Integrated DNA Technologies Research Articles

P456: A LONG READ SEQUENCING AND CRISPR-CAS9 BASED APPROACH FOR RAPID COPY-NUMBER ALTERATION AND STRUCTURAL VARIATION DETECTION IN HEMATOLOGIC MALIGNANCIES

Background: Current standard karyotyping methods are labor-intensive and have severe limitations in terms of resolution and duration to results. For hematologic malignancies, but not restricted to, this represents a relevant obstacle in translating molecular findings into time critical treatment decisions. Aims: To develop a sequencing based approach for rapid and scalable cytogenetics that can be applied to a wide spectrum of hematologic malignancies to detect clinically relevant molecular markers within 48hrs Methods: For copy number alteration detection, whole genome sequencing was performed on a GridION sequencer (Oxford Nanopore Technologies, ONT, using the SQK-LSK109 kit) in a cohort of 53 hematologic neoplasms, including acute myeloid leukemia (AML, n=18), chronic lymphocytic leukemia (CLL, n=8), multiple myeloma (MM, n=17) and pediatric acute lymphocytic leukemia (ALL, n=10) samples. For the identification of balanced alterations, transcriptome sequencing data were generated using ONT (SQK-DCS109 kit), and we applied an in-house developed analysis pipeline based on minimap2 alignment followed by Blast with an ensuing filtering algorithm based on orientation, inter-read distance and length filtering. For targeted sequencing of structural aberrations, we developed a CRISPR-Cas9 based tiling approach using sgRNA pools (Integrated DNA Technologies, IDT). This approach allows to cover AML relevant targets such as t(8;21), the KMT2A (MLL) breakpoint area, and the FLT3-ITD region as well as lymphoma relevant regions such as the immunoglobulin heavy chain locus (library preparation was performed using SQK-CS9109 kit). Results: For copy number profiling a median whole-genome coverage of 2.6 was reached (range 0.27-7 fold). ONT sequencing and conventional karyotyping approaches showed a high concordance with Pearson correlation coefficients >0.95 for copy number alteration comparisons between conventional cytogenetics and ONT sequencing results for all investigated disease entities. Regarding the detection of structural aberrations transcriptome sequencing and fusion gene analysis using the above described analysis and filtering pipeline we could e.g. reliably detect the t(9;22) translocation from the K562 cell line and the t(8;21) translocation from Kasumi-1. Additionally, using this method we were able to correctly identify the primary AML sample harboring a t(8;21) translocation that was among the n=12 AML patient samples. In addition to an RNA-based work-flow, we established a CRISPR-Cas9 based DNA enrichment approach for the detection of recurrent structural aberrations from specified genomic DNA loci. The enrichment of genomic regions of interest using sgRNA libraries led e.g. to a median coverage of 44 reads (range 5-136-fold) of the t(8;21) region-of-interest in primary AML samples, which allow the detection of the exact coordinates of the breakpoints. Summary/Conclusion: Long read based sequencing based copy number alteration and structural variation detection based on combination of different long read sequencing approaches like low-coverage whole genome sequencing, transcriptome sequencing and CRISPR-Cas9 based sequencing of genomic loci of interest represents a highly promising tool for high resolution and high speed cytogenetics that has the potential to overcome many limitations of conventional cytogenetics.

Automation of hybridization and capture based next generation sequencing library preparation requires reduction of on-deck bead binding and heated wash temperatures.

Capture-based library preparation for next generation sequencing (NGS) offers a balance between sequencing depth and bioinformatics cost of analysis. Liquid handling automation enhances the reliability of the library preparation process by reducing sample-to-sample variation and substantially enhances throughput, particularly when it can be employed in a 'walk-away' fashion with limited hands-on interaction. This requires complex series of mixing and heating steps like those utilized in capture chemistries to happen on the liquid handler. While developing liquid handling automation for Integrated DNA Technologies (IDT) xGen Exome, Illumina TruSight Oncology 500, and Personal Genome Diagnostics (PGDx) elio Plasma Resolve chemistries on the PerkinElmer Sciclone liquid handler, we found that applying the capture temperatures recommended for manual library preparation results in low yield on automation. To restore the final library yield, we reduced bead binding and/or heated wash temperatures of the Peltier heaters on the liquid handlers by about 10°C. Since this applied across three unique capture-based chemistries, we consider this a generalizable principle of automating capture on the Sciclone. We hypothesize that this is driven by the very different thermodynamic environments represented by a sealed plate on a thermal cycler and a plate with a lid on a Peltier heater. This phenomenon should be considered when automating NGS library preparation on PerkinElmer Sciclone instruments.

Systematic comparative analysis of strand-specific RNA-seq library preparation methods for low input samples

Despite the recent precipitous decline in the cost of genome sequencing, library preparation for RNA-seq is still laborious and expensive for applications such as high throughput screening. Limited availability of RNA generated by some experimental workflows poses an additional challenge and increases the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies—Swift RNA and Swift Rapid RNA, presently offered by Integrated DNA Technologies (IDT) —alongside the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark gene expression in these kits, at input quantities ranging between 10 to 500 ng. We found normalized read counts between all treatment groups to be in high agreement. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits offer shorter workflow times enabled by their patented Adaptase technology. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways directly attributable to input mRNA amount.

Higher Prevalence of Multi-Drug Resistant Gonococcal Infections Among Patients with Prior Gonococcal and Chlamydia Infections

Abstract Introduction/Objective Neisseria gonorrhoeae (NG) is the 2nd most common cause of sexually transmitted infection worldwide. WHO data from 2009-14 showed widespread resistance to ciprofloxacin (97%), azithromycin (81%), and oral cefixime and injectable ceftriaxone (66%). We have investigated local epidemiology of antimicrobial resistance in PCR-confirmed NG by chromosomal multi-locus sequence typing and analysis. Methods/Case Report Clinical specimens of 85 patients who tested positive for NG using automated PCR (COBAS 4800; Roche Diagnostics) were used in this study. The samples were amplified by PCR using 10 primers (Integrated DNA Technologies, USA) targeting seven antibiotic resistance-associated loci in the bacterial chromosome (penA, mtrR, ponA, porB, gyr, parC and 23s rRNA) and sequenced on a 48-channel ABI 3730 Sanger sequencer (Thermo Fisher). The resulting sequences were analyzed using the NG Sequence Typing for Antibiotic Resistance (NG-STAR) database for matching alleles, correlating to clinical resistance. Results were analyzed by Chi-square or Fisher’s exact tests. Results (if a Case Study enter NA) Most patients positive for NG were non-Hispanic Black males (60%), with mean age of 25 years (range: 14-53 years). 23.5% had Chlamydia trachomatis (CT) co-infection, and 29.4% had other sexually transmitted infections, such as syphilis, HIV and HPV. 15.0% had previous NG infection, and 12% had previous CT infection. Sixty-five samples (76.5%) yielded at least one gene locus with a DNA sequence with >95% similarity in the NG-STAR database. About half (50.6%) of these sample had confirmed resistance mutations, 5 of which indicated resistance to multiple antibiotics. All specimens with porB and gyr mutations were resistant to penicillins, ceftriaxone, cefixime, erythromycin, tetracycline and ciprofloxacin. Most of those with mtrR mutations were resistant to ciprofloxacin. Most of those with mutations (with or without resistance) had previous NG infection. Significant correlation was observed between those with at least 1 mutated gene locus and CT co-infection (p=0.0001). Conclusion A significant proportion of our patients tested positive for resistance mutations, a majority of whom had previous NG infection. There was significant correlation between the presence of mutations and CT co-infection. These findings warrant utilization of culture and sensitivity testing in patients with previous NG and CT co-infection.

Targeted-Capture Sequencing Approach to V(D)J Rearrangement, Immunoglobulin (Ig) Translocation and Mutation Detection in Multiple Myeloma (MM): Potential Applications in Minimal Residual Disease (MRD) Analysis and Comparison to Multiparameter Flow Cytometry (MFC) in the Myeloma Canada Research Network (MCRN) 001 Study with Intravenous Busulfan and Melphalan (BuMel) Conditioning Followed By Lenalidomide Maintenance in Newly Diagnosed Multiple Myeloma (NDMM)

Targeted-Capture Sequencing Approach to V(D)J Rearrangement, Immunoglobulin (Ig) Translocation and Mutation Detection in Multiple Myeloma (MM): Potential Applications in Minimal Residual Disease (MRD) Analysis and Comparison to Multiparameter Flow Cytometry (MFC) in the Myeloma Canada Research Network (MCRN) 001 Study with Intravenous Busulfan and Melphalan (BuMel) Conditioning Followed By Lenalidomide Maintenance in Newly Diagnosed Multiple Myeloma (NDMM)

CRISPR/Cas9-Mediated Diacylglycerol Kinase Knockout Potentiates Anti-Tumor Efficacy of Human Chimeric Antigen Receptor T-Cells

IntroductionTumors can evade immune surveillance by deactivating T cells via various immunosuppressive ligands and soluble factors. The inhibitory signals from tumors turn on or off specific signaling pathways of T cells, making T-cell therapy inefficient. Many efforts for efficient adoptive T-cell therapy have focused on blocking or inhibiting CD28/B7 pathway, signal2, by targeting PD1 or CTLA4, but robust therapeutic outcomes have remained challenging. Diacylglycerol (DAG) is an essential signal transduction molecule cascading CD3 signal, signal1, and DGK is an enzyme that phosphorylates DAG to phosphatidic acid (PA). In many tumors, DGK was found to be highly expressed in tumor infiltrating lymphocytes (TILs) and inhibition of DGK recover the dysfunction of TILs. In this study, we sought to invigorate the T-cell function by deleting Diacylglycerol kinase (DGKs) using CRISPR/Cas9. We found that ablation of DGKs potentiates anti-tumor function and proliferation of CAR-T cell, by strengthening the CD3 distal signals. Also, DGKαζ (two isoforms) knockout (KO) CAR-T showed no significant elevation of exhaustion markers, and was less responsive to immunosuppressive soluble factors, such as TGF-beta and prostaglandin E2. Collectively, these findings highlight the utility of DGK KO by CRISPR/Cas9 to reinforce the effector function of T-cell. The observed biological effects of DGK KO may also yield new strategies for improving the efficacy of T-cell therapy in combination with signal 2 checkpoint inhibitors in clinical applications.MethodsHuman pan-T cells were purchased from STEMCELL TECHNOLOGIES. X-VIVO15 media was supplemented with hIL-2 (50 U/mL) and hIL-7 (5 ng/mL) for culture, and activation was performed by using anti-CD3/28 Dynabeads (ThermoFisher SCIENTIFIC) at bead-to-cell ratio of 3:1. After 24 hours of activation, T-cells were mixed with 139-CAR lentivirus plates coated with Retronectin for 48 hours. 40 μg of recombinant S. pyogenes Cas9 (Toolgen) and 10 μg of chemically synthesized tracr/crRNA (Integrated DNA Technologies) were electroporated using 4D-Nucleofecter (Lonza).For in vitro assays, U87vIII, pre-stained with Cell Trace (ThermoFisher Scientific), was co-cultured with 139 CAR-T at indicated ratio, supplemented with or without TGF-β1 at 10 ng/mL and PGE2 at 0.5 ug/mL. Following co-incubation, cells were stained with 7-aminoactinomycin for cytotoxicity assays. Sample were acquired on Attune NxT Acoustic Focusing Cytometer, and analyzed with FlowJo. Cytotoxicity was calculated as [(%lysis sample-% lysis minimum)/(%lysis max [100%]-%lysis minimum]×100%. The supernatant of co-culture was analyzed to measure IL-2 and IFN-γ using ELISA Kit(Biolegend). For proliferation assays, distribution of CellTrace in 139 CAR-T cells was assessed using flow cytometry.ResultsIn following experimental scheme (Fig 1A), delivery of single Cas9/gRNA Ribonucleoprotein (RNP) complex targeting DGKα or DGKζ resulted in 75.9% and 93.5% of Indel, respectively in 139 CAR-T (Fig 1B). To generate DGKα and DGKζ double-negative 139 CAR-T cells, two gRNAs targeting DGKα and DGKζ were electroporated, which resulted in 49.2% and 92.4% indel, respectively (Fig 1B). No significant off-targets were detected by targeted deep-sequencing (Fig 1C). DGKα, DGKζ, and DGKαζ KO 139 CAR-T showed dramatic increase in cytotoxicity, multiple cytokine production, and proliferation capacity compared to wild-type 139 CAR-T (Fig 2A, B, C). Interestingly, DGKαζ KO 139 CAR-T showed further increase in cytokine release compared to DGKα or DGKζ single KO one, implying a synergistic effect of DGKα and DGKζ. The increase in effector function of DGK KO 139 CAR-T attributed to amplified CD3-distal signal, ERK1/2, and higher CAR expression after antigen exposure (Fig 3A, B). Despite of the strong activation signals in DGKs KO 139 CAR-T, no significant tonic activation was observed as indicated by basal IFN-γ secretion level (Fig 4A). Also, in comparison with 139 CAR-T, PD1 and TIM3 expression were not elevated in DGKs KO 139 CAR-T, suggesting that DGKs KO does not facilitate T-cell exhaustion after long term antigen exposure (Fig 4B). In contrast that anti-tumor effect of 139 CAR-T was markedly impaired with treatment of signal1 immunosuppressive inhibitors such as TGF-β1 and PGE2, DGKαζ KO 139 CAR-T maintained its cytotoxicity and cytokine release in the presence of the inhibitory soluble factors. (Fig 5 A, B) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Myeloid Mixed Chimerism and Relapse in Myelofibrosis after Allogeneic Stem Cell Transplant

Background:Allogeneic stem cell transplant (SCT) remains the only potential curative treatment for myelofibrosis (MF). However, relapse after transplant is one of the leading causes of death. The role of mixed myeloid chimerism (MMC) in predicting post-transplant molecular and morphologic relapse is not well studied.Methods: Using a large single-center database, all consecutive patients with primary or secondary MF who underwent first allogeneic SCT in the period 2005-2015 were included in the study. Patients were defined to have MMC if they were found to have less than 95% of donor myeloid cells at any point over the duration of their follow up. Peripheral blood chimerism analysis was performed using 8 highly polymorphic microsatellite markers (Integrated DNA Technologies, Coralville, IA) in a multiplex PCR assay. The primary end point was to assess the correlation of MMC with any evidence of morphologic and/or molecular relapse.Results: A total of 85 patients (47 males, 38 females) were identified with a median age at transplant of 57 years (27-74). Among patients with molecular data (n=80), 60% had positive pre-transplant molecular markers. A total of 36 (43%) patients with full chimerism data (n=83) had MMC. All patients with MMC developed morphologic and/or molecular relapse, except for two; the first patient converted to full donor chimerism after immunosuppression reduction and the second patient had progressive myeloid chimerism and graft failure. Among patients with positive molecular testing and available chimerism data (n=46), 22 patients had MMC, of which 95% (n=21) had molecular relapse. Similarly, for patients with available chimerism data and negative pre-transplant molecular testing (n=32), 93% (n=14) of the 15 patients with MMC experienced eventual morphologic relapse. On the other hand, none of the patients with full donor myeloid chimerism (n=47) progressed during the study period, except for one patient with mixed T-Cell chimerism. With a median follow up of 49 months, the 5-year PFS was 7% for those with MMC vs 62% for patients with full donor myeloid chimerism (p<0.0001).Conclusion: Given the high sensitivity of chimera PCR DNA testing and its strong association with molecular and morphologic relapse, we speculate that myeloid chimerism testing, alone or combined with clonal molecular testing, would serve as one of the earliest and best markers to predict MRD and relapse in myelofibrosis post-transplant. DisclosuresOran:Astex: Research Funding; AROG: Research Funding; Celgene: Research Funding. Bose:Incyte Corporation: Honoraria. Pemmaraju:LFB: Consultancy, Honoraria; stemline: Consultancy, Honoraria, Research Funding; novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Honoraria; cellectis: Research Funding; roche diagnostics: Consultancy, Honoraria; affymetrix: Research Funding; abbvie: Research Funding. Verstovsek:Galena BioPharma: Research Funding; Blueprint Medicines Corp: Research Funding; CTI BioPharma Corp: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Gilead: Research Funding; Astrazeneca: Research Funding; Roche: Research Funding; Bristol Myers Squibb: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Lilly Oncology: Research Funding; Pfizer: Research Funding; Promedior: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; CTI BioPharma Corp: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Astrazeneca: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Research Funding; Blueprint Medicines Corp: Research Funding; Genentech: Research Funding; Promedior: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Lilly Oncology: Research Funding.

Optimization of SARS-CoV-2 laboratory testing in a rural healthcare facility in the United States

Background: The diagnostic testing for SARS-COV-2 (COVID-19) presented a profound challenge to the entire world, dominating the concern of most governments and public health systems, particularly rural community hospitals in the United States. Indiana University of Pennsylvania (IUP) in partnership with Indiana Regional Medical Center (IRMC) began on site, same-day COVID-19 testing in efforts to not only combat the challenges that health providers faced in rural Indiana community but also help to strengthen global diagnostic capacity.Methods: Clinical samples were collected as dry swabs from the nasopharyngeal (NP) regions and processed in phosphate buffer saline (PBS). The crude RNA was directly tested using real-time (RT) reverse transcription quantitative polymerase chain reaction (RT-qPCR) with PrimeDirect probe RT-qPCR Mix (Takara Bio USA) and optimized with probe-primer sets [Integrated DNA Technologies (IDT)].Results: Validation experiments with dry swabs from NP clinical samples showed no difference in the testing accuracy to those collected in viral transport medium or universal transport medium. Extraction of COVID-19 RNA in PBS reduced processing time of a batch of 50 NP clinical samples from 6 hours to an hour. This allowed for rapid diagnostic testing of nearly 200 clinical samples per day. Optimization of analytical variables helped to detect virus loads up to 2.0 copies/μl during routine diagnostic testing.Conclusions: During an infectious outbreak, the ideal response by public health authorities is rapid testing. The collaboration between IUP and IRMC attests to the importance of teamwork between local initiatives to detect and prevent further spread within a rural community.

Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR.

The gold standard of molecular pathogen detection is the quantitative polymerase chain reaction (qPCR). Modern qPCR instruments are capable of detecting 4-6 analytes in a single sample: one per optical detection channel. However, many clinical applications require multiplexing beyond this traditional single-well capacity, including the task of simultaneously testing for SARS-CoV-2 and other respiratory pathogens. This can be addressed by dividing a sample across multiple wells, or using technologies such as genomic sequencing and spatial arrays, but at the expense of significantly higher cost and lower throughput compared with single-well qPCR. These trade-offs represent unacceptable compromises in high-throughput screening scenarios such as SARS-CoV-2 testing. We demonstrate a novel method of detecting up to 20 targets per well with standard qPCR instrumentation: high-definition PCR (HDPCR). HDPCR combines TaqMan chemistry and familiar workflows with robust encoding to enable far higher levels of multiplexing on a traditional qPCR system without an increase in cost or reduction in throughput. We utilize HDPCR with a custom 20-Plex assay, an 8-Plex assay using unmodified predesigned single-plex assays from Integrated DNA Technologies and a 9-Plex pathogen panel inclusive of SARS-CoV-2 and other common respiratory viruses. All three assays were successful when tested on a variety of samples, with overall sample accuracies of 98.8, 98.3, and 100%, respectively. The HDPCR technology enables the large install base of qPCR instrumentation to perform mid-density multiplex diagnostics without modification to instrumentation or workflow, meeting the urgent need for increased diagnostic yield at an affordable price without sacrificing assay performance.

Abstract PS19-25: Metastatic TNBC is highly associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism programmation

Abstract Background: Triple negative breast cancer (TNBC) is characterized by heterogeneous metastatic and metabolic properties, which difficult the diagnosis and development of specific treatment. Therefore, the understanding of the mitochondria´s role in the different grades of TNBC could be relevant for the design of novel specific TNBC treatments. This research aimed to explore the mitochondria morphology and gene expression of proteins related to oxidative and non-oxidative metabolism in metastatic and non-metastatic TNBC cell lines. Methods: We performed a gene expression analysis of mitochondrial biogenesis, EMT and principal metabolism-related genes (Integrated DNA Technologies, CA, USA) in three breast cancer cell lines HCC-1395, MDA-MB-231 and MCF-12A. After the gene expression qPCR data mining, in sílico analisis were performed using confocal microscopy (Leica Microsystems,WZL, DE) in the cell lines where mitochondrial distribution, morphology, function and ROS production were measured. Results: The metastatic TNBC-cell line showed a preference for fatty acid biosynthesis and glycolytic metabolism since overexpression of genes related to both pathways was observed. These metabolism features were accompanied by a fused mitochondrial morphology and lower mitochondrial activity since was observed less mitochondrial density accompanied by the downregulated expression of biogenesis-related genes such as PGC1α. In contrast, the non-metastatic TNBC-cell line presented a hyperfragmented mitochondria, a stress associated mitochondrial morphology accompanied by upregulated expression of mitochondrial biogenesis-related genes, both characteristics related to the higher ROS production observed in this cell line. Conclusions: Metastatic TNBC was characterized by a mitochondrial function and morphology similar to control ranges with a metabolic gene program directed to glycolysis and FA usage. While mitochondria of the non-metastatic TNBC cell line was characterized by a higher density and potential, related to an increase of mitochondrial biogenesis, which in turn was associated with higher levels of ROS and with the consequence of a hyperfragmented mitochondrial morphology. These characteristics can provide a better understanding of the metastasis observed in TNBC and contribute to the development of a specific and personalized TNBC therapy. Key words: Triple Negative Breast Cancer, Mitochondria, Cell-metabolism. Citation Format: Claudia D Aguayo-Millán, Perla Perez-Trevino, Sandra Santuario-Facio, Alberto Camacho, Noemí Garcia, Rocio Ortiz-Lopez. Metastatic TNBC is highly associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism programmation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-25.

Abstract PS5-24: Novel genomic variants and pathways associated with baseline serum thymidine kinase 1 levels in HR-positive HER2-negative MBC patients commencing palbociclib and letrozole

Abstract Background: Cyclin dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in hormone-receptor (HR)-positive metastatic breast cancer (MBC), but progression of disease is inevitable. Serum thymidine kinase-1 (TK1) is a secreted marker of proliferation that is prognostic in patients (pts) with HR-positive, HER2-negative MBC and may be predictive of ET and CDK 4/6i response. PROMISE (NCT0281902) is a prospective study enrolling women with HR-positive MBC starting palbociclib (Pb) + letrozole (L) (1st line) or Pb + fulvestrant (2nd line). We undertook a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome in order to identify novel genomic variants and pathways associated with an early decline in TK1 (measured after 2 months) and PFS. Additionally, patient derived xenografts/organoids were generated at baseline and progression to test new therapeutic approaches to overcome resistance to CDK4/6i and ET. We report the initial association between the baseline genomic landscape and baseline TK1 levels. Methods: FFPE tumor biopsies were obtained for DNA/RNA sequencing (TempusTM) and blood samples for TK1 (Divitum® assay, Biovica) were collected pretreatment (pre-Pb) and after 2 cycles of Pb + ET (post-Pb2). Both whole-exome (exome capture) sequencing (WES) and RNA-Seq used the Integrated DNA Technologies xGen Exome Research Panel v1.0 capture kit. TK1+ disease was defined as &amp;gt; 200 Du/L and TK1- disease as below limit of detection up to 200 Du/L. We tested the association between genomic and transcriptomic characteristics with baseline TK1 data in pretreatment samples where both WES and RNA-seq and TK1 was available. The data were analyzed using bioinformatics pipelines for somatic and germline mutations and copy number alterations. The current analysis focuses on baseline 1st-line pre-Pb omics data in conjunction with baseline TK1 levels. The database was locked for analysis on 5/29/2020. Results: Thirty-three pts (median age: 59 yrs.) were evaluable, with paired samples for TK1 in 32. Six pts had TK1+ disease pre-Pb and post-Pb2. Twenty-two pts had TK1- disease pre-Pb and post-Pb2. Four pts had a decrease in TK1 after 2 cycles of treatment that altered the classification from TK1+ to TK1-. Both baseline RNA seq and serum TK1 (n=16) were available for 4 TK1+ and 12 TK1- pts. In this group, 476 genes were differentially regulated (398 upregulated; 78 downregulated). Pathway analysis demonstrated enrichment in complement and coagulation cascade pathway, PPAR signaling pathway, and metabolism-related pathways related to up-regulation of CYP and UGT gene families. Further testing for the association of WES data with baseline TK1+ (n=8) and TK1- (n=16) disease demonstrated somatic copy number variations on chromosomes 6, 11, 12 and 15. CDK4 copy number gains were observed in 3/8 TK1+ pts and 0/16 TK1- pts. We also observed that somatic mutations (LOH, copy number and/or SNV/INDELs) were more prevalent in the TK1+ compared to the TK1- pre-Pb group in several cancer-associated genes (FAS [p=0.06] PTEN, PIK3CB, NAB2, SOX9 and FAT1 [p=0.08], TP53, and MAP2K4 [p=0.22]). Conversely, we also noted that 6/7 pts with GATA3 mutations had TK1- disease (p=0.23). Conclusions: Using a comprehensive “omics” approach, our data suggest that a secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the first line treatment of HR+ MBC is associated with established and novel genes and pathways associated with prognosis of pts receiving ET and CDK 4/6i. Analysis of on-treatment (after 2 cycles) tumor RNA seq and its association with change in TK1 as well as data from the 2nd-line cohort will be presented at the meeting. Citation Format: Ciara C O Sullivan, Krishna R Kalari, Vera J Suman, Peter T Vedell, Ann Moyer, Abraham D Eyman Casey, Jason Sinnwell, Xiaojia Tang, Kevin Thompson, Alvaro Moreno-Aspitia, Donald W Northfelt, Minetta C Liu, Tufia C Haddad, Saranya Chumsri, Prema Peethambaram, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Adrian Nordmark, Mattias Bergqvist, Brendan P McMenomy, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Novel genomic variants and pathways associated with baseline serum thymidine kinase 1 levels in HR-positive HER2-negative MBC patients commencing palbociclib and letrozole [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-24.

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.

BackgroundDue to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.MethodsHere, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.FindingsWe report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.InterpretationThe results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.FundingThis work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

Multiplexed Functional Assessment of Genetic Variants in CARD11

SummaryGenetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Correction of the Energetic Defects in Pyruvate Kinase Deficiency through Genome Editing in Hematopoietic Stem and Progenitor Cells

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene that lead to a reduction of the erythroid pyruvate kinase (RPK) protein activity, which causes an energetic imbalance in PKD erythroid cells. This disease is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. In selected cases, allogeneic Hematopoietic Stem Cell Transplantation has been shown to correct the disorder. Therefore, autologous HSCT of genetically corrected cells will offer a durable and curative therapeutic option. In fact, a global Phase I clinical trial (clinicaltrials.gov #NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy with severe PKD. Looking for a guided integration of the exogenous therapeutic DNA sequences, herein we conducted a gene editing approach to correct PKD in human Hematopoietic Stem Cells (HSCs). We developed a knock-in approach to insert either a TurboGFP expression cassette or a promotor-less therapeutic codon optimized RPK cDNA (coRPK) at the genomic starting site of the PKLR gene, a gene editing strategy that will correct most of the mutations present in PKD patients. This gene editing approach combines RNP nucleofection and adeno-associated viral vector (AAV6) mediated delivery of homologous donors. In order to assess the safety of the proposed gene editing approach, we performed GUIDE-Seq and rhAmpSeqTM analyses of different single guide RNAs targeting the PKLR starting site. We found PKLR sgRNAs that showed a high targeting efficiency, up to 40% targeted hematopoietic progenitors (in vitro semisolid colony forming units) and a safety profile, with no off-targets detected above threshold values (0.1%) and in the absence of cellular toxicity, when applied to healthy cord blood CD34+ (CB-CD34+) cells. These gene-edited CB-CD34+ cells engrafted efficiently in both primary and secondary immunodeficient NSG recipient mice, demonstrating the gene editing in long-term hematopoietic repopulating HSCs. Furthermore, we evaluated the therapeutic potential of this gene editing strategy to restore the energetic imbalance in erythroid cells derived from PKD patients. CD34+ cells from 4 different PKD patients, were purified, nucleofected with PKLR sgRNA and transduced with AAV-coRPK donor. In vitro differentiated erythroid cells derived from edited PKD CD34+ expressed coRPK mRNA and restored their energetic defect up to normal values obtained in in vitro differentiated erythroid cells from healthy donor cells. Moreover, gene edited mobilized Peripheral Blood CD34+ (mPB-CD34+) cells from a PKD patient engrafted efficiently in immunodeficient NBSGW mice, having human cells that showed the specific integration of coRPK donor 2 months post-transplant. Overall, these results demonstrate the feasibility and safety of PKLR gene editing in human HSPCs and, therefore, its potential clinical application for the treatment of PKD patients. Disclosures Dever: Integral Medicines: Current Employment. Turk:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bianchi:Agios Pharmaceuticals: Other: Scientific Advisor. Behlke:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents &amp; Royalties, Research Funding. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents &amp; Royalties, Research Funding.

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

The presence of dog meat is a crucial issue because dog meat is non-halal meat for Muslims. The objective of this study was to design and validate species-specific primer for the identification of dog meat DNA in meatball using real-time polymerase chain reaction (real-time PCR). The specific primer targeting mitochondrial cytochrome c oxidase subunit 1 (CO1) was validated. The specific primers used were designed using Integrated DNA Technologies (IDT) software and subjected to NCBI BLAST procedure. The candidate primers were tested for specificity study using several DNAs from fresh meat of pork, chicken, beef, lamb, and rat. The method was also validated by determining several parameters of linearity, sensitivity, precision, and efficiency. The results showed that primer could amplify specifically DNA target at an optimized annealing temperature of 56.6 °C. The limit of detection (LoD) obtained was 5 ng DNA, corresponding to 2.5% of dog meat in a meatball. The repeatability evaluation, expressed with relative standard deviation (RSD), and efficiency value was in the acceptable range (RSD &lt; 25% and efficiency (90–105%). This method was successfully used for the analysis of marketed samples. Real-time PCR can be used as a standard method in halal authentication analysis through DNA analysis.

Abstract 5863: Improved conversion in extraction, library construction, and capture improve sensitivity for variants in liquid biopsy samples

Abstract Demonstrate performance of a complete automation and reagent workflow for analysis of cfDNA from bodily fluids. The efficient extraction of cfDNA from bodily fluids is a unique challenge due to the very low concentrations of nucleic acid. The extraction process along with library preparation is a laborious workflow, where human variability can lead to increased variability in the downstream analysis. Integrated DNA Technology (IDT) and Beckman Coulter (BC) have teamed up to provide a complete automation and reagent workflow for analysis of low frequency variants in cfDNA. The Apostle MiniMax™ High Efficiency Isolation Kit from BC provides complex, utilized magnetic nanoparticles to effectively capture cfDNA. IDT's library prep kit utilizes novel chemistry to maximize conversion, suppress adapter-dimer formation, reduce chimera rates, and facilitate double strand consensus analysis to call ultra-low frequency variants. Finally, IDT's xGen™ hybrid capture products maintain high library diversity and on-target rates to enable low frequency variant calling regardless of panel size. The Biomek i5 and i7 Hybrid workstations bring out the best performance from these reagents. The Biomek NGS workstations protocol is written with a modular design with safe stop points, making it customizable for each lab. The automated protocol uses Beckman's Demonstrated Method Interface tools which include: Biomek Method Launcher to run the method without going into Biomek software, Method Options Selector to choose the run parameters with a user friendly interface, Guided labware Setup to set the deck with labware based on the run parameters, DeckOptix Final Check software to help reduce deck setup errors. We demonstrate the performance of this complete workflow with a range of plasma inputs (4-8 mL). Using control samples with known variant frequencies, the workflow yields high library complexity, 100% positive predictive value, and reliable detection of &amp;lt;0.5% mutant allele frequency variants. With real cfDNA, the workflow demonstrates both high cfDNA and sequencing library yields along with high library complexity. The combination of these reagents on the Biomek workstations provides a robust and reproducible solution for the analysis of cfDNA. Citation Format: Nicole Roseman, Shilpa Parakh, Hsiao-Yun Huang, Kevin Lai, Timothy Barnes, Lyn Lewis, Ushati Das Chakravarty, Anastasia Potts, Alisa Jackson, Amy Yoder, Jessica Sheu, Tzu-Chun Chen. Improved conversion in extraction, library construction, and capture improve sensitivity for variants in liquid biopsy samples [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 5863.

Interpret with caution: An evaluation of the commercial AusDiagnostics versus in-house developed assays for the detection of SARS-CoV-2 virus

IntroductionThere is limited data on the analytical performance of commercial nucleic acid tests (NATs) for laboratory confirmation of COVID-19 infection. MethodsNasopharyngeal, combined nose and throat swabs, nasopharyngeal aspirates and sputum was collected from persons with suspected SARS-CoV-2 infection, serial dilutions of SARS-CoV-2 viral cultures and synthetic positive controls (gBlocks, Integrated DNA Technologies) were tested using i) AusDiagnostics assay (AusDiagnostics Pty Ltd); ii) in-house developed assays targeting the E and RdRp genes; iii) multiplex PCR assay targeting endemic respiratory viruses. Discrepant SARS-CoV-2 results were resolved by testing the N, ORF1b, ORF1ab and M genes. ResultsOf 52 clinical samples collected from 50 persons tested, respiratory viruses were detected in 22 samples (42 %), including SARS CoV-2 (n = 5), rhinovirus (n = 7), enterovirus (n = 5), influenza B (n = 4), hMPV (n = 5), influenza A (n = 2), PIV-2 (n = 1), RSV (n = 2), CoV-NL63 (n = 1) and CoV-229E (n = 1). SARS-CoV-2 was detected in four additional samples by the AusDiagnostics assay. Using the in-house assays as the "gold standard", the sensitivity, specificity, positive and negative predictive values of the AusDiagnostics assay was 100 %, 92.16 %, 55.56 % and 100 % respectively.The Ct values of the real-time in-house-developed PCR assay targeting the E gene was significantly lower than the corresponding RdRp gene assay when applied to clinical samples, viral culture and positive controls (mean 21.75 vs 28.1, p = 0.0031). ConclusionsThe AusDiagnostics assay is not specific for the detection SARS-CoV-2. Any positive results should be confirmed using another NAT or sequencing. The case definition used to investigate persons with suspected COVID-19 infection is not specific.

CRISPR gene-engineered CYBBko THP-1 cell lines highlight the crucial role of NADPH-induced reactive oxygen species for regulating inflammasome activation

Summary CRISPR-Cas9 engineered CYBBko THP-1 cell lines display an inflammatory profile with increased IL-1β, IL-6 and TNF-α secretion as consequence of NADPH-induced ROS deficiency. This phenotype is reminiscent of the one observed in phagocytes from CGD patients.

Endosomal TLR-8 Senses microRNA-1294 Resulting in the Production of NFḱB Dependent Cytokines.

The primary function of toll-like receptor 8 (TLR-8) is the detection of viruses and other microbial pathogens. Recent evidence suggests that TLR-8 also senses host microRNAs (miRNAs) and implicate TLR-8 in autoimmune disorders. This study examined the interaction between miR-1294 and TLR-8. We first performed a BLAST search to identify miRNAs with the same sequences as two core motifs of miR-1294. Next, we examined NFḱB activation induced by the binding of miR-1294 mimic to endosomal TLR-8. HEK-Blue™ hTLR-8 cells (Invivogen), a HEK293 cell line co-transfected with human TLR-8 gene, were incubated with miR-1294 mimic. A TLR-8 agonist ssRNA40, was used as a positive control. Using the same experimental set up, we also examined the effects of miR-1294 and its two core motifs (Integrated DNA Technologies) on IL-8, IL-1β, and TNFα. Data were analyzed using t-test or one-way ANOVA and Dunnets post-hoc test. Using miRCarta we identified 29 other mature human miRNAs or their precursors which contain the same core motifs as miR-1294. Our data show that miR-1294 activates NFḱB in cells expressing TLR-8 (p < 0.05). miR-1294, and its core motifs induce expression of IL-8, IL-1β, and TNFα via TLR8 activation (p < 0.05). This constitutes a novel mechanism by which endosomal TLR-8 senses host miRNAs resulting in the release of pro-inflammatory cytokines and thus potentially contributing to autoimmune disorders.