Association of apolipoprotein E4 to the number of cerebral microbleeds in patients recovered from COVID-19

Background: COVID-19 has been associated with cerebral microbleeds (CMB). Previously, an association of ApoE4 with COVID-19 severity and CMBs in autopsy was found. In this study, we investigated if carrying the Apoe4 allele relates to the number of CMBs in magnetic resonance imaging (MRI) in patients recovered from COVID-19. Material(s) and Method(s): Adult patients recovered from COVID-19 and a control group without a history of COVID-19 was recruited. Exclusion criteria were major neurologic disease, developmental disability or pregnancy. The participants underwent brain MRI 6 months after infection, and a blinded neuroradiologist analyzed the findings. ApoE was genotyped using a microarray. Statistical analysis was performed using the statistical software R. A negative binomial model was chosen based on the distribution of CMBs. Result(s): Of the 216 subjects that underwent MRI, 168 consented to genetic testing, additionally 2 patients were excluded due to extensive CMBs and 1 due to diffuse axonal injury. We included 113 COVID-19 patients (49 ICU-treated, 29 ward-treated and 35 home-isolated) and 52 controls. The most prevalent comorbidities were hypertension, asthma and diabetes. CMBs was found in 47 subjects, with the number of CMBs ranging from 0 to 26. The ApoeE4 allele was carried by 37%, equally distributed among the groups. After adjustment, age (aRR = 1.06, p = 0.007) and COVID-19 (aRR = 2.59, p = 0.038) were independently associated with CMBs. The ApoE4 allele (aRR = 2.16, p = 0.07, CI = 0.94-5.10) was not significant. Conclusion(s): Age and previous COVID-19, but not possession of the ApoeE4 allele, were independently associated with the number of CMBs.

Exploration of potential mechanism of SARS-CoV-2 induced immune inflammatory response leading to progression of atherosclerosis and prediction of traditional Chinese medicine for preventing and treating based on bioinformatics.

Objective To explore the core targets and important pathways of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) induced atherosclerosis (AS) progression from the perspective of immune inflammation, so as to predict the potential prevention and treatment of traditional Chinese medicine (TCM). Methods Microarray data were obtained from the Gene Expression Omnibus (GEO) database for coronavirus disease 2019 (COVID-19) patients and AS patients, and the "limmar" and "Venn" packages were used to screen out the common differentially expressed genes (DEGs) genes in both diseases. The gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were performed on the common DEGs to annotate their functions and important pathways. The two gene sets were scored for immune cells and immune function to assess the level of immune cell infiltration. The protein-protein interaction (PPI) network was constructed by STRING database, and the CytoHubba plug-in of Cytoscape was used to identify the hub genes. Two external validation datasets were introduced to validate the hub genes and obtain the core genes. Immuno-infiltration analysis and gene set enrichment analysis (GSEA) were performed on the core genes respectively. Finally the potential TCM regulating the core genes were predicted by Coremine Medical database. Results A total of 7898 genes related to COVID-19, 471 genes related to AS progression;And 51 common DEGs, including 32 highly expressed genes and 19 low expressed genes were obtained. GO and KEGG analysis showed that common DEGs, which were mainly localized in cypermethrin-encapsulated vesicles, platelet alpha particles, phagocytic vesicle membranes and vesicles, were involved in many biological processes such as myeloid differentiation factor 88 (MyD88)-dependent Toll-like receptor signaling pathway transduction, interleukin-8 (IL-8) production and positive regulation, IL-6 production and positive regulation to play a role in regulating nicotinamide adenine dinucleotide phosphate oxidase activity, Toll-like receptor binding and lipopeptide and glycosaminoglycan binding through many biological pathways, including Toll-like receptor signaling pathways, neutrophil extracellular trap formation, complement and coagulation cascade reactions. The results of immune infiltration analysis demonstrated the state of immune microenvironment of COVID-19 and AS. A total of 5 hub genes were obtained after screening, among which Toll-like receptor 2 (TLR2), cluster of differentiation 163 (CD163) and complement C1q subcomponent subunit B (C1QB) genes passed external validation as core genes. The core genes showed strong correlation with immune process and inflammatory response in both immune infiltration analysis and GSEA enrichment analysis. A total of 35 TCMs, including Chuanxiong (Chuanxiong Rhizoma), Taoren (Persicae Semen), Danggui (Angelicae Sinensis Radix), Huangqin (Scutellariae Radix), Pugongying (Taraxaci Herba), Taizishen (Pseudostellariae Radix), Huangjing (Polygonati Rhizoma), could be used as potential therapeutic agents. Conclusion TLR2, CD163 and C1QB were the core molecules of SARS-CoV-2-mediated immune inflammatory response promoting AS progression, and targeting predicted herbs were potential drugs to slow down AS progression in COVID-19 patients.Copyright © 2023 Editorial Office of Chinese Traditional and Herbal Drugs. All rights reserved.

SARS-CoV-2 Host Receptors Expression in Human Pancreatic Islets Revealed Upregulation of ACE2 in Diabetic subjects

Background: It is well established that diabetic patients infected with COVID-19- are at higher risk of developing severe symptoms that may lead to death. Such observation argues for the possibility that SARS-CoV-2 may target and infect pancreatic islets. SARSCoV- 2 is thought to enter the cells through the binding of viral spike S1 protein to ACE2. The cellular entry process includes priming of the S protein by TMPRSS2 and ADAM17, which facilitate the binding and promote ACE2 shedding. To date, no conclusive evidence has emerged to address the expression of TMPRSS2 and ADMA17 or the interaction between SARS-CoV-2 and human pancreatic islets. Method(s): Microarray and RNA-sequencing (RNA-seq) expression data from human islets were used to profile the expression pattern of ACE2, ADAM17, and TMPRSS2 in diabetic and non-diabetic subjects. Result(s): Pancreatic islets express all three receptors regardless of diabetes status. ACE2 expression was significantly elevated in diabetic islets than non-diabetic. Female donors showed to have higher ACE2 expression compared to males, whereas ADAM17 and TMPRSS2 were not affected by gender. No difference in the expression of the three receptors in young (<=40 years old) compared to old (>=60 years old) islets. Obese donors (BMI>30) showed significantly higher expression levels of ADAM17 and TMPRSS2 as compared to non-obese (BMI<25). Expression of TMPRSS2 was associated positively with HbA1c and inversely with age, while ADAM17 and TMPRSS2 were associated positively with BMI. Muscle and subcutaneous adipose tissues showed similar expression of the three receptors in diabetic and nondiabetic donors. Conclusion(s): ACE2 expression is increased in diabetic human islets. More studies are warranted to understand the permissiveness of human pancreatic beta-cells to SARS-Cov-2 and whether variations of ACE2 expression could explain the severity of COVID-19 infection between diabetics and non-diabetic patients.

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Trisomy 18p detected in DNA from saliva but absent in DNA from blood

Molecular diagnostic testing using DNA from saliva specimens markedly increased recently due to the ease of sample collection, compared to peripheral blood, during the COVID-19 pandemic. Published literature suggests that DNA from saliva is primarily composed of epithelial cells (70-90%), with the remainder being primarily leukocytes (10-30%). Here, we describe a case followed by our clinicians since 2007 characterized by developmental delay, autism, a somewhat coarse face with full cheeks, up-slanting palpebral fissures, thin corpus callosum, and a full-scale IQ of 60. This patient had an extensive work-up including high-resolution blood chromosome analysis, FISH for 22q microdeletion, three separate microarrays (various platforms), FMR1 molecular analysis, urine oligosaccharides analysis, an autism gene panel by NGS, and whole-exome sequencing, none of which identified a satisfactory diagnosis. These tests were performed on two peripheral blood samples collected at different times. Recently, a new microarray was ordered on a saliva sample from this patient, and an apparently non-mosaic gain of 18p was detected. The possibility of a sample swap was eliminated by comparing the SNP genotype of the saliva sample to the previously tested blood sample. The limit of detection for mosaicism in genomic microarrays is around 20%, so it is possible that the 18p duplication was present at a level undetectable by microarray in the peripheral blood samples. This case suggests that the differences between DNA obtained from saliva and peripheral blood may be, in some cases, more drastic than previously recognized. By testing primarily with DNA from peripheral blood, significant mosaic abnormalities may go undetected. Copyright © 2022

Novel Lateral Flow-Based Assay for Simple and Visual Detection of SARS-CoV-2 Mutations.

Identification of the main SARS-CoV-2 variants in real time is of interest to control the virus and to rapidly devise appropriate public health responses. The RT-qPCR is currently considered to be the reference method to screen SARS-CoV-2 mutations, but it has some limitations. The multiplexing capability is limited when the number of markers to detect increases. Moreover, the performance of this allele-specific method may be impacted in the presence of new mutations. Herein, we present a proof-of-concept study of a simple molecular assay to detect key SARS-CoV-2 mutations. The innovative features of the assay are the multiplex asymmetric one-step RT-PCR amplification covering different regions of SARS-CoV-2 S gene and the visual detection of mutations on a lateral flow DNA microarray. Three kits (Kit 1: N501Y, E484K; Kit 2: L452R, E484K/Q; Kit 3: K417N, L452R, E484K/Q/A) were developed to match recommendations for surveillance of SARS-CoV-2 variants between January and December 2021. The clinical performance was assessed using RNA extracts from 113 SARS-CoV-2-positive samples with cycle thresholds <30, and results demonstrated that our assay allows specific and sensitive detection of mutations, with a performance comparable to that of RT-qPCR. The VAR-CoV assay detected four SARS-CoV-2 targets and achieved specific and sensitive screening of spike mutations associated with the main variants of concern, with a performance comparable to that of RT-qPCR. With well-defined virus sequences, this assay can be rapidly adapted to other emerging mutations; it is a promising tool for variant surveillance.

Characterization of TF positive small extracellular vesicles in SARS-CoV- 2 infection

Background: Coronavirus-2 (SARS-CoV- 2) infection causes a sustained prothrombotic state driven by a massive Tissue Factor (TF) expression in circulating platelets, leukocytes and microvesicles (MVs). Whether also circulating small extracellular vesicles (sEVs), in addition to large MVs (MVs), can contribute to this hypercoagulable scenario through TF expression is not yet known, mainly due to methodological issues in detecting and sizing the smallest vesicles. Aim(s): To characterize TF expression and activity in circulating sEVs, compared to that of MVs, of COVID-19 patients during acute phase infection and after symptom remission. Method(s): MVs and sEVs were isolated by plasma differential centrifugation from 10 COVID-19 patients enrolled at acute phase infection (T0) and at six-month- follow- up (T1). Ten healthy subjects (HS) were analyzed as controls. sEVs were counted by Nano Tracking Assay. In sEVs TF expression was analyzed within CD9/CD63/CD81pos events by imaging flow cytometry (IFC) and ExoViewTM microarray, while TF activity by FXa generation assay. TF expression and activity in MVs were evaluated for comparison. Result(s): By IFC analysis COVID-19 patients at T0 have about 1.5-and 4-fold higher number of TFpos-sEVs and -MVs, respectively, compared to HS, with a trend toward reduction to physiological levels at T1. By microarray analysis sEVs behaved similarly (36 +/- 12 and 25 +/- 10 TFpos-spots at T0 and T1, respectively;p = 0.0281). sEVs-associated TF is functionally active thus able to partially support FXa formation as sEVs preincubation with TF-neutralizing antibody reduced FXa generation by ~30%. However, although sEVs number is significantly higher compared to that measured for MVs (~600-fold in HS), functional activity of sEV is one-third lower compared to that of MVs. Conclusion(s): These data suggest that, in COVID-19 patients, the altered procoagulant phenotype could also be supported by TF carried by sEVs, although their functional activity is significantly lower than that of MVs.

UNDERSTANDING THE NEED FOR A BROAD DIFFERENTIAL DIAGNOSIS: A CASE OF PNEUMONIC TULAREMIA

SESSION TITLE: Disseminated Bacterial Infections SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/18/2022 10:15 am - 11:10 am INTRODUCTION: Tularemia is a rare infectious disease caused by Francisella Tularensis that typically affects the skin, eyes, lymph nodes, and lungs. There are a variety of forms of tularemia with varying rates of contagiousness and mortality. Respiratory tularemia has a high mortality rate if left untreated and presents with non-specific viral like symptoms occurring in conjunction with respiratory symptoms: cough, hemoptysis, and pleuritic chest pain. In this COVID ARDS era, it is important to evaluate a broad differential diagnosis. Therefore, the authors describe a patient presenting with flu-like respiratory symptoms whom was ultimately was diagnosed with acute respiratory distress syndrome (ARDS) due to F. Tulerensis. CASE PRESENTATION: A 44-year-old male presented with a four-day history of night sweats, shortness of breath, a productive cough which progressed to hemoptysis, and oliguria. Prior to admission, his initial symptoms were treated as chronic sinusitis with varied antibiotics. Social history including tobacco abuse and deer hunting 1 month prior to presentation. Vitals were stable except for tachycardia, hypoxia, and tachypnea. Laboratory findings were significant for AKI, lactic acidosis, mild transaminitis, hyperbilirubinemia, and leukocytosis with predominant neutrophilia. Thoracic CTA showed bilateral diffused pulmonary edema without evidence of pulmonary embolism. Due to the patient's worsening respiratory status, he was intubated for support. The patient progressed to Severe ARDS per Berlin Criteria eventually requiring pronation and continuous paralyzing. Bronchoscopy was performed with bronchial lavage. Bacterial, viral, and fungal cultures did not show growth while vasculitic work-up was negative. Empiric antibiotic treatment did not show improvement until the patient was diagnosed with F. Taularensis via serological testing with an IgM of 20 U/mL, and patient was transitioned to gentamycin. Ultimately, the patient was extubated, transitioned to oral doxycycline, and discharged home. DISCUSSION: Approximately 250 cases of tularemia are reported to CDC each year. Respiratory tularemia has a mortality rate up to 30% if not treated. For this reason, F. tularensis is a potential biological weapon and is categorized as a Group A pathogenic agent. Serological testing may be negative early in disease progression;therefore, early inflammatory markers with clinical suspicion are essential to diagnose the disease early in its course. DNA microarray has high specificity and sensitivity for rapid diagnosis of tularemia while being cost effective. After prompt diagnosis, intravenous aminoglycosides;such as gentamycin or streptomycin;must be started. CONCLUSIONS: In the above case, we illustrate the gradual onset and rapid patient deterioration when treatment is delayed;yet, there is rapid recovery once appropriate treatment is used. Reference #1: 1. Ranjbar, Reza, Payam Behzadi, and Caterina Mammina. "Respiratory tularemia: Francisella tularensis and microarray probe designing.” The open microbiology journal 10 (2016): 176. Reference #2: 2. Akhvlediani, N., I. Burjanadze, D. Baliashvili, T. Tushishvili, M. Broladze, A. Navdarashvili, S. Dolbadze et al. "Tularemia transmission to humans: a multifaceted surveillance approach.” Epidemiology & Infection 146, no. 16 (2018): 2139-2145. Reference #3: 3. Tularemia in British Columbia: A case report and review. Issue: BCMJ, vol. 52, No. 6, July August 2010 (Pages 303- 307). Megan Isaac-Renton, BSc, Muhammad Morshed, PhD, SCCM Eleni Galanis, MD, MPH, FRCPC Sunny Mak, MSc Vicente Loyola, MD, FRCPC, Linda M.N. Hoang, MD, MHSc, FRCPC DISCLOSURES: No relevant relationships by Munish Adhikari No relevant relationships by Ashma Ul Husna No relevant relationships by Yan Jiang No relevant relationships by Divya Kharel No relevant relationships by Gregory Polcha

A Miniaturized Silicon Lab-on-Chip for Integrated PCR and Hybridization Microarray for High Multiplexing Nucleic Acids Analysis.

A silicon lab-on-chip, for the detection of nucleic acids through the integrated PCR and hybridization microarray, was developed. The silicon lab-on-chip manufactured through bio-MEMS technology is composed of two PCR microreactors (each volume 11.2 µL) and a microarray-hybridization microchamber (volume 30 µL), fluidically connected by buried bypass. It contains heaters and temperature sensors for the management and control of the temperature cycles during the PCR amplification and hybridization processes. A post-silicon process based on (i) plasmo-O2 cleaning/activation, (ii) vapor phase epoxy silanization, (iii) microarray fabrication and (iv) a protein-based passivation step was developed and fully characterized. The ssDNA microarray (4 rows × 10 columns) composed of 400 spots (spot size-70 ± 12 µm; spot-to-spot distance-130 ± 13 µm) was manufactured by piezo-dispense technology. A DNA microarray probe density in the range of 1310 to 2070 probe µm-2 was observed, together with a limit of detection of about 19 target µm-2. The performances of the silicon lab-on-chip were validated by the detection of the beta-globin gene directly from human blood. Remarkable sensitivity, multiplexing analysis and specificity were demonstrated for the detection of beta-globin and mycobacterium tuberculosis sequences.

Microarray-Based Transcriptome Analysis of Peripheral Blood Mononuclear Cells in Lung Cancer Patients

: Lung cancer is the most commonly occurring cancer in men worldwide. To search for new biological markers of this pathology, the transcriptome of the blood mononuclear cells from patients and healthy donors (residents of Kemerovo oblast, Russia) was studied using SurePrint G3 Human Gene Expression microarray technology. A total of 288 differentially expressed genes were identified, including 108 up-regulated genes and 180 down-regulated genes. Functional enrichment analysis using the WebGestalt resource and different databases (Gene Ontology, KEGG, and Reactome) indicated changes in the expression profiles of genes involved in the processes of immune response, protein synthesis, cell cycle control, and apoptosis. Analysis of protein–protein interactions using the STRING algorithm made it possible to identify functional clusters of gene products with different expression levels.

Microarray patch-based adenovirus vaccines enable effective skin immunization

Recombinant live adenovirus (Ad) vectors represent a readily modular vaccine construct that can be engineered to include any antigen of interest, thereby enabling rapid vaccine development against a myriad of infectious pathogens, including coronaviruses. However, current approaches used for delivery, storage, and distribution of Ad vaccines hinder their full potential for effective global immunization campaigns. Here, we developed simple, effective, practical, and needle-free Ad vaccines based on microarray patches (MAPs) to enable sustainable mass vaccination against SARS-CoV-2. Rational formulation of live Ads with nonreducing sugars and mechanically strong carbohydrates into dissolvable MAPs enabled effective skin-targeted delivery of Ads and efficient cutaneous transduction as determined by in vivo live imaging of mice following skin application of MAPs integrating Ad vectors with a reporter gene. Immunogenicity assessment of dissolving MAP-based Ad vectors encoding SARS-CoV-2 proteins in mice demonstrated that skin immunization via MAP delivery of Ad-vectored COVID-19 vaccines elicited robust antigen-specific antibody responses, and these antibodies led to virus-specific neutralization activities, which were enhanced compared to those obtained with traditional intramuscular immunization. Furthermore, MAP delivery of Ad-vectored vaccines elicited potent cell-mediated immune responses, including polyfunctional virus-specific CD8+ and CD4+ T-cell responses in spleens and lungs of immunized mice, as determined by intracellular cytokine staining and flow cytometry, as well as antigen-specific cytotoxic T-cell responses in spleens of mice, as determined by lytic assay. Collectively, our results suggest that dissolvable MAPs could enable the development of skin-targeted Ad-based vaccines that may increase the effectiveness of global immunization programs for SARS-CoV-2 and other existing or future pathogens.

Gene expression pattern in severely progressing covid-19 patients is related to diabetes mellitus type 1: A functional annotation analysis

Aims: The aim of this study was to extract the signaling mediators or biological pathways that link covid-19 to other diseases such as type 1 diabetes mellitus (T1DM). Methods: Microarray data of covid-19 (GSE164805) was extracted from Gene Expression Omnibus (GEO) and analyses were performed by R package and GEO2R. Functional enrichment analysis was done to extract enriched molecular pathways (MP), biological process (BP) and molecular function (MF). Then commonly up- and down-regulated genes in covid-19 and T1DM were extracted by comparing deferentially expressed genes (DEGs) of GSE164805 and GSE9006. Results: Down-regulated DEGs in the severely progressing covid-19 patients (SPCP) had a link to T1DM. Major histocompatibility system (MHC) class II, gamma interferon (IFNγ), and IL-1B were enriched in extracted pathway that leads to T1DM. In addition, comparing extracted DEGs from GSE164805 and GSE9006 indicated that MTUS1, EGR1 and EGR3 are the genes that are up-regulated in both SPCP and T1DM. Conclusion: The findings of this study indicate that coincidence of SARS-COV-2 infection and T1DM may increase the severity of both diseases. Although covid-19 reduced the T cell mediated immune response, but increased mediators of T-cell signaling pathway such as IL-1 in both diseases. This could potentiate the inflammation response and worsens the severity of covid-19 cytokine storm or increase the resistance to insulin.

MACHINE LEARNING ANALYSIS of DNA METHYLATION in COVID-19 DISEASE

Background: SARS-CoV-2 infection has resulted in over 219 million confirmed cases of COVID-19 with 4.5 million fatalities, highlighting the importance of elucidating mechanisms of severe disease. Here we utilized machine learning (ML) technologies to identify DNA methylation footprints of COVID-19 disease from publicly available data. Methods: Genome-wide DNA methylation of SARS-CoV-2 infected and uninfected patients using Illumina HumanMethylationEPIC microarray platform from whole blood was publicly available through NCBI Gene Expression Omnibus. A training cohort (GSE167202) consisting of 460 individuals (164 COVID-19-infected and 296 non-infected) and an external validation dataset (GSE174818) consisting of 128 individuals (102 COVID-19-infected and 26 non-COVID with pneumonia diagnosis) were obtained. COVID-19 severity score (SS) was classified as follows: 0. uninfected;1. released from department to home;2. admitted to in-patient care;3. progressed to ICU;and 4. death. Participants were then dichotomized by SS=0 or SS≥3. Raw data was processed using ChAMP in R 4.1.1, resulting in over 850,000 methylation sites per sample for analysis. Beta values were logit transformed to M values using CpGTools in Python 3.8.8. JADBio AutoML platform was leveraged to analyze these datasets with the goal of identifying a methylation signature indicative of COVID-19 disease. Results: From our training cohort, JADBio utilized LASSO feature selection (penalty=1.5) to identify 4 unique methylation sites capable of carrying the predictive weight of a classification random forest trained on 100 trees with Deviance splitting criterion (minimum leaf size=3). The average area under the curve of receiver operator characteristic (AUC-ROC) of the model was 0.933 (95% confidence interval [0.885, 0.970]), while the average area under the precision-recall curve (AUC-PRC) of 0.965 [0.932, 0.986]. When COVID-19 mild infections (SS = 1 or 2) were returned to the training dataset as an internal control, the model retained its predictive power (AUC-ROC=0.985, AUC-PRC=0.992). When applied to our external validation, this model produced an AUC-ROC of 0.901 with an AUC-PRC of 0.748. Conclusion: We developed a Random Forest Classification model capable of accurately predicting COVID-19 infection leveraging JADBio AutoML platform. These results enhance our understanding of epigenetic mechanisms used by SARS-CoV-2 in disease pathogenesis and identify potential therapeutic targets.

The utility of using genomic testing on needle core biopsies during the COVID-19 pandemic: Molecular characterization, risk stratification, neoadjuvant outcome, and future implications

Introduction: The COVID-19 pandemic has caused an extraordinary challenge for global health. New guidelines were implemented, including postponing non-essential surgical procedures to conserve resources. In response, the COVID19 Pandemic Breast Cancer Consortium expert opinion suggested the use of core biopsies for genomic testing to help triage patients for surgical vs. systemic treatment. To better understand how expedited genomic results could impact peri-operative care, we performed a pre-operative quality project to assess testing Mammaprint (MP), a 70-gene risk of recurrence assay, and Blueprint (BP), an 80-gene molecular subtyping assay, on core biopsies. Here we report our experience with MP and BP testing on core biopsies, and the correlation between test results and response to neoadjuvant therapy.Design: From April to December 2020, all core biopsies with a breast carcinoma diagnosis from our clinic (300 patients) were routinely sent for MP and BP testing as part of a rapid result program that was initiated to see whether test results could be obtained in time and whether they would lead to more informed pre-operative treatment decisions. Unstained slides were sent for genomic and receptor testing concurrently. When genomic results differed from IHC/FISH results or suggested a different S treatment plan vs. clinical factors alone, we referred to this as "reclassification." For those patients who completed their neoadjuvant chemotherapy, we grouped them by their genomic results and by their conventional IHC/FISH/Clinical classification, and compared the outcome. Results: MP and BP results from core biopsy were available for 96.6% of patients (n=290/300). The average time from biopsy to test results was 10 days, and the average lab turnaround time was 5 days. Results were available for tumor conference discussions 100% of the time. MP and BP re-classified 84 of 300 patients (28%) from conventional IHC/FISH subtyping, and reclassified 42 of 195 patients (22%) of patients from their risk category based on traditional clinical factors (Table-1). Of the 38 patients with available post-neoadjuvant therapy outcome, 13 patients (34%) achieved pathologic complete response (pCR). 16 patients were classified as Her-2 enriched by IHC/FISH of which 9 (56%) achieved pCR. MP/BP aligned with the IHC/FISH Her-2 enriched classification in 11/16 patients, while 5 patients were reclassified to Luminal B by MP/BP. Of the 11 patients with concordant IHC/FISH and MP/BP results, 8 achieved pCR (73%), while one of the 5 cases reclassified to Luminal B achieved pCR (20%). Of the patients who were classified Luminal by IHC (9 patients), one patient achieved pCR (11%), and of the patients classified luminal (A or B) by MP/BP (12 patients) two achieved pCR (16%). Of the IHC-triple negative patients and genomic basal patients, three patients achieved pCR in each group (23% and 20%, respectively). Conclusion: Performing MP/BP tests on core needle biopsies hold a high success rate. Incorporating test results into peri-operative discussions may result in better-informed decisions about treatment planning and timing of surgery versus systemic therapy. A higher rate of pCR was seen in the MP/BP Her-2 enriched group compared to the IHC/FISH Her-2 enriched group. Although this workflow was designed to triage patients during the COVID pandemic, this approach has great potential beyond the pandemic.

Genomic testing on breast cancer core biopsy material is feasible and provides useful treatment information

Background: Genomic profiling assays for invasive breast cancer provide useful predictive and prognostic information and are performed most commonly on surgical resection specimens. Obtaining the molecular profile at the time of initial core needle biopsy is ideal because it could provide information that could significantly alter preoperative decision-making and avoid unnecessary treatments. In January 2020, the breast center began a 3 month pilot program that would reflexively send core biopsy material on all newly diagnosed patients for genomic testing using the MammaPrint (MPT) and BluePrint (BPT) assays regardless of receptor status. The goals were to determine feasibility and examine the impact to patient care. Methods: Breast core biopsy tissue was triaged by an attending pathologist as soon as slides were available. If invasive carcinoma with at least 3 mm in linear extent of tumor with 20% cellularity was identified, materials were immediately sent to Agendia for MPT/BPT testing even before ER/PR/HER2 testing was available to the pathologist. Clinicopathologic information, turn-around time (TAT) and adequacy data were tracked. The impact was discussed regularly at breast tumor board to determine if the results led to altered decision-making that could reduce unnecessary interventions and time to initial treatment. Results: 445 core biopsy specimens were sent for genomic testing. Of those 442, (97%) Syielded genomic results with an average TAT from biopsy to genomic result of 11 calendar days. MPT identified 233 (53%) cases that were low risk and 209 (47%) cases that were high risk. BPT showed that 60 (14%) cases were Basal, 18 (4%) cases were HER2, and 364 (82%) cases were Luminal. Further analysis of the Luminal subgroup demonstrated that 154 (42%) were Luminal A low risk, 78 (21%) were Luminal A Ultralow risk and 132 (36%) were Luminal B High Risk. Analysis by race demonstrated a significantly higher percentage of high risk tumors in African-American women including a higher percentage of basal cancers (26%) as compared to Caucasian women (10%). Of note, 51 patients in the pilot study had additional OncotypeDX (ODX) testing on subsequent surgical resection. There was fair correlation between the assays with the majority of the low risk MPT having low risk ODX scores (<=25) and the high risk MPT having high risk ODX scores (>25), though MPT identified more patients as being high risk. The COVID-19 pandemic altered plans to assess time to treat and treatment interventions as initially intended. However, the knowledge of genomic result enhanced the ability to triage patients, allowing those patients with low risk tumors to begin endocrine therapy and delay surgery. Use of preoperative hormone therapy was considered more often in place of neoadjuvant chemotherapy in patients with low genomic risk ER+/HER2-patients. Because of the continued feedback indicating positive impact, the pilot ultimately was extended to 11 months to allow time for formal implementation. Conclusions: Genomic testing using the MPT/BPT assay on core biopsy samples with at least 3 mm yields results 97% of the time with an average TAT of 11 days from biopsy date to result. The genomic information at the time of initial diagnosis impacted patient care most notably in the ER+ setting. The results led to the immediate implementation of direct reflex testing of all newly diagnosed ER+ and HER2-or IHC 2+ cancers by pathology after the initial pilot phase.

Torus Synestia Nucleic Acid Analysis Platform for Fast, High Multiplex Analysis of Nucleic Acids with Single-Nucleotide Discrimination Level

Background. Nucleic acid amplification testing (NAAT) is an essential tool both for biomedical research and for clinical molecular diagnostics. Currently, there are multiple NAAT platforms available, each offering certain performance and utility advantages and disadvantages as compared to each other. Next generation NAAT platforms aim to deliver increased target detection sensitivity and specificity, low limits of target detection, quantitative high multiplex target capacity, rapid time to results, and simple sample-to-answer workflow. Methods. Here we describe the Torus Synestia System, a NAAT platform capable of rapid, highly multiplexed amplification and detection of both DNA and RNA targets. The platform comprises a small, portable (~ 2kg) amplification and detection device and a disposable single-use cartridge housing a PCR amplification chamber with an integrated label-free microarray for real-time data acquisition and interpretation. The platform offers a 30-min turnaround time with a detection limit of 10 DNA/RNA molecules per assay and single nucleotide discrimination. Results. We demonstrate the Synestia System performance and utility with three distinct molecular applications: 1) detection of 20 genetic loci and 30 single nucleotide polymorphisms in human genomic DNA;2) detection and genotyping of 43 unique bacterial species associated with human urinary tract infections;and 3) detection and profiling human respiratory viral pathogens including SARS-CoV-1/2, seasonal coronaviruses, Influenza A/B, and human respiratory syncytial viruses. In addition, the single-nucleotide specificity of our label-free microarray probes allowed for robust identification and discrimination of newly emerging SARS-CoV-2 lineages, such as B.1.1.7 (a.k.a. UK), B.1.351 (a.k.a. South African), P.1 (a.k.a. Brazilian), and B.1.617 (a.k.a. Indian). Conclusion. The Torus Synestia System has broad applicability in both clinical and research environments. We are confident that the Torus Synestia System will revolutionize syndromic diagnostics at the point of care (PoC) and lead to improved response times during future epidemic and pandemic pathogen outbreaks.

Comparing MammaPrint and BluePrint results between core needle biopsy and surgical resection breast cancer specimens

Goals: The COVID-19 pandemic continues to strain healthcare systems globally. The ESMO COVID-19 adapted recommendations1 advocate for the use of pre-operative/neoadjuvant endocrine therapy as a strategy to defer surgery by 6–12 months in clinical stage I-II breast cancers to prioritize resources for patients that require urgent care. Accurate risk assessment is an integral component of this prioritization process. Adjuvant studies such as MINDACT showed that up to 46% of clinically high risk tumors were classified as genomic Low Risk with MammaPrint, and still have excellent outcomes at 8-yrs with endocrine therapy alone, highlighting the potential for overtreatment if using clinical-risk alone. Here, gene expression results with MammaPrint (MP) and BluePrint (BP) were compared between pre-operative core needle biopsy (CNB) and postoperative surgical resection (SR) specimens to evaluate test performance across specimen type. Methods: 10,574 routine diagnostic samples from outside the US submitted to Agendia between Jan 2017 and Oct 2020 were included in this study.MP and BP testingwere processed according to standard FFPE microarray procedures. MP was used to stratify samples into Ultra LowRisk (UL), LowRisk (LR), and High Risk (HR). BPwas used to classify samples into Basal, Luminal or HER2-type. MP Index (MPI) distribution on BP defined Luminal-type tumors were compared between CNB and SR samples. Comparative “logistics metrics” (avg. turnaround time [TAT] and success rate) were also assessed between these specimen types. Results: 10% of samples were CNB and 90% were SR (Table 1). BP Basal, Luminal and HER2-type distributions were 2%, 97%, and 1% respectively for CNB samples and 1%, 98.6%, and 0.4% respectively for SR samples. Within Luminal-type tumors (majority of the samples), the frequency of UL, LR, and HR results were 14%, 61%, and 39% for CNB, and 13%, 58%, and 42% for SR, respectively. Overall, MP Index distributions were similar between samples tested from CNB vs. SR. Average TAT and success rate % between CNB and SR were similar (Table 2).(Table Presented)Definitions: Turnaround Time (TAT) is calculated from the time a specimen is received at the laboratory to the time a result is available. Success % excludes test failures due to insufficient RNA yield % and sub-optimal RNA quality, and evaluates the total number of specimens that have met the pre-requisite 30% minimum invasive tumor requirement that have a valid result. Conclusion(s): The frequency of each MP risk group as well as the distribution pattern of MP Index were essentially identical between CNB and SR samples, indicating comparable performance regardless of specimen type. With timely TAT and no meaningful difference in MPI distribution between CNB and SR specimens, pre-operative use of MP+BP genomic testing on CNB can be incorporated into the preoperative treatment decision making process. Conflict of Interest: Employee of Agendia, equity/stock ownership interest.

Inflammasome activation in Kawasaki disease and multisystem inflammatory syndrome in children

Introduction: Multisystem Inflammatory Syndrome in Children (MIS-C) is a newly identified syndrome elicited by infection of acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although MIS-C shares several clinical features with Kawasaki disease (KD), a greater magnitude of systemic inflammation is usually associated with MIS-C. Inflammasome activation induces the secretion of pro-inflammatory cytokines, IL-1β and IL-18 and the inflammatory form of cell death, pyroptosis. To provide mechanistic insights into MIS-C and KD, we compared the expression and activation of the inflammasome in blood samples from MIS-C and KD patients. Methods: Expression levels of canonical and non-canonical inflammasome components, including NLRP3, CASP1, CASP4, CASP5, IL1B, and the inflammatory mediator, TIFA in whole blood from KD and convalescent patients were analyzed from microarray datasets. The expression of these inflammasomerelated genes was further examined in whole blood samples from MIS-C, KD, KD shock syndrome (KDSS) and convalescent patients using RT-qPCR. Inflammasome activation and TIFA expression were validated in granulocytes of febrile control, KD and MIS-C patients by Western blotting. Results:TIFA, NLRP3, CASP1, CASP4, CASP5, IL1B were upregulated in whole blood from MIS-C, KD, and KDSS patients as compared to convalescent patients. However, the differences were not significant among diseases. Although gene expression profiles were similar in KD, KDSS and MIS-C whole blood RNA, the processing of canonical and non-canonical inflammasome caspases, caspase-1, and caspase-4 were only observed in granulocytes isolated from MIS-C patients, but not KD and febrile controls. Moreover, TIFA was upregulated along with the activation of the inflammasome in granulocytes of MIS-C patients. Conclusions: Our results suggest that activation of inflammasomes, especially non-canonical inflammasome induction in granulocytes, is a hallmark of MIS-C, and differentiates it from KD.

SOCS1 Haploinsufficiency Presenting As Incidental Refractory Thrombocytopenia in a Pediatric Patient with Inflammatory Symptoms and History of Sars Cov-2 Infection

BACKGROUND: Immune thrombocytopenia purpura (ITP) has a complex pathogenesis and may be a primary diagnosis or secondary to an underlying condition 1. Evaluation for underlying diagnoses in patients presenting with atypical features of classic ITP is key, as this can impact treatment decisions, therapy response, and prognosis. Genetic variants that predispose patients to ITP are especially important to investigate as patients may be at risk for additional autoimmune phenomenon or malignancy. The SARS CoV-2 pandemic has added further complexity as reports suggest the infection can lead to autoimmunity in those with genetic predispositions 2,3. Loss of the suppressor of cytokine signaling 1 (SOCS1) function has been described to manifest with autoinflammatory syndrome, with or without immunodeficiency 4,5. Reports of autoimmunity developing in patients with SOCS1 haploinsufficiency after SARS CoV-2 infection are documented, including multi-system inflammatory syndrome (MIS-C) 2. A proposed mechanism of this virus-triggered autoimmunity includes a transient innate and adaptive immunodeficiency 3. This raises the question whether patients harboring genetic variants with risk of autoimmunity are placed at an even higher risk for ITP in the wake of SARS-CoV2 infection. CASE PRESENTATION: We present a 6-year-old female with isolated thrombocytopenia of 4,000/uL identified during evaluation for severe arthralgias unresponsive to corticosteroid treatment (maximum dose 1mg/kg/day) over a 6-month period. Laboratory results at presentation were consistent with ITP, including presence of platelet autoantibodies. Evaluation revealed hypocellularity for age (~50%) on bone marrow evaluation as well as elevated IgE (2080 kU/L), with IgA, IgM, and IgG levels within reference range. She had a remote history of SARS CoV-2-like illness and SARS CoV-2 antibodies were found present in serologic assay, without a history of vaccination. Genetic testing, including chromosomal microarray from peripheral blood and marrow, was included in the diagnostic workup given concern for a history of developmental delays with macrocephaly and necessity to rule-out malignancy with the patient noted to have a 5 mega-base deletion at 16p13.2p13.11, which includes the SOCS1 gene. Comprehensive next generation sequencing for additional immune dysregulation/primary immunodeficiency associated variants was unremarkable. Functional studies of surface expression of interferon-inducible genes (CD169 (SIGLEC-1)) and STAT1 phosphorylation via analysis of CD14+ monocytes revealed excess interferon signaling previously described in patients with SOCS1 haploinsufficiency (Figure 1). Measurements of B-cell-activating factor were also found to be extremely elevated at 6432 pg/mL. The patient's ITP course was complicated by hematuria, melena and refractory platelet response to first line therapy consisting of intravenous immunoglobulin 1 g/kg x2 doses and 2 mg/kg/day prednisolone. She required escalation to high dose methylprednisolone (30mg/kg), rituximab 375 mg/m 2/weekly x4 doses, and concurrent romiplostim (2 doses) for control of thrombocytopenia and bleeding manifestations. Her rheumatologic symptoms subsided with initiation of corticosteroids, and she has subsequently completed a prolonged corticosteroid taper. She currently has a normal platelet count with non-steroidal anti-inflammatory therapy utilized for arthralgia management with plan to transition to JAK inhibition for maintenance therapy. CONCLUSION: This case highlights the potential impact of investigating for susceptibility genes for ITP with consideration for broader testing including targeted next generation sequencing panels or microarray analysis in patients with atypical ITP presentations or response to therapy, as knowledge of this patient's underlying genetics led to earlier treatment and use of alternative agents. Additionally, the case adds the novel finding of bone marrow hypocellularity to the clinical phenotype of SOCS1 haploinsufficiency, as this has not yet been reported and contributes to the literature on the relationship of autoimmunity and SARS CoV-2 infections in patients with predisposing genetic variants. [Formula presented] Disclosures: Walkovich: Horizon Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees;Pharming: Honoraria, Membership on an entity's Board of Directors or advisory committees;Swedish Orphan Biovitrum AB (Sobi): Consultancy, Honoraria;X4 Pharmaceuticals: Other: Local PI for clinical trial involving mavorixafor and patients with neutropenia.