. To address this, we have developed a 2-step diagnostic test to identify NTRK1, NTRK2, NTRK3, ROS1, and ALK rearrangements in formalin-fixed paraffin-embedded clinical specimens. This test is comprised of immunohistochemistry screening using a pan-receptor tyrosine kinase cocktail of antibodies to identify samples expressing TrkA (encoded by NTRK1), TrkB (encoded by NTRK2), TrkC (encoded by NTRK3), ROS1, and ALK followed by an RNA-based anchored multiplex polymerase chain reaction NGS assay. We demonstrate that the NGS assay is accurate and reproducible in identification of gene rearrangements. Furthermore, implementation of an RNA quality control metric to assess the presence of amplifiable nucleic acid input material enables a measure of confidence when an NGS result is negative for gene rearrangements. Finally, we demonstrate that performing a pan-receptor tyrosine kinase immunohistochemistry staining enriches detection of the patient population for gene rearrangements from 4% to 9% and has a 100% negative predictive value. Together, this 2-step assay is an efficient method for detection of gene rearrangements in both clinical testing and studies of archival formalin-fixed paraffin-embedded specimens.

Detection of Acute HIV-1 Infection by RT-LAMP.

A rapid, cost-effective diagnostic test for the detection of acute HIV-1 infection is highly desired. Isothermal amplification techniques, such as reverse-transcription loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for the development of a rapid nucleic acid amplification test (NAAT) because they are quick, easy to perform and do not require complex, dedicated equipment and laboratory space. In this study, we assessed the ability of the HIV-1 RT-LAMP assay to detect acute HIV infection as compared to a representative rapid antibody test and several FDA-approved laboratory-based assays. The HIV-1 RT-LAMP assay detected seroconverting individuals one to three weeks earlier than a rapid HIV antibody test and up to two weeks earlier than a lab-based antigen/antibody (Ag/Ab) combo enzyme immunoassay (EIA). RT-LAMP was not as sensitive as a lab-based qualitative RNA assay, which could be attributed to the significantly smaller nucleic acid input volume. To our knowledge, this is the first demonstration of detecting acute HIV infection using the RT-LAMP assay. The availability of a rapid NAAT, such as the HIV-1 RT-LAMP assay, at the point of care (POC) or in laboratories that do not have access to large platform NAAT could increase the percentage of individuals who receive an acute HIV infection status or confirmation of their HIV status, while immediately linking them to counseling and medical care. In addition, early knowledge of HIV status could lead to reduced high-risk behavior at a time when individuals are at a higher risk for transmitting the virus.