PLUS Kit Research Articles

Paired comparison of the analytical performance between the Oncomine™ Comprehensive Assay v3 and whole-exome sequencing of ovarian cancer tissue

BackgroundNext-generation sequencing (NGS) has been implemented in clinical oncology as a personalized medicine tool to identify targetable genetic alterations and to guide treatment decisions. However, the optimal NGS test strategy and target genes for clinical use are still being discussed. The aim was to compare the performance of the Oncomine™ Comprehensive Assay v3 (OCAv3) (targeted gene panel) and whole-exome sequencing (WES) to investigate somatic single and multiple nucleotide variants and small indels in ovarian cancer patients.Methods and resultsGenomic DNA was isolated from fresh frozen samples of five high-grade serous (HGSC) and three clear cell ovarian (oCCC) cancer patients. Exome sequencing libraries were prepared by using the Ion AmpliSeq Exome RDY kit, whereas libraries for OCAv3 were prepared using by Ion AmpliSeq™ Library Kit Plus. Sequencing was performed using the Ion S5XL System (Thermo Fisher Scientific). When including only variants classified as pathogenic, likely pathogenic or unknown significance based on ClinVar database verdicts and comparing overlapping regions covered both by the OCAv3 assay and WES, 23 variants were detected by both assays. However, OCAv3 detected additionally two variants: ARID1A: p.Gln563Ter and TP53: p.Ser261ValfsTer84 that have not passed WES filtering criteria due to low coverage.ConclusionsWith the present treatment possibilities, OCAv3 panel testing provided higher diagnostic yield due to better coverage. Our study emphasizes that WES, although offering the potential to identify novel findings in genes not covered by OCAv3, might overlook variants in genes relevant for OC.

Alternative genome sequencing approaches of SARS-CoV-2 using Ion AmpliSeq Technology

A thorough understanding of SARS-CoV-2 genetic features is compulsory to track the ongoing pandemic across multiple geographical locations of the world. Thermo Fisher Scientific USA has developed the Ion AmpliSeq SARS-CoV-2 Research Panel for the targeted sequencing of SARS-CoV-2 complete genome with high coverage and lower error rate. In this study an alternative approach of complete genome sequencing has been validated using different commercial sequencing kits to sequence the SARS-CoV-2. Amplification of cDNA with the SARS-CoV-2 primer pool was performed separately using two different master mixes: 2X environmental master mix (EM) and Platinum™ PCR SuperMix High Fidelity master mix (PM) instead of 5X Ion AmpliSeq™ HiFi Mix whereas NEBNext® Fast DNA Library Prep Set for Ion Torrent™ kit was used as an alternative to Ion AmpliSeq Library Kit Plus for other reagents. This study demonstrated a successful procedure to sequence the SARS-CoV-2 whole genome with average ∼2351 depth and 98.1% of total the reads aligned against the reference sequence (SARS-CoV-2, isolate Wuhan-Hu-1, complete genome). Although genome coverage varied, complete genomes were retrieved for both reagent sets with a reduced cost. This study proposed an alternative approach of high throughput sequencing using Ion torrent technology for the sequencing of SARS-CoV-2 in developing countries where sequencing facilities are low. This blended sequencing technique also offers a low cost protocol in developing countries like Bangladesh.

LincRNA-p21 Promotes Cellular Senescence by Down-regulating the Wnt/β-catenin Pathway in MPP+-treated SH-SY5Y Cells.

Long intergenic non-coding RNA-p21 (lincRNA-p21) plays a critical role in various senescence-associated physiological and pathological conditions. We aimed to explore the senescence-associated effects of lincRNA-p21 in 1-methyl-4-phenylpyridinium (MPP+) treated neuroblastoma SH-SY5Y cell line as a therapeutic target. The RNA expression levels of lincRNA-p21, p53, p16, and telomere length were examined with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The Telo TAGGG™ Telomerase PCR ELISA PLUS Kit was used to determine telomerase activity. Cellular viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay. Western blot was performed to analyze β-catenin protein expression. Besides, oxidative stress was evaluated by J-aggregate-forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC‑1) stain, fluorescence spectrophotometry, colorimetric assay, and malondialdehyde (MDA) formation. This research demonstrated that MPP+ caused a distinct increase in the expression of LincRNA-p21 in SH-SY5Y cells. MPP+ induced cellular senescence with decreasing cellular proliferation and viability, increasing expression levels of senescence-associated makers such as genes p53 and p16, accompanied by significantly decreasing telomere length and telomerase activity. At the same time, these effects were abolished by silencing lincRNA-p21 with small interfering RNA (siRNA). On the contrary, β-catenin silencing contributes to reversing anti-senescent effects caused by lincRNA-p21 silencing. Moreover, modifying lincRNA-p21 exerted an anti-senescent influence depending on decreasing oxidant stress. Our study showed that in the treatment of MPP+, lincRNA-p21 might serve a role in the SH-SY5Y cell senescence by modulating the Wnt/β-catenin pathway, as well as increasing oxidant stress. Thus, trying to target lincRNA-p21 may have important therapeutic and practical implications for PD.

Sequencing the SARS-CoV-2 Genome from Stool Samples of Post-acute Cases Implicates a Novel Mutation Associated with Reduced Antibody Neutralization.

Whole-genome SARS-CoV-2 sequencing tools are crucial for tracking the COVID-19 pandemic. However, current techniques require sampling of actively infectious patients following COVID-19 testing to recover enough SARS-CoV-2 RNA from the nasopharyngeal passage, which rapidly clears during the first few weeks of infection. A prospective assessment of the viral genome sourced from recovered non-infectious patients would greatly facilitate epidemiological tracking. Thus, we developed a protocol to isolate and sequence the genome of SARS-CoV-2 from stool samples of post-acute SARS-CoV-2 patients, at timepoints ranging from 10-120 days after onset of symptoms. Stool samples were collected from patients at varying timepoints post-convalescence, and viral DNA was isolated and sequenced using the QIAamp Viral RNA Mini Kit (Qiagen Inc.) and Ion Ampliseq™ Library Kit Plus (Life Technologies Corporation). Capacity of neutralizing antibodies in patient plasma was tested using a Luminex panel (Coronavirus Ig Total Human 11-Plex ProcartaPlex™ Panel, ThermoFisher). Of 64 samples obtained from post-acute patients, 21 (32.8%) yielded sufficient material for whole-genome sequencing. This allowed us to identify widely divergent phylogenetic relativity of the SARS-CoV-2 genome from post-acute patients living in the same households and infected around the same time. Additionally, we observed that individuals who recovered from infection expressed varying degrees of antibodies against SARS-CoV-2 structural proteins that corresponded to distinct variants. Interestingly, we identified a novel point mutation in the viral genome where infected patients expressed antibodies with a significantly reduced capacity to neutralize the virus in vitro relative to that of those infected with the wild-type strain. Altogether, we demonstrate a protocol to successfully sequence the SARS-CoV-2 genome from stool samples from patients up to 4 months post-infection, which can be applied to studies that assess the relationship between variants and immune response post-hoc and safe monitoring of the SARS-CoV-2 genome during the pandemic.

The Effect of Ascorbic Acid and Nicotinamide on Panton-Valentine Leukocidin Cytotoxicity: An Ex Vivo Study.

Background: Panton−Valentine Leukocidin sustains a strong cytotoxic activity, targeting immune cells and, consequently, perforating the plasma membrane and inducing cell death. The present study is aimed to examine the individual effect of ascorbic acid and nicotinamide on PVL cytotoxicity ex vivo, as well as their effect on granulocytes viability when treated with PVL. Materials and Methods: The PVL cytotoxicity assay was performed in triplicates using the commercial Cytotoxicity Detection Kit PLUS (LDH). LDH release was measured to determine cell damage and cell viability was measured via flow cytometry. Results and discussion: A clear reduction in PVL cytotoxicity was demonstrated (p < 0.001). Treatment with ascorbic acid at 5 mg/mL has shown a 3-fold reduction in PVL cytotoxicity; likewise, nicotinamide illustrated a 4-fold reduction in PVL cytotoxicity. Moreover, granulocytes’ viability after PVL treatment was maintained when incubated with 5 mg/mL of ascorbic acid and nicotinamide. Conclusions: our findings illustrated that ascorbic acid and nicotinamide exhibit an inhibitory effect on PVL cytotoxicity and promote cell viability, as the cytotoxic effect of the toxin is postulated to be neutralized by antioxidant incubation. Further investigations are needed to assess whether these antioxidants may be viable options in PVL cytotoxicity attenuation in PVL-associated diseases.

Next generation sequencing for the formalin-fixed paraffin-embedded samples of ovarian cancer in Vietnam

Ovarian cancer is one of the most popular causes of mortality among women, and the prevalence of ovarian cancer is increased. Early diagnosis of this disease via genetic variant testing is one potential strategy for enhancing treatment and disease outcome. In this study, we used the formalin-fixed, paraffin-embedded (FFPE) samples of ovarian tumor tissues from Vietnamese patients to detect pathogenic variants in BRCA1/BRCA2 via next-generation sequencing. DNA was extracted using QIAamp DNA FFPE Tissue Kit, and then its quality was assessed using BioDrop and Qubit. The BRCAaccuTestTM PLUS kit and Illumina MiSeqDx instrument were used for both library preparation and sequencing. All samples had passed the A260/280 ratio cut-off for DNA purity and the requirement of DNA concentration. Excepted for the 1st time, the percentage of ≥ Q30 was more than 80%, while the density was approximately 1,200 K/mm2, while the phasing and prephasing (%) metrics were satisfied to be less than 0.1%. 5 pathogenic variants in BRCA1/BRCA2, including both single nucleotide polymorphisms and indels were successfully detected using NgeneAnalySysTM software.

Abstract 437: Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel

Abstract Background: The rapid spread of COVID-19 has resulted in an urgent need for effective diagnostic and therapeutic strategies against SARS-CoV-2. Next-generation sequencing (NGS) is a powerful tool in the identification and characterization of this pathogen and genomic information may aid in understanding the mechanisms of therapeutic resistance, vaccine escape, virulence, and pathogenicity. The Ion AmpliSeq SARS-CoV-2 Research Panel is a targeted NGS solution that facilitates sequence analysis of the SARS-CoV-2 genome. Paired with a bioinformatics assembly and variant calling pipelines, this assay allows for accurate characterization of the dominant SARS-CoV-2 variant. This assay’s performance was analytically validated for the detection of mutations (substitutions, insertions, and deletions) in RNA derived from nasopharyngeal (NP) swabs. Method: The Ion AmpliSeq SARS-CoV-2 Research panel consists of two primer pair pools generating 237 amplicons specific to the SARS-CoV-2 virus. Reverse transcription of the RNA was performed using the SuperScript VILO cDNA Synthesis kit. Library preparation was then completed using the Ion AmpliSeq Library Kit Plus kit. The final library was quantified, normalized, pooled, and sequenced. Raw sequencing data was aligned to the AmpliSeq SARS-CoV-2 Research panel, using the MN908947.3 reference genome. Variants were called using the Torrent Variant Caller and annotated using the COVID19AnnotateSnpEff plugin. The reference-guided iterative assembler IRMA was used to produce a single consensus sequence consisting of the reference genome sequence modified to include sequence variations supported by the reads. The Pangolin COVID-19 lineage assigner software tool was used to assign SARS-CoV-2 lineage. Analytical validation was completed using controls (Twist Biosciences, BEI Resources, ATCC) and RNA derived from NP swabs. Accuracy and specificity were examined by evaluating the correctness of calling true negative variants compared to false positive and all other variant calls, respectively. Precision and limit of detection (LoD) were examined by evaluating the concordance of variants across replicate samples. Limit of Blank (LoB) was calculated as the 95th percentile of reads per amplicon in the negative samples. Results: Accuracy of base calling, specificity, and precision were 100% for SNVs, insertions, and deletions above 25% allele frequency. LoD was determined to be 576 viral copies/mL. LoB was determined to be 202 reads per amplicon. Pangolin lineage assignment was 100% for all samples. Conclusions: This panel accurately characterizes SARS-CoV-2 variants, allowing for accurate consensus sequence generation, mutation annotation, and lineage assignment. Citation Format: Desiree Unselt, Katherine Knudsen, Christopher Rounds, Janet Doolittle-Hall, Fernando Torres, Jennifer Sims, Jennifer Mason. Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 437.

SARS-CoV-2 Whole-Genome Sequencing by Ion S5 Technology-Challenges, Protocol Optimization and Success Rates for Different Strains.

The COVID-19 pandemic demonstrated how rapidly various molecular methods can be adapted for a Public Health Emergency. Whether a need arises for whole-genome studies (next-generation sequencing), fast and high-throughput diagnostics (reverse-transcription real-time PCR) or global immunization (construction of mRNA or viral vector vaccines), the scientific community has been able to answer all these calls. In this study, we aimed at the assessment of effectiveness of the commercially available solution for full-genome SARS-CoV-2 sequencing (AmpliSeq™ SARS-CoV-2 Research Panel and Ion AmpliSeq™ Library Kit Plus, Thermo Fisher Scientific). The study is based on 634 samples obtained from patients from Poland, with varying viral load, assigned to a number of lineages. Here, we also present the results of protocol modifications implemented to obtain high-quality genomic data. We found that a modified library preparation protocol required less viral RNA input in order to obtain the optimal library quantity. Concurrently, neither concentration of cDNA nor reamplification of libraries from low-template samples improved the results of sequencing. On the basis of the amplicon success rates, we propose one amplicon to be redesigned, namely, the r1_1.15.1421280, for which less than 50 reads were produced by 44% of samples. Additionally, we found several mutations within different SARS-CoV-2 lineages that cause the neighboring amplicons to underperform. Therefore, due to constant SARS-CoV-2 evolution, we support the idea of conducting ongoing sequence-based surveillance studies to continuously validate commercially available RT-PCR and whole-genome sequencing solutions.

Challenges in obtaining high-quality data from a custom-made panel for the next generation sequencing (NGS) using Ion Torrent GeneStudio™ S5 platform

The goal of this part of the study was to optimize the sequencing procedure for 16 human genes and their regulatory regions that might be associated with differential immunological response to COVID-19. The study was performed on 60 COVID-19 patients from the General Hospital of Tešanj, Bosnia and Herzegovina, categorized into three groups of mild, moderate, and severe clinical manifestation, based on the diagnosis by the residential physician. Target coding sequences and their regulatory regions were amplified for the following genes: HLA-A, HLA-B, HLA-C, ACE2, IL-6, IL-4, TMPRSS2, IFITM3, IL-12, RIG-I/DDX58, IRF-7, IRF-9, IL-1B, IL-1A, CD55, and TNF-α. DNA was isolated from the whole blood samples stored at -20°C for six months using QIAamp® DNA Mini Kit according to manufacturer’s instructions. Since NGS analysis of target genomic regions was performed on the Ion Torrent GeneStudio™ S5 platforms, libraries were prepared using Ion AmpliSeq™ Library Kit Plus according to manufacturer’s instructions in a protocol optimized for low-quality DNA. Due to dissatisfactory sequencing results, further protocol optimization steps were employed through separating two primer pools, increasing the number of PCR cycles, and decreasing the annealing temperature for the primer pool which showed poorer amplification results. In the end, 36 samples produced optimal results, while the remaining 24 samples will be re-sequenced following repeated sample collection and DNA isolation, accompanied by additional protocol modifications.

Detection of Microsatellite Instability in Colorectal Cancer Patients With a Plasma-Based Real-Time PCR Analysis.

A microsatellite instability (MSI) test is crucial for screening for HNPCC (Hereditary nonpolyposis colorectal cancer; Lynch syndrome) and optimization of colorectal cancer (CRC) treatment. Mismatch repair (MMR) deficiency is a predictor for good response of immune checkpoint inhibitors in various malignancies. In this study, we evaluated the results of a newly developed plasma-based real-time PCR kit for the detection of MSI in CRC patients. We assessed a peptide nucleotide acid (PNA) probe-mediated real-time PCR test (U-TOP MSI Detection Kit Plus) that determines MSI status by using amplicon melting analysis of five markers (NR21, NR24, NR27, BAT25, and BAT26) from plasma. Eighty-four CRC patients (46 dMMR and 38 pMMR) with colorectal cancer were analyzed. The concordance rate of MSI status assessment between the plasma kit and IHC was 63.0% in dMMR patients (29/46), but in the pMMR evaluation, a 100% (38/38) concordance rate was observed. In the evaluation of the performance of a custom tissue U-TOP MSI Detection Kit and plasma kit in 28 patients, sensitivity, specificity, PPV (positive predictive value) and NPV (negative predictive value) of plasma kit were 68.4, 100, 100, and 44.4%, respectively, with the tissue U-TOP MSI Detection Kit. Our results demonstrate the feasibility of a non-invasive and rapid plasma-based real-time PCR kit (U-TOP MSI Detection Kit Plus) for the detection of MSI in colorectal cancer.

Benchmarking Single-Cell mRNA–Sequencing Technologies Uncovers Differences in Sensitivity and Reproducibility in Cell Types With Low RNA Content

Single-cell RNA sequencing (scRNA-seq) has the ability to classify each cell and determine the transcriptomic profile of specific cell types and cells of a given disease state; however, sensitivity of the gene count for each cell can be a critical component to the success of a single-cell study. The recently introduced SMART-Seq Single Cell PLUS Kit (SSsc PLUS) claims to provide higher sensitivity and reproducibility versus popular methods for the sequencing analysis of single cells. Here, the cDNA-generation component of the kit, SMART-Seq Single Cell Kit (SSsc), was compared with the popular homebrew protocol, Smart-seq2, and its update, Smart-seq3. The SMART-Seq Library Prep Kit from SSsc PLUS was benchmarked against a commonly used scRNA-seq library preparation method, Illumina Nextera XT. Finally, the SSsc chemistry was tested in both full and fractional volumes on 2 popular liquid-handler devices to investigate whether the high sensitivity was maintained in miniaturization. We demonstrate that SSsc PLUS outperforms these other full-length methods in convenience, sensitivity, gene identification, and reproducibility while also offering full compatibility with automation platforms.

The utility of p16Ink4a/Ki-67 dual immunostain to triage women with Hr-HPV positive and negative Pap smear results (NILM) in Harare, Zimbabwe

Background: Women who test Hr-HPV+/NILM have a 3-7% risk harboring of undetected underlying precancerous cervical lesions. The biomarkers: p16 ink4a and Ki67 are surrogate markers of cell-cycle dysregulation mediated by transforming HPV infections. Therefore, the p16 ink4a /Ki67 dual immunostain can be used to identify women with transforming HPV infections; these women have a higher risk of harboring undetected cervical precancerous lesions. Objectives: (1) To determine the proportion of women with prior Hr-HPV+/NILM screening results who are positive for p16 ink4a/Ki67 dual immunostain. (2) To compare Hr-HPV persistence rates between women with positive and negative immunostain results after 12 months. Design: Cross sectional descriptive study. Setting: Cimas Medical Laboratories and Parirenyatwa Central Hospital Laboratory. Subjects: Residual LBC samples from women with prior HR-HPV+/NILM results. Materials and Methods: Cell blocks were prepared from the residual LBC samples using the plasma-thrombin method. A paraffin serial section was cut from the cell blocks and stained manually with a mix of two monoclonal antibodies against p16Ink4a and Ki-67 proteins using the CINtec PLUS kit. The slides were evaluated using the binary rating score system: positive or negative. Follow-up Hr-HPV persistence rates were compared using an independent t-test. Results: Of the 82 residual LBC samples evaluated: 17.1% [14/82] were positive and 82.9% [68/82] were negative for the p16 ink4a/Ki67 dual immunostain. The 12 month follow up attrition rate was 75.6% [62/82]. The follow up Hr-HPV persistence rates in p16 ink4a/Ki67 dual positive and negative women were 72.0% [8/11] and 47.1% [24/51] respectively. These two Hr-HPV persistence rates were distinct (p=0.01). Conclusions: Approximately 20% of patients with Hr-HPV+/ NILM screening results are positive for the p16Ink4a/Ki67 dual immunostain. Hr-HPV infections in p16Ink4a/Ki67 dual immunostain positive women are more likely to persist compared to women with negative dual immunostain results. Recommendation: p16Ink4a/Ki67 dual immunostain should be done on all patients with Hr-HPV+/ NILM results to stratify them according to the risk of harboring cervical precancerous lesions.

Clonal Evolution in CLL

Clonal Evolution in CLL

SNHG1/miR-186/FUT8 regulates cell migration and invasion in oral squamous cell carcinoma.

Recently, lncRNAs are associated with the progression and development of various cancers. We aimed to explore the effects of lncRNA SNHG1 on the proliferation, apoptosis, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. Quantitative real-time PCR (RT-qPCR) was used for measurement of SNHG1 in OSCC cells. Cell proliferation, apoptosis, migration, and invasion were detected by CCK-8 assay, flow cytometry, Cell Death Detection ELISA PLUS kit, and transwell assays. Dual-luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were used to clarify the relationship between SNHG1 and miR-186. SNHG1 was overexpressed in OSCC cells. SNHG1 silencing prevented cell proliferation and increased the incidence of apoptosis, DNA fragments, cleaved-caspase 3, and Bax protein levels. Cell migration and invasion were reduced after SNHG1 deletion, and MMP2 and MMP9 protein levels were decreased. SNHG1 overexpression promoted cell survival, migration, and invasion, reduced DNA fragments formation. Mechanistically, we demonstrated that SNHG1 could directly bind to miR-186 and positively regulated α1, 6-fucosyltransferase (FUT8) level. Functional investigation showed that miR-186 depletion reversed the roles of SNHG1 silencing in cell proliferation, apoptosis, and migration. Taken together, our findings illuminated that SNHG1 regulated cell proliferation, migration, and invasion by sponging miR-186 to depress FUT8 expression.

Detection of SARS-CoV-2 Virus on Stainless-Steel Surfaces: AOAC Performance Tested MethodSM 022102.

BackgroundThe SureFast® SARS-CoV-2 PLUS Test is a reverse transcription qPCR (RT-qPCR) assay for the direct, qualitative detection of novel coronavirus (SARS-CoV-2) RNA from stainless-steel environmental sample swabs.ObjectiveTo validate the SureFast SARS-CoV-2 PLUS Kit as part of the AOAC Research Institute’s Emergency Response Validation Performance Tested Method(s)SM program.MethodThe SureFast SARS-CoV-2 PLUS Kit was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms) using the ThermoBLAST program. The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the US Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020).ResultsResults of the in silico analysis demonstrated 99.99% selectivity of the method in being able to detect target sequences of the known CoV-2 genomes and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the PCR detection reference method.ConclusionsThe SureFast SARS-CoV-2 PLUS Kit is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on stainless-steel contact surfaces.HighlightsSureFast SARS-CoV-2 PLUS test method is highly specific for primer/probe binding to the E target genome region for the SARS-CoV-2 virus, 99.99% binding specificity using in silico analysis.

Experimental and Mathematical Optimization of a Pooling Test for Detection of SARS-CoV-2 in a Population with Low Viral Load.

BackgroundA pooling test is a useful tool for mass screening of coronavirus disease 2019 (COVID-19) in the pandemic era. We aimed to optimize a simple two-step pooling test by estimating the optimal pool size using experimental and mathematical validation.Materials and MethodsExperimental pools were created by mixing one positive respiratory sample with various numbers of negative samples. We selected positive samples with cycle threshold (Ct) values greater than 32 to validate the efficiency of the pooling test assuming a high likelihood of false-negative results due to low viral loads. The positivities of the experimental pools were investigated with a single reverse-transcription polymerase chain reaction (RT-PCR) using the U-TOP™ COVID-19 Detection Kit Plus (Seasun Biomaterials, Daejeon, Korea). We used the Dorfman equation to calculate the optimal size of a pooling test mathematically.ResultsViral RNA could be detected in a pool with a size up to 11, even if the Ct value of a positive sample was about 35. The Dorfman equation showed that the optimal number of samples in a pool was 11 when the prevalence was assumed to be 0.66% based on the test positivity in Daejeon, Korea from April 1, 2020 to November 10, 2020. The efficiency of the pooling test was 6.2, which can save 83.9 of 100 individual tests.ConclusionEleven samples in a pool were validated optimal experimentally assuming a prevalence of 0.66%. The pool size needs modification as the pandemic progresses; thus, the prevalence should be carefully estimated before pooling tests are conducted.

Abstract P4-09-13: Spectrum analysis of the inherited mutations of BRCA1/-2 genes that associated with high risk breast cancer in Saudi population

Abstract Background: Breast cancer (BC) is the most common prevalent cancer among female worldwide, despite the advances in molecular biology. In Saudi Arabia, the disease represents 19.9% of the total cancer-related deaths in women. Inherited mutations of BRCA1/-2 are well documented to be associated with higher risk of breast cancer. Aim: To investigate the role of inherited mutation in BRCA1/-2 genes that associated with breast cancer in Saudi population. Methodology: A total of 41 FFPE samples were collected from BC patients admitted to KFSHRC-Jeddah from 2013 to 2018. Genomic DNA was extracted using the Qiagen FFPE kit. A100ng of each DNA samples were used for library preparation using Ion AmpliSeq™ Library Kit Plus with Saudi Inherited Cancer Panel. Ion Torrent Proton platforms were used for DNA sequencing on Ion Torrent PGM using Ion PI™ Hi-Q™ Sequencing 200 Kit. Results: A total of 26 distinct heterozygous BRCA1/-2 variants were identified in Saudi population, 15 variants in BRCA1 gene and 11 in BRCA2 gene. In BRCA1, one variant was nonframeshift deletion and 11 were missense, 3 variants likely benign, while in BRCA 2, one was stopgain and 6 were missense and 4 variants likely benign. Conclusion: Screening of BRCA1/-2 genes mutations helps in understanding the risk associated with mutations of other forms of cancer in Saudi population. The preliminary data is used as an establishing of a high-throughput NGS molecular profiling for Saudi breast cancer patients Citation Format: Yousef MohammedRabaa Hawsawi, Abdelrazak Meliti, Edward Cupler, Turki Sobahy, Samar Zailaie, Mohammed Baghdadi, Hosam Alardati. Spectrum analysis of the inherited mutations of BRCA1/-2 genes that associated with high risk breast cancer in Saudi population [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-13.

MiR‑34a derived from mesenchymal stem cells stimulates senescence in glioma cells by inducing DNA damage.

Insights into the roles of microRNAs (miRNAs/miRs) in development and disease, particularly in cancer, have made miRNAs attractive tools and targets for novel therapeutic approaches in the treatment of glioma. miR‑34a, as a well‑known tumor suppressor miRNA, is closely related with cellular senescence. Mesenchymal stem cells (MSCs) are a major component of the tumor microenvironment and possess the ability to deliver exogenous miRs to glioma cells to exert anti‑tumor effects. The present study investigated whether modified MSCs with miR‑34a possess an anti‑tumor function in glioma cells. A Transwell system was used to co‑culture U87 glioma cells and MSCs overexpressing miR‑34a, and cell proliferation and senescence assessed. The expression of senescence‑related genes p53, Cdkn1a, and Cdkn2c were tested using reverse transcription‑quantitative polymerase chain reaction and protein expression levels of sirtuin 1 (SIRT1) and γ‑H2A histone family, member X were detected by western blotting. Telomerase activity of U87 cells was examined using the Telo TAGGG Telomerase PCR ELISA PLUS kit. The results demonstrated that the delivered exogenous miR‑34a from MSCs significantly decreased expression of the target gene SIRT1. In addition, the delivered miR‑34a decreased the proliferation of glioma cells and provoked the expression of senescence‑related genes p53, Cdkn1a, and Cdkn2c. In addition, upregulation of miR‑34a induced DNA damage, shortened telomere length and impaired telomerase activity. However, these pro‑senescent effects were reversed by forced SIRT1 upregulation. In conclusion, the results demonstrated a novel role for miR‑34a, inducing glioma cell senescence, whereas miR‑34a modulation of SIRT1, inducing DNA damage, is crucial for miRNA replacement therapy in glioma treatment.

Mesenchymal stem cells confer resistance to doxorubicin-induced cardiac senescence by inhibiting microRNA-34a.

Doxorubicin (DOXO) is a chemotherapeutic agent widely used in the treatment of various types of cancer. However, cardiotoxicity is a major side effect of DOXO therapy due to the ability of this compound to induce cardiac cellular senescence. It is well known that microRNA (miR)-34a serves a role in cardiac dysfunction and ageing, and that it is involved in several cellular processes associated with DOXO-induced cardiotoxicity. Furthermore, mesenchymal stem cell (MSC)-based therapies have been reported to modulate cellular senescence. In the present study, the Transwell system was used to co-culture H9c2 cells and MSCs, and cell proliferation and viability were assessed. The expression of senescence-related genes, p53 and p16, and telomere length were analyzed using reverse transcription-quantitative polymerase chain reaction (PCR), and the protein expression levels of situin 1 (SIRT1) were detected by western blotting. Additionally, telomerase activity of H9c2 was examined using the Telo TAGGG Telomerase PCR ELISA PLUS kit. The present study revealed that, in the presence of DOXO, H9c2 cells were in senescence, as characterized by a low proliferation rate, poor viability and a marked increase in the expression of p53 and p16. By contrast, when co-cultured with MSCs in the presence of DOXO, the proliferation and viability of H9c2 cells increased. Additionally, the expression of p53 and p16 decreased, and increased length of telomere and telomerase activity was also observed. Additionally, the mechanism underlying the anti-senescence function of MSCs was revealed to involve the miR-34a-SIRT1 axis, confirmed by overexpressing miR-34a using a miR-34a mimic or silencing SIRT1 using small interfering RNA, which abolished the anti-senescence effect of MSCs on DOXO-treated H9c2 cells. Taken together, the results of the present study suggest that MSCs may rejuvenate H9c2 cells from a state of DOXO-induced senescence by increasing SIRT1 expression, there by inhibiting miR-34a. Therefore, treatment with MSCs may have important therapeutic implications in the restoration of cardiotoxicity in patients with cancer undergoing treatment with DOXO.

Keratin 23 promotes telomerase reverse transcriptase expression and human colorectal cancer growth

The overexpression of human telomerase reverse transcriptase (hTERT) has been associated with the proliferation and migration of colorectal cancer (CRC) cells. We investigated the roles of KRT23 and hTERT in promoting CRC cell proliferation and migration. We verified the relationship between KRT23 and hTERT in CRC using streptavidin-agarose pulldown and chromatin immunoprecipitation (ChIP) assays. One hundred and fifty-four human CRC specimens were analyzed using immunohistochemistry. The roles of KRT23 and hTERT in cell growth and migration were studied using siRNA and lentiviruses in vivo and in vitro. Western blot and wound scratch analyses were used to determine the signaling pathway for KRT23-mediated activation of CRC growth and migration. Telomerase activity was measured by using the TeloTAGGG Telomerase PCR ELISA PLUS Kit. We identified KRT23 as a new hTERT promoter-binding protein. Patients with high KRT23 and hTERT expression had markedly shorter overall survival. Overexpression of KRT23 upregulated the expression of hTERT protein, hTERT promoter-driven luciferase and telomerase activity in CRC. Conversely, inhibition of KRT23 by a KRT23-specific siRNA repressed the endogenous hTERT protein, the expression of hTERT promoter-driven luciferase and telomerase activity. Overexpression of KRT23 also promoted CRC proliferation and migration. By contrast, KRT23 inhibition significantly inhibited tumor cell growth in vitro and in vivo. KRT23 promoted cancer stem cell properties and increased the expression of CD133 and CD44. These results demonstrate that KRT23 is an important cellular factor that promotes CRC growth by activating hTERT expression and that KRT23 is a potential novel therapeutic target for CRC.