Panel Of Probes Research Articles

Development of a microfluidic biosensor for the diagnostics and typing of <i>Mycobacterium Tuberculosis</i>

Background. Despite on the general trend towards decreasing the incidence of newly diagnosed active forms of tuberculosis, the situation with spreading of this disease in Russian Federation remains extremely tense. At the same time, the diagnosis is carried out according to the standard scheme, which takes about a month; another month takes test formulation for drug sensitivity. Thus, the development of new methods for diagnostics and typing of mycobacteria, as well as practice implementation of these developments is an urgent direction. Modern developments in the field of microfluidic technologies open up great opportunities in this direction. Aim. Development of a method for identification and typing of Mycobacterium tuberculosis using a label-free biosensor on surface waves in a one-dimensional photonic crystal (PC SM biosensor). Methods. Oligonucleotide probes were selected and synthesized as DNA targets for M. tuberculosis typing. The photonic crystal surface was modified with aqueous solutions of (3-aminopropyl)triethoxysilane, Leuconostoc mesenteroides dextrans and bovine serum albumin. Experiments were carried out using a PC SM biosensor. Results. Sequences of detecting oligonucleotide probes were selected for spoligotyping of M. tuberculosis on the PC SM biosensor. Modification of their 3'-ends was carried out in order to create extended single-stranded regions that are not subject to the formation of secondary structures and facilitate hybridization with a single-stranded DNA target. Several series of experimental modifications of the PC surface were carried out by using L. mesenteroides dextrans with different functional groups (including detection of the modification results real time) with simultaneous registration of the increment layer size and volume refractive index of the mixture, which excludes the use of a reference cell. Other experiments were carried out to detect the specific binding of biotinylated oligonucleotide probes to the modified PC surface. Conclusions. A technique for the design of probes was developed and a model system of oligonucleotides for the detection of single-stranded DNA using a PC biosensor was proposed. The developed technique of modification of the PC surface with dextrans from L. mesenteroides, which allows to increase the sensitivity of detection of oligonucleotides using the PC SM biosensor. This approach will further expand the panel of diagnostic probes, including identification of resistance markers.

Method for Determining the Profile of Antibiotic Resistance Genes in the <I>Vibrio cholera</I> Strains by RT-PCR

The aim of the work was to design and carry out experimental studies of a set of reagents to identify the spectrum of genes that determine the resistance of the Vibrio cholerae strains to antibacterial drugs.Materials and methods. V. cholerae strains isolated from humans and environmental objects during epidemiological complications and the cholera-free period were included in the study. Sensitivity to antimicrobial drugs was evaluated by the disk diffusion method. Whole genome sequencing was performed on an Illumina MiSeq. The profile of resistance genes was determined based on a comparison with the ResFinder database. The temperature regime, the composition of the reaction mixtures, and the reaction parameters were optimized; the specificity, sensitivity and reproducibility of the constructed prototype test system were measured.Results and discussion. The spectrum of antibiotic resistance and the profile of resistance genes were determined for the studied strains. To develop multiplex PCR, we selected the most common in the V. cholerae populations genes, which are responsible for resistance to tetracycline (tetA), streptomycin (strA), florfenicol/ chloramphenicol (floR) and trimethoprim/sulfamethoxazole (two variants of the dihydrofolate reductase gene: dfrA1 and dhfR), as well as SXT element integrase gene (int). In the reaction, markers were specifically detected in accordance with the genomic resistance profile, which correlates with the phenotypic manifestation of resistance determined by the disco-diffusion method. The sensitivity of the developed panel of primers and probes for V. cholerae strains was 103 –104 CFU/ml. Therefore, taking into account the specificity, rapidity and simplicity of the reaction, the developed system of primers and probes can be successfully applied for a preliminary assessment of the resistance of the V. cholerae strains to antimicrobial agents.

The Cytogenetic Aberrations and Their Prognostic Value in 181 Untreated Patients with Lymphoplasmacytic Lymphoma/ Waldenström Macroglobulinemia in China

Background: lymphoplasmacytic lymphoma/ Waldenström macroglobulinemia (LPL/WM) is a rare B cell proliferative malignancy characterized by immunoglobulin M monoclonal gammopathy and /or bone marrow infiltration by lymphoplasmacytic cells. Cytogenetic aberrations are now the main prognostic predicators in some B-NHLs such as CLL. However, their prognostic valve in LPL/WM remains controversial and recurrently chromosomal abnormalities have rarely been reported. Furthermore, the genetic basis of LPL/WM in China is also unclear.Methods: A panel of DNA probes was used to detect cytogenetic aberrations by fluorescence in situ hybridization (FISH), including RB1/D13S25 at 13q14, ATM at 11q22, TP 53 at 17p13, CEP12 and IGH translocation at 14q32, MYB at 6q23. Chromosomes were identified by G-banding and karyotypes were described according to the International System for Human Cytogenetics Nomenclature (ISCN, 1995). Totally, 181 untreated LPL/WM patients were enrolled in this study.Results: The median age of 181 patients was 61 years (range 32-87). The male to female ratio was 2.75 (133/48). 90% and 15.7% patients showed an elevated serumβ2 microglobulin and LDH. The percentage of hepatomegaly, splenomegaly, lymphadenopathy and B symptom were 15.3%, 35.2%, 50% and 27.3%, respectively. Almost half patients were in the high risk ISSWM group. The chromosomal karyotypes were successful in 153/181 (84.5%) cases, of which 15 patients showed clonal abnormalities. 8 of the 15 patients with abnormal chromosome were complex. The main aberrations of chromosomal karyotypes were 6q deletion (3/15, 20%), and loss of sex chromosomes (3/15, 20%). The other abnormalities include trisomy 12, trisomy 4, trisomy 3, 20q deletion, 17p deletion and so on. All the 6q deletions patients were in the complex karyotype group. Moreover, the trisomy 12 detected by FISH were always accompanied with chromosomal karyotypes abnormalities. Of 181 patients, 139 patients had FISH detection, of which 25 patients (18%) had FISH abnormalities. The most common aberrations were 6q deletion (6/28, 21.4%), trisomy 12 (5/59, 8.5%), 17p deletion (8/131, 6.1%). 13q deletion, gain 1q21and 11qdeletion were 4.6% (6/130) and 2.8% (3/36) and 2% (2/99), respectively. About 5.4% (7/130) patients had IGH gene translocation. Although it was higher in our study, this aberration always accompanied with other clones. Besides, 17p deletion was also more frequent in complex clone group, while trisomy 12 was more common in the single clone group. In LPL/WM, the chromosomal abnormities was an adverse predictor for both progress-free survival (PFS) and overall survival (OS). The median PFS and OS for patients with this abnormity was 62 months (62m vs 69m, p=0.022) and 75 months (75m vs 109m, p=0.033), which was significantly higher than normal patients. In different FISH groups, only TP 53 deletion had a poor effect on both PFS and OS in LPL/WM patients. The median PFS and OS were significantly shorter than those without the abnormality (median PFS 34m vs 77m, p=0.008; median OS 34m vs 135m, p=0.006). However, the 6q deletion and trisomy 12 did not significantly influence both PFS and OS. In addition, we found that the number of different clones had an effect on the survival of patients. The complex clone group showed shorter PFS and OS than the single clone group (p<0.05).Conclusion: Although the detection of cytogenetic aberration is rare in Chinese patients, it plays an essential role in diagnosis and prognostication. DisclosuresNo relevant conflicts of interest to declare.

Identifying High-Risk Phenotypes Using Three-Way Feature Interactions in Multiple Myeloma: An Analysis of Publically Available Mmrf Commpass Study

The introduction of next generation sequencing has exponentially increased the number of potential disease targets from a limited panel of probes to markers across the entire genome. Previous studies of NGS data in multiple myeloma have identified key features associated with outcome in additive models, typically showing involvement of TP53, gain of 1q, MYC translocations, and others.We modified the multi-factor dimensionality reduction method (MDR) method, a computationally efficient method for detecting non-linear patterns of gene-gene interactions in genome-wide association studies (GWAS) first proposed by Richie et al, to identify three-way interactions significantly associated with outcome in MM. Specifically, we altered the ranking strategy of the survival adapted Cox-MDR method of Lee et al from balanced accuracy (average of sensitivity and specificity) to the absolute sum of deviance residuals. Due to the high level of censoring (94 events in 559 cases), balanced accuracy ranking favored interactions that identified few high risk cases and failed to validate well on external data.The publically available MMRF CoMMpass trial database (release version IA10) was utilized as primary training set for our MDR method. We included in our comparisons 164 RNA-SEQ genes with significant association with overall survival, the 20 most abundant non-synonymous, non-IG variants, and 24 estimated whole chromosome arm copy number gains or loss calls from the long insert WGS data of 559 baseline cases with outcome and clinical data (median follow-up 1.8 years). We searched for all three-way interactions across 210 total features resulting in the ranking of over 1.5 million unique three-way interactions, adjusted for age, ISS, creatinine level, and treatment by modified MDR. The top three-way RNA-expression interactions from our discovery phase were validated on 540 cases with GEP microarray and clinical data from UAMS Total Therapy 2 and 3 with median follow-up of 4.66 years (publically available data from GSE2658, GSE31161, and GSE24080).Of the top 100 three-way gene expression interactions discovered that we could validate on Affymetrix GEP data, 84 were significant in the UAMS TT2 and TT3 data set (log rank p-value < 0.05). HMGB3, a proposed inhibitor of myeloid differentiation, and DDX51, known to be differentially methylated in ALL, were the most commonly involved genes in the top significant interactions across both data sets. Pathway analysis of the 150 most frequently observed genes across the top 5,000 interactions in CoMMpass revealed an enrichment of DNA replication and cell cycle associated genes: RFC5, MCM2, MCM3, MCM4, MCM6, CCNA2, CDK2, etc.Within the top interactions that included whole arm copy number variants, gain of 1q and loss of 13q were the most frequently observed. We note that the top three-way interactions with gain of 1q paired exclusively with ALYREF, a transcriptional promoter, and one additional gene. Of the top interactions that included a SNV, mutations of DIS3 and DNAH5 were the most frequently observed. Many of the top interactions including DIS3 mutations paired with HMGB3 or TRIB3, a negative regulator of NF-kB and sensitizer to apoptosis, and one additional gene.MDR models are capable of identifying complex interactions of features that are not strictly additive. We observed a variety of interaction models, and present below an example where presence of 2 or more features resulted in overall negative outcome while presence of 1 or fewer did not. This situation was observed for the interaction of high expression of HMGB3, DDX51, and PSMB2, a bortezomib sensitizer in MM cell lines, where high expression in 2 or more of these genes imparted a negative outcome in CoMMpass and TT2/TT3 data sets. In the TT2/TT3 data set, cases identified as high risk from this interaction were enriched for the PR subtype and GEP70 HR.This modified MDR method is an effective tool at mining large genomic data sets for all k -way interactions associated with outcome among binary or multi-level features, adjusted for clinical covariates. Our modifications to MDR method allow us to identify more relevant interactions and increase reliability in validation data. This method could potentially further knowledge of the complex relationships between key features in genomic landscape of MM by discovering novel interactions that identify high-risk phenotypes. [Display omitted] DisclosuresBergsagel:Phosplatin Therapeutics: Research Funding.

Presence of Immunoglobulin Heavy Chain Gene (IGH) Translocations in Chronic Lymphocytic Leukemia Is Related to Poor Prognosis.

Background: Genetic analysis has an important role in the diagnosis, classification, and management of patients with CLL. Although translocations involving the IGH locus are well characterized in B-cell malignancies such as follicular cell lymphoma, their frequency in CLL are less well defined and little is known about their prognostic significance. We studied a series of CLL patients using a FISH probe set which included an IGH probe and reviewed clinical data and disease course of CLL patients with IGH translocations.Methods: FISH was performed on blood using a DNA probe set designed to detect common chromosome anomalies associated with CLL and translocations involving IGH. Patients with an IGH abnormality were further studied using FISH probes sets for IGH and cyclin D1, BCL-2, BCL-3, BCL-6, BCL-11a, c-MYC, MALT-1, c-MAF, FGFR-3 or PAX-5. Between 5/15/03 and 5/15/07, samples from 4,976 patients with a presumed lymphoproliferative disorder were analyzed by FISH. Of these, 316 patients (6.3%) had translocations involving the IGH locus. Sixty-three (21%) of the 316 patients with IGH translocations had been seen at Mayo Clinic Rochester and had clinical and laboratory data for detailed review and analysis of associations to the FISH results.Results: We identified 39 patients with CLL and IGH translocations. Identified partner genes were: BCL-2 (n=22), BCL-3 (n=7), CMYC (n=2), MALT-1 (n=1). The partner gene was not identified despite using above mentioned panel of partner gene probes in 7 patients. Median age at time of diagnosis of patients with IGH translocations was 62 (45-82), 29 patients were males. The median time to initial treatment (TTT) of all 39 patients with IGH translocations was 3.1 years. The median TTT for patients with IGH/BCL-2 was 3.6 years; 70% of patients required treatment by 5 years. The median TTT for IGH/BCL-3 patients was significantly shorter, 1.5 years, p=0.017, with all patients requiring therapy 3 years from diagnosis (see Figure 1).Conclusion: The presence of IGH translocations in CLL appears to be associated with rapid disease progression and the need for an early initiation of therapy. The IGH partner gene seems to be important; IGH/BCL-3 fusion is associated with a worse prognosis than IGH/BCL-2. Further studies on the biological significance of IGH translocations in CLL are warranted and continue at our institution. [Display omitted]

Multiplexed live-cell profiling with Raman probes

Single-cell multiparameter measurement has been increasingly recognized as a key technology toward systematic understandings of complex molecular and cellular functions in biological systems. Despite extensive efforts in analytical techniques, it is still generally challenging for existing methods to decipher a large number of phenotypes in a single living cell. Herein we devise a multiplexed Raman probe panel with sharp and mutually resolvable Raman peaks to simultaneously quantify cell surface proteins, endocytosis activities, and metabolic dynamics of an individual live cell. When coupling it to whole-cell spontaneous Raman micro-spectroscopy, we demonstrate the utility of this technique in 14-plexed live-cell profiling and phenotyping under various drug perturbations. In particular, single-cell multiparameter measurement enables powerful clustering, correlation, and network analysis with biological insights. This profiling platform is compatible with live-cell cytometry, of low instrument complexity and capable of highly multiplexed measurement in a robust and straightforward manner, thereby contributing a valuable tool for both basic single-cell biology and translation applications such as high-content cell sorting and drug discovery.

HLA-DRB1 mismatch-based identification of donor-derived cell free DNA (dd-cfDNA) as a marker of rejection in heart transplant recipients: A single-institution pilot study

Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.

Comprehensive Structure–Activity Profiling of Micheliolide and its Targeted Proteome in Leukemia Cells via Probe-Guided Late-Stage C–H Functionalization

The plant-derived sesquiterpene lactone micheliolide was recently found to possess promising antileukemic activity, including the ability to target and kill leukemia stem cells. Efforts toward improving the biological activity of micheliolide and investigating its mechanism of action have been hindered by the paucity of preexisting functional groups amenable for late-stage derivatization of this molecule. Here, we report the implementation of a probe-based P450 fingerprinting strategy to rapidly evolve engineered P450 catalysts useful for the regio- and stereoselective hydroxylation of micheliolide at two previously inaccessible aliphatic positions in this complex natural product. Via P450-mediated chemoenzymatic synthesis, a broad panel of novel micheliolide analogs could thus be obtained to gain structure–activity insights into the effect of C2, C4, and C14 substitutions on the antileukemic activity of micheliolide, ultimately leading to the discovery of “micheliologs” with improved potency against acute myelogenic leukemia cells. These late-stage C–H functionalization routes could be further leveraged to generate a panel of affinity probes for conducting a comprehensive analysis of the protein targeting profile of micheliolide in leukemia cells via chemical proteomics analyses. These studies introduce new micheliolide-based antileukemic agents and shed new light onto the biomolecular targets and mechanism of action of micheliolide in leukemia cells. More broadly, this work showcases the value of the present P450-mediated C–H functionalization strategy for streamlining the late-stage diversification and elucidation of the biomolecular targets of a complex bioactive molecule.

Germline and somatic mutation profile in Cancer patients revealed by a medium-sized pan-Cancer panel

Gene mutation detection and the resulted precision-medicine therapy is transforming clinical practice. Here, we report the use of a custom-developed, medium-sized, pan-cancer probe panel for the detection of somatic and germline mutations. We used a hybridization capture-based NGS assay for targeted deep sequencing of all exons and selected introns of 181 key cancer driver genes, covering both inherited risks and somatic mutations. We performed paired-variant calling on tumor samples and their matched normal samples. We processed clinical patient samples of formalin-fixed, paraffin embedded tumors (FFPE samples) and cell-free peripheral blood (cfDNA samples). We found germline mutations of inherited cancer risk at 9%; and discovered a novel germline mutation in BRCA1. Somatic mutation rate in driver genes is at 73.1%, much higher than previously reported. On recommending precision-medicine therapeutics, we achieved 91.6% for patients with FFPE samples.

A cohort study of men infected with COVID-19 for presence of SARS-CoV-2 virus in their semen

IntroductionWhether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen and transmitted sexually is a vital question that has, thus far, been inconclusive. Prior studies, with limited numbers, have included men in various stages of infection with most in the recovery phase of the illness. The timing of test results and severity of illness has made recruiting study participants a significant challenge. Our pilot study will examine semen from men with a recent diagnosis of COVID-19 as well as those in the convalescent phase to determine if SARS-CoV-2 can be detected and its relationship, if any, with the severity of the disease.MethodsEighteen men with a median age of 32 (range, 24-57) who tested positive for COVID-19 by rt-PCR analysis were enrolled and provided a semen sample. The study group demonstrated symptoms of COVID-19 ranging from asymptomatic to moderate and none required hospitalization. Samples were subjected to viral RNA extraction and then processed by real-time RT-PCR using the US Centers for Disease Control and Prevention (CDC, USA) panel of 2019-Novel Coronavirus (2019-nCoV) primers and probes to detect the presence of SARS-CoV-2 RNA.ResultsLength of time from diagnosis to providing a specimen ranged from 1 to 28 days (median, 6 days). Fifteen participants were symptomatic and three were asymptomatic, including recovering men, at the time of semen collection. No SARS-CoV-2 was detected in any of the semen samples.ConclusionBased on these preliminary results and consistent with prior findings, we suggest SARS-CoV-2 is not present in semen during the acute or convalescent phase of COVID-19.

Genotyping and Characterization of HPV Status, Hypoxia, and Radiosensitivity in 22 Head and Neck Cancer Cell Lines.

Simple SummaryCell lines are widely used for research, but even commonly used cell lines may not be well characterized, making validation of published data and generalizing the results to the clinic problematic. Here, we have established a head and neck squamous cell carcinomas (HNSCC) cell line database. We believe that these cell lines are suitable models to study HNSCC disease in vitro and in vivo and may provide a valuable tool for investigators wishing to study the relation between e.g., hypoxia and radiosensitivity in HNSCC.To study head and neck squamous cell carcinomas (HNSCC) in vitro, a large variety of HNSCC cell lines have been developed. Here, we characterize a panel of 22 HNSCC cell lines, thereby providing a tool for research into tumor-specific treatment options in HNSCC. Both human papillomavirus (HPV) positive and HPV negative tumor cell lines were collected from commercial and collaborative sources. Short tandem repeat profiling was used to confirm or characterize the identity of the cell lines. Targeted sequencing was performed using a standard pathology single molecule Molecular Inversion Probe panel to detect mutations for 23 tumor suppressors and oncogenes. HPV status, p16 status, radiosensitivity data, and hypoxia data are summarized from all cell lines. We detected HPV transcripts in five cell lines, all of which overexpressed p16. One HPV negative cell line was also p16 positive. We detected mutations in KIT (SCCNij185), PIK3CA (SCCNij185), and CDKN2A (UT-SCC-5 and UT-SCC-38). TP53 mutations were the most frequent, occurring in 16/22 cell lines. HPV infection and TP53 mutations were almost mutually exclusive, with the exception of 93-VU-147T. The cell lines exhibited a wide range of sensitivities towards hypoxia and irradiation. Here, we provide a description of a set of frequently used HNSCC cell lines with diverse characteristics as found in HNSCC patients.

Titin mutation in circulatory tumor DNA is associated with efficacy to immune checkpoint blockade in advanced non-small cell lung cancer.

BackgroundOnly a fraction of patients with advanced non-small cell lung cancer (NSCLC) respond well to immune checkpoint blockade (ICB) therapy. Here, we investigated whether Titin (TTN) mutation, which has been demonstrated to be a predictive biomarker in tissue-based analysis, can identify patients with a greater likelihood in response to ICB based on circulatory tumor DNA (ctDNA) sequencing.MethodsIn this retrospective analysis, 92 patients with advanced NSCLC from two independent cohorts who received ICB treatment were included. A probe panel covering all exons of TTN was developed and validated to detect TTN mutation in ctDNA. Baseline plasma samples were collected and subjected to ctDNA sequencing with the TTN probe panel.ResultsOf the 92 patients, 28.3% harbored TTN mutation in their baseline ctDNA. Progression-free survival was significantly improved in patients with the mutated TTN (212 days and 334.5 days for cohort 1 and 2) compared to those without the mutation (113 days and 147 days for cohort 1 and 2). Objective response to ICB treatment (40% for TTNmut and 15.8% for TTNwt in cohort 1; 50% for TTNmut and 23.4% for TTNwt in cohort 2) was common in patients with mutated TTN. Stratified analysis showed a generally predictive potential of TTN mutation in patients with advanced NSCLC.ConclusionsThe presence of mutated TTN in pre-treatment peripheral blood was associated with favorable objective response and survival with ICB administration. Therefore, circulatory TTN mutation may be applicable for guiding ICB immunotherapy in patients with NSCLC.

Abstract PS16-02: Moving in the fast lane: Test design and validation to produce up-to-date hereditary breast and gynecologic cancer tests

Abstract Introduction Germline genetic testing is increasingly relevant to breast and gynecologic (GYN) cancer clinicians for monitoring and managing high-risk patients. In particular, multi-gene panels (MGPs) can identify unsuspected cancer syndromes and variants that may become clinically significant. Effective MGPs must be comprehensive and up-to-date. For the current study, we used a probe panel designed for targeted enrichment of 4,500 genes associated with various inherited diseases to develop and validate a 66-gene comprehensive hereditary cancer panel, including subsets of genes associated with breast and GYN cancers. Materials and Methods Genomic DNA was extracted and taken through next generation sequencing (NGS) library preparation to be sequenced on an Illumina NovaSeq instrument. Targeted capture-based enrichment with a long-range PCR (LR-PCR) component was used to interrogate all protein-coding exons, intron-exon splice sites (+/-10bp), as well as clinically relevant deep intronic, 5’UTR, and 3’UTR regions for single nucleotide variants (SNVs) and insertions/deletions (indels) of all genes of interest. Copy number variations (CNVs) were also interrogated for all applicable regions. Data analysis was performed using a proprietary in-house bioinformatics variant analysis pipeline. For validation, samples from the Coriell Repository and more than 100 unique de-identified genomic DNA specimens from whole blood and saliva were analyzed for 17,911 variants in 508 genes. Variants were previously identified by orthogonal methods (in-house Sanger sequencing, CLIA validated NGS assays, and microarray). The well-characterized Genome in a Bottle (GIAB) NA12878 and Ashkenazim Trio samples (NA24149, NA24385, and NA24143) were also included. The analytic sensitivity (Positive Percent Agreement, %PPA) and specificity (Technical Positive Predictive Value, %TPPV and Negative Percent Agreement, %NPA) were determined for each variant type (SNV, indel, and CNV). The 66-gene hereditary cancer panel includes genes that confer ≥2-fold increased risk or 5% lifetime risk for developing cancer (APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN1B, CDKN2A (p16, p14), CHEK2, DICER1, EGFR, EPCAM, FANCA, FANCC, FANCM, FH, FLCN, GALNT12, GREM1, HOXB13, MAX, MEN1, MET, MITF, MLH1, MRE11 (MRE11A), MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NTHL1, PALB2, PMS2, POLD1, POLE, POT1, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RECQL, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMARCA4, SMAD4, STK11, SUFU, TMEM127, TP53, TSC1, TSC2, VHL, and XRCC2). Of these, 30 genes increase the lifetime risk of breast and/or GYN cancers and are also distributed among smaller phenotype-specific panels. Results The analytical sensitivity (%PPA) for SNVs and indels was 100.0% and 97.8% for CNVs. The overall specificity for SNVs, indels, and CNVs was &amp;gt;99.0%. The %TPPV for SNVs, Indels, and CNVs was 100.0%, 99.3%, and 100.0%, respectively. The %NPA for SNVs, Indels, and CNVs was 100.0%. The %PPA, %TPPV, and %NPA for LR-PCR was 100.0%. Conclusion Validation of the 66-gene hereditary cancer panel demonstrated high analytical sensitivity and specificity. As additional gene-cancer associations are established, using an already designed and developed comprehensive 4,500 gene panel will expedite the process of updating panel tests to include relevant candidate genes, allowing clinicians and patients to benefit from up-to-date and comprehensive testing. Citation Format: Taraneh E Angeloni, Anindya Bhattacharya, Linda L Cheng, Hansook K Chong, Kelli M Conlan, Christopher D Elzinga, Anna Gerasimova, David Grover, Andrew Grupe, Michael Hua, Domagoj Hodko, Krista Kazmierkiewicz, Rebecca E Nakles, Camille R Nery, Renius Owen, Dana M Goos-Root, Charles M Rowland, Alla Smolgovsky, Elaine C Weltmer, Ke Zhang, Felicitas L Lacbawan. Moving in the fast lane: Test design and validation to produce up-to-date hereditary breast and gynecologic cancer tests [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 PS16-02.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping

There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called “capture RASL-seq” (cRASL-seq), which enables highly sensitive (down to ~1–100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

SARS-CoV-2 Epitope Mapping on Microarrays Highlights Strong Immune-Response to N Protein Region.

A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155–71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155–171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

Accuracy Comparisons of Channel Emulation Methods for Two-dimensional Uniform and Three-dimensional Sectored Multi-probe Anechoic Chamber

Multiple-input-multiple-output (MIMO) over-the- air (OTA) testing has been seen as a promising solution for evaluation of the radio performance of MIMO devices. In this work, we compare the accuracy of two channel emulation methods, i.e., the prefaded signal synthesis (PFS) and the plane wave synthesis (PWS), in two-dimensional (2D) uniform and 3D sectored multi-probe anechoic chamber (MPAC), respectively. The PWS method is proven to be more accurate than the PFS for 2D uniform MPAC system. However, for 3D sectored MPAC system, the PFS method emerged to be better than the PWS method. To explain these seemingly contradicting findings, both the required number of active probes and the leakage of power spectrum are considered in this paper. It is found that the PWS method has higher emulation accuracy than the PFS method when the number of active probes becomes sufficiently large, whereas the PFS is more robust to the undersampling due to the limited number of active probes in practical 3D sectored MPAC system. Moreover, when the number of active probes is particularly small (less than the number of clusters in the probe panel), the emulation accuracy of the PWS method outperform its counterpart.

423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

BackgroundCOVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome.MethodsThe SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis.ResultsThe SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens.ConclusionThe SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease.DisclosuresDorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

Rational molecular design of a reversible BODIPY-Based fluorescent probe for real-time imaging of GSH dynamics in living cells

Marring the reversible covalent chemistry with BODIPY dye, which is a superfamily of fluorophores with striking photophysical performances, would enable a panel of diverse dynamic fluorescent probes for biomedical applications. Herein we show that structural manipulation of BODIPY allows rational tuning of α-site or meso-site activation as well as the spectral response toward nucleophiles. By rational molecular design, we have obtained a highly specific and reversible GSH probe, αBD-GSH, which exhibits a tremendously fast and dynamic fluorescence response within the wide physiological GSH concentration range of 0–8 mM. We successfully applied αBD-GSH to real-time imaging of intracellular GSH dynamics in different cell lines. In light of the remarkable photophysical properties and synthesis flexibility of BODIPY dyes, the current findings will help to design more reversible BODIPY-based fluorescent probes targeting various bio-species.

Prevalence of Genetic Abnormalities in Patients with Multiple Myeloma and Its Clinical Relevance in a Developing Country like India

Introduction Multiple myeloma (MM) is a malignancy involving terminally differentiated plasma cells. Its incidence in India is about 0.7/1,00,000 population amounting to about 6,800 new cases a year A number of genomic aberrations are associated with MM, most of which confer prognostic significance. Cytogenetic abnormalities are a part of R-ISS score for prognostication which stratifies presence of del(17p), t(4;14) or t(14;16) as stage 3, mSMART is another risk stratification tool which divides MM into high risk and standard risk groups based on genetic aberrations. Hence it is evident that determining the genetic abnormality in MM is important. however, due to limited resources genetic testing is not routinely done and the data in the Indian population is limited. Objective: To estimate the prevalence of molecular cytogenetic abnormalities by Fluorescent in situ hybridization (FISH) analysis in patients with MM and to assess the co-relation with response to induction chemotherapy, relapse and overall survival. Material and Methods: 64 patients were included from January 2016 to December 2019 and followed up till June 2020. Interphase FISH study was performed either at diagnosis or at relapse, on bone marrow aspirate with panel of probes consisting of CKS1B (1q21-22), CDKN2C (1p32.3), D13S319 (13q14.2/13q34), IGH (14q32.33), p53 (17p13.1) and trisomy (5p15/9q22/15q22) (trisomies are considered as hyperdiploidy in this study). Plasma cell purification techniques were not applied prior to FISH analysis. Patients were divided into 2 risk groups; 1) high risk group with presence of del17p, del13q, amplification 1q, del1p and two or more aberrations with either of these and 2) standard risk group with presence of hyperdiploidy or no genetic abnormality. There was no difference in chemotherapy regimen between the 2 groups; 46 (71.8%) received bortezomib-thalidomide-dexamethasone, 10 (15.6%) received bortezomib-cyclophosphamide-dexamethasone, 2(3.1%) received bortezomib-lenalidomide-dexamethasone, 1(1.5%) received daratumumab-bortezomib-dexamethasone and 5(7.8%) received 2 drug chemotherapy. Patients who did not complete minimum follow up of 6 months either due to death or lost to follow up were excluded from the study. Institutional Ethics Committee's approval was taken. Results: Mean age of the population was 60.33 years and male to female ratio was 1.65. 46.87%, 28.13% and 25% of the study population were in the age group of ≤ 60, 61 - 65 and ≥66 years respectively. 12.3%, 43.8% and 43.8% were in R ISS stage 1, 2 and 3 respectively. FISH analysis was done on 61 out of 64 patients (remaining 3 were excluded due to hemodilute bone marrow sample). 22 (36.1%) patients had abnormal genetic aberration on FISH analysis with 10 (16.39%) having two or more abnormalities. The frequency of genetic aberrations was as follows; amplification 1q (13/61, 21.31%), del13q (9/61, 14.75%), hyperdiploidy (7/61, 11.47%), del17p (4/61, 6.55%), IgH rearrangement (3/61, 4.91%), and del1p (1/61, 1.6%). All 3 patients with IgH rearrangement had associated one or more high-risk genetic aberration and hence were included in high risk group. 31.1% of the patients were high risk and 68.9% were standard risk. The response to induction chemotherapy, incidence of relapse, time to 1st relapse and total number of relapses are shown in (table;1) and there was no significant difference between high risk and standard risk group. Overall survival in standard risk group at 2 and 5 years was 89.6% ± 5.8% and 78.6% ± 13.9% and in high risk group was 60.7% ± 13.2% and 29.5% ± 23.1% respectively (p value: 0.762). Overall survival was significantly lower in age group ≥66 years as compared to age group ≤ 60 years and 61 - 65 years (p value 0.001) and it was also significantly lower in R ISS stage 3 as compared to R ISS stage 1 and 2 (p value 0.006). Conclusion: More than one third patients of MM (36.1%) showed genetic abnormality, amplification 1q being the most frequent. Overall survival was significantly lower in older age group and R ISS stage 3 patients. Response to induction chemotherapy and relapse rate were similar in high and standard risk groups. Although overall survival was lower in high risk group, it was statistically not significant. This study highlights the importance of FISH analysis for disease stratification and prognostication which should be routinely practiced. Disclosures No relevant conflicts of interest to declare.

Duplex Sequencing with Patient-Specific Hybrid Capture Panels Reveals Ultra-Low Frequency Measurable Residual Disease in Pediatric Acute Myeloid Leukemia

Sensitive and specific detection of measurable residual disease (MRD) after treatment in pediatric acute myeloid leukemia (AML) is prognostic of relapse and is important for clinical decision making. Mutation-based methods are increasingly being used, but are hampered by the limited number of common driver gene mutations to target as clone markers. Additional targets would greatly increase MRD detection power. However, even in cases with many AML-defining mutations, it is the limited accuracy of current molecular methods which establishes the lower bounds of sensitivity. Here we describe an ultrasensitive approach for disease monitoring with personalized hybrid capture panels targeting hundreds of somatic mutations identified by whole genome sequencing (WGS), and using extremely accurate Duplex Sequencing (DS) in longitudinal samples. In a pilot cohort of 13 patients we demonstrate detection sensitivities several orders of magnitude beyond currently available single locus testing or less accurate sequencing. With multi-target panels, overall power for MRD detection is cumulative across sites. For example, if a patient has MRD at a true frequency of 1/30,000, sequencing a single mutant site to 10,000x molecular depth would be unlikely to detect MRD. However, sequencing 10 sites each to 10,000x would effectively total 100,000x informative site depth, increasing power to &amp;gt;95%. However, standard sequencing assays are insufficiently accurate to achieve this theoretical limit of detection (LOD). DS enables accurate detection of individual variants to &amp;lt;10-5 with an error rate &amp;lt;10-7 and, thus, can achieve MRD sensitivities below one-in-one-million. Marrow aspirates were collected from 13 uniformly treated pediatric AML patients at time of diagnosis (TOD), during treatment (end of induction, EOI), in remission (end of therapy, EOT), and at relapse. 9/13 patients relapsed. DNA from TOD was analyzed by WGS. Germline variants were excluded and somatic single nucleotide variants (SNVs) were targeted by a custom probe panel designed for each patient. An average of 170 SNVs were targeted per patient (range 53-200). More than 90% of the SNVs were noncoding. Longitudinal samples were then analyzed with DS, which compares sequences from both strands of each DNA molecule to eliminate technical noise and reveal biological mutation signal with extreme accuracy and sensitivity. A median of 82% of WGS SNVs were validated by DS in the TOD DNA, and the vast majority of those were also present at relapse. Relapsers had more SNVs at diagnosis than non-relapsers. EOT samples were sequenced to an average Duplex molecular depth of 29,400x, with a maximum of 61,283x. The figure below shows time course plots tracking SNVs at diagnosis, EOT and relapse for 2 patients. Among mutations validated in TOD samples, a median of only 8 (5%) were detected per EOT sample (range 0-66 mutations). MRD was detected in 8/9 relapsers. Targeting 1 or even 10 SNVs would therefore have missed MRD in the majority of these patients. Among relapsers, median EOT SNV VAF was 0.069%. The lowest single VAF detected per EOT sample ranged from 0.036% to 0.002%. The presence of an SNV at diagnosis and relapse implies that it must truly be present at EOT, whether or not it is detected. Therefore, if a small minority of leukemic mutations are detected at EOT, the true overall MRD frequency is much lower than the LOD at any single site. In the only relapser where MRD was not detected, targeted SNVs were present at diagnosis and relapse, so additional sequencing depth at EOT would eventually reveal ultra-low frequency mutations. Among patients that did not relapse by the end of the study, median VAF at EOI (the latest time point DNA available) was 0.0258%. Therefore, non-relapsers have a lower median VAF at EOI than relapsers do even later at EOT, potentially indicating very early on that treatment is more successful. This study shows excellent performance of DS-based assays for detecting MRD with patient-specific panels. We have demonstrated that among large panels of validated somatic SNVs present at time of diagnosis, a median of 5% are identified at EOT in eventual relapsers. DS detected MRD in 8/9 patients, and at a median VAF well below the limit of detection of any other sequencing technology. Comprehensive personalized hybrid selection panels coupled with DS represents a powerful option for MRD monitoring in pediatric AML and potentially other cancers. Figure Disclosures Higgins: TwinStrand Biosciences: Current Employment. Lo:TwinStrand Biosciences: Current Employment. Hipp:TwinStrand Biosciences: Current Employment. Valentine:TwinStrand Biosciences: Current Employment. Williams:TwinStrand Biosciences: Current Employment. Radich:TwinStrand Biosciences: Research Funding. Salk:TwinStrand Biosciences: Current Employment.