Panel Kit Research Articles

Investigation of the Effect of the COVID-19 Pandemic Period on Respiratory Tract Viruses at Istanbul Medical Faculty Hospital, Turkey.

Aim: Respiratory viruses significantly impact public health, contributing to high morbidity and mortality rates in both children and adults. This study evaluates the distribution and incidence of respiratory tract viruses in our hospital from 2019 to 2022, focusing on changes post-COVID-19 pandemic. Material and Methods: Utilizing molecular methods, we analyzed nasopharyngeal swabs with the FTD Respiratory Pathogens 21 kit and the QIAStat Dx Respiratory Panel kit at Istanbul Faculty of Medicine. A total of 1186 viruses were detected in 2488 samples (47.6% of the total) examined with the FTD Respiratory Pathogens 21 kit between 2019 and 2022. Results: It was determined that the detection rates were 52.8% in 2019, 44.3% in 2020, 50.0% in 2021, and 40.0% in 2022. Notable changes in prevalence were observed for pandemic influenza A (IAV-H1N1pdm2009), parainfluenza virus (PIV)-3, rhinovirus (RV), and respiratory syncytial virus (RSV)-A/B (p < 0.05). RV consistently showed the highest detection rates across all years (17.6% to 7.9%). Additionally, 1276 viruses were detected in 1496 samples using the QIAStat DX kit, with 91.3% positivity in 2021 and 78.6% in 2022, highlighting the kit's effectiveness in rapid diagnosis. Conclusions: This study enhances understanding of respiratory virus epidemiology during and after the pandemic, emphasizing the need for ongoing surveillance and strategic public health measures to address the evolving landscape of respiratory infections.

B-207 Clinical Evaluation of ExiStation™ FA 96 RV for the detection of human respiratory viruses in human nasopharyngeal swab specimen

Abstract Background There has been growing demand for fully automated multiplex testing of respiratory viruses in molecular diagnostics following the COVID-19 pandemic. ExiStation™ FA 96 RV developed by BIONEER is a multiplex real-time RT-PCR assay. It is designed for the simultaneous detection and differentiation of SARS-CoV-2, Influenza A, Influenza B, and RSV RNA in human nasopharyngeal swab specimens from individuals showing signs and symptoms of respiratory viral infection. ExiStation™ FA 96 automates the entire process from RNA extraction to real-time RT-PCR for up to 96 samples at once within 100 minutes. Moreover, the system is equipped to decap sample tubes, eliminating the need for handling sample transport media. In this study, the clinical performance of the ExiStation™ FA 96 RV assay was evaluated using nasopharyngeal swab samples. Methods Nasopharyngeal swab samples, totaling 859 samples, are collected according to the procedures by the clinical institution in France and preserved in Virus transport media. Once anonymized and randomly allocated, the swab transport media tube, without capping procedure, are directly placed onto the ExiStation™ FA 96. Subsequently, both extraction and real-time RT-PCR are automatically processed. Clinical sensitivity, specificity, and agreement are analyzed by comparing results with confirmatory reagents SARS-CoV-2 RT-qPCR Reagent Kit, PKAmp™ Respiratory SARS-CoV-2 RT-PCR Panel Kit, and Bosphore SARS-CoV-2/Flu/RSV Panel Kit, as well as comparator reagent Allplex™ Respiratory Panel 1 and Allplex™ SARS-CoV-2 Assay. Results The comparison between confirmatory reagents and the ExiStation™ FA 96 RV resulted in a clinical sensitivity of at least 96.4% (164/164 for SARS-CoV-2, 64/65 for Influenza A, 74/75 for Influenza B, and 53/55 for RSV) and a clinical specificity of 100% (500/500 for all targets). When compared with the comparator reagent, the overall agreement and Cohen's kappa value were both determined to be above 99.1% and 0.989, respectively (664/664 for SARS-CoV-2, 564/565 for Influenza A, 573/575 for Influenza B, and 550/555 for RSV). Additionally, any discrepant samples identified in comparison with the comparator reagent were verified through sequencing analysis. Upon sequencing analysis on a total of 8 samples and aligning the confirmed results with the analysis content, no alterations observed in the analysis of clinical sensitivity and specificity. Conclusions The evaluation of the ExiStation™ FA 96 RV assay showed outstanding clinical performance in detecting four respiratory viruses, SARS-CoV-2, Influenza A, Influenza B, and RSV, with high sensitivity and specificity. This study supports that the ExiStation™ FA 96 RV assay is a reliable tool for simultaneous molecular diagnostics of various respiratory infections in clinical laboratories.

The ATOM-Seq sequence capture panel can accurately predict microsatellite instability status in formalin-fixed tumour samples, alongside routine gene mutation testing

Microsatellite instability (MSI) occurs across a number of cancers and is associated with different clinical characteristics when compared to microsatellite stable (MSS) cancers. As MSI cancers have different characteristics, routine MSI testing is now recommended for a number of cancer types including colorectal cancer (CRC). Using gene panels for sequencing of known cancer mutations is routinely performed to guide treatment decisions. By adding a number of MSI regions to a small gene panel, the efficacy of simultaneous MSI detection in a series of CRCs was tested. Tumour DNA from formalin-fixed, paraffin-embedded (FFPE) tumours was sequenced using a 23-gene panel kit (ATOM-Seq) provided by GeneFirst. The mismatch repair (MMR) status was obtained for each patient from their routine pathology reports, and compared to MSI predictions from the sequencing data. By testing 29 microsatellite regions in 335 samples the MSI status was correctly classified in 314/319 samples (98.4% concordance), with sixteen failures. By reducing the number of regions in silico, comparable performance could be reached with as few as eight MSI marker positions. This test represents a quick, and accurate means of determining MSI status in FFPE CRC samples, as part of a routine gene mutation assay, and can easily be incorporated into a research or diagnostic setting. This could replace separate mutation and MSI tests with no loss of accuracy, thus improving testing efficiency.

Pathogen kinetics and detection by next-generation sequencing in pediatric complicated pneumonia

Pediatric pneumonia can be severe and result in empyema. Next-generation sequencing (NGS) may broadly detect pathogens though, optimal timing and impact of sample type on diagnostic yield is unknown. This is a prospective, single-center pilot study of children aged 3 months through 17 years admitted to the PICU with a primary diagnosis of complicated pneumonia. Plasma, endotracheal, nasopharyngeal, and pleural fluid samples were collected at three time points during hospitalization. After nucleic acid extraction, combined libraries were enriched with an NGS enrichment panel kit (RPIP, Illumina), sequenced and quantitative organism detections were analyzed. NGS identified the same bacterial pathogen as traditional testing in all samples, regardless of antibiotic pre-treatment or time collected. Conventional culture methods only identified the pathogen reliably in invasively obtained pleural fluid or endotracheal aspirates. Future application of NGS may allow for non-invasive pathogen detection at a broader range of time points and more targeted antibiotic coverage.

New Variants Identified by Next-Generation Sequencing in Polycystic Kidney Disease Patients.

Polycystic kidney disease (PKD) is a common inherited disease characterized by multiple cysts in kidneys and various extra renal manifestations. Molecular diagnosis plays a crucial role in confirming both the clinical diagnosis and preimplantation genetic diagnosis furthermore, selecting appropriate treatment options. This study aimed to expand the understanding of genetic mutations in patients with polycystic kidney disease and to improve the management of patients. The study included 92 patients with a clinical diagnosis of PKD based on renal ultrasound criteria. Targeted next-generation sequencing was performed using a custom panel kit. Of the 92 patients included in the study, pathogenic/likely pathogenic variants of the PKD1, PKD2 genes were detected in 37 patients (40.2%), while 8 patients (8.6%) had variants with uncertain clinical significance. After the additional assessment of pathogenic/likely pathogenic variants, it was found that 15 of the variants in PKD1 and 2 of the variants in PKD2 have not been reported in the literature previously. Additionally, pathogenic variants, 5 of which were novel, have been identified in different genes in 8 patients. This study presented the largest patient cohort conducted in Turkey. These findings were significant in expanding our understanding of the genetic variations associated with polycystic kidney disease. The study contrıbuted the literature data on polycystic kidney disease by reporting important findings that could pave the way for further investigations in the diagnosis, treatment, and management of the affected patients.

Unraveling Copy Number Alterations in Pediatric B-Cell Acute Lymphoblastic Leukemia: Correlation with Induction Phase Remission Using MLPA.

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy occurring in children. Copy number alterations (CNA) like PAX5, CDKN2A/2B, PAR1 Region, ETV6, IKZF1, BTG1, and RB1 gene deletion are important genetic events that define and prognosticate B-cell ALL. Thus, this study aimed to evaluate associations of CNA with induction phase remission status in childhood B-cell ALL. This study was observational with a cross-sectional design at the Dharmais Cancer Hospital, Harapan Kita Mother and Children Hospital, and Tangerang Regional Public Hospital. We evaluated 74 pediatric B-cell ALL cases with 1-18-year-olds. Genomic DNA was analyzed by Multiplex Ligation Dependent Probe Amplification Assay (MLPA). This study used the P335 ALL-IKZF1 panel kit, which contains several ALL-related genes. The patient's clinical and laboratory characteristics were collected from medical records from January to December 2019. We observed gene copy number alteration in children with B-Cell ALL. PAX5 was the most commonly observed gene deletion, followed by CDKN21/2B, ETV6, IKZF1, BTG1, RB1, and PAR1 Region. Based on gene mutations, only the PAX5 had a significant association with the remission status of pediatric B-cell ALL (p-value <0.05; OR = 3.91). It showed that patients with PAX5 gene mutations have 3.9 times the risk of no remission and/or relapse compared to those without PAX5 gene mutations. Patients with mutations in the PAX5 gene have a higher chance of not achieving remission and/or experiencing relapse than those without such mutations. The MLPA method can be utilized for examining copy number alterations, which is valuable for achieving more precise stratification in diagnosis.. Further research is needed to expand upon this finding.

A universal pipeline to combine spatial transcriptomics, proteomics, and diagnostic H&amp;E assays on a single tissue section to study tissue microenvironment.

e14657 Background: Spatial multi-omics approaches, including spatial transcriptomics (ST) and spatial proteomics (SP), that elucidate the complex tissue microenvironment (TME) are gaining traction in cancer research studies. However, combining these approaches onto one tissue section remains challenging. ST methods visualize, locate and quantify RNA targets, evaluating the transcriptome of the TME at the subcellular level while SP methods stain and visualize protein biomarkers at the cellular level. Here we show a pipeline that combines these ST and SP methods with Hematoxylin and Eosin (H&amp;E) approach on a single tissue section to achieve single slide multi-omics data with high resolution tissue morphology images. Methods: 5µm formalin-fixed paraffin-embedded sections of human lung cancer samples were placed on a Xenium (10x Genomics, Pleasanton, CA) slides and treated to expose RNA molecules. Probe hybridization was performed with a human lung panel kit containing 289 barcoded DNA padlock probes. Downstream ligation and amplification generated copies of the barcode and thereafter, the Xenium Analyzer performed imaging and data collection. The same tissue section was then stained and imaged in COMET (Lunaphore Technologies SA, Switzerland) using sequential immunofluorescence (seqIF) method to detect 40 protein targets in situ. Serial sections of the specimens were used for direct H&amp;E and seqIF staining for comparative validation. Results: Multi-omics images generated from Xenium and COMET were overlayed, jointly analyzed and interpreted. Staining specificity of the protein targets for the post-Xenium slides remain comparable with the direct COMET-stained slide. 40-plex COMET staining intensity in post-Xenium slide was minimally weaker compared to direct COMET-stained slide. Additionally, H&amp;E staining of the post-Xenium/COMET slide and direct COMET-stained slide were comparable to the direct H&amp;E-stained section, demonstrating discernable tissue landscape features despite a fainter hematoxylin stain. Overall, tissue integrity and cell morphology were well-preserved across the different workflows performed on each section, and the protein targets remain relatively stable for labelling after in situ RNA detection process. Conclusions: We present a pipeline that combines two spatial omics approaches with the diagnostic standard H&amp;E staining on a single tissue section, allowing the same cells to be assessed for their histological, proteomic, and transcriptomic information. A visual comparison of images showed minimal compromise to cellular morphology and protein stability at the end of the pipeline compared to direct staining of serial sections. This pipeline could be potentially generalized to most of the ST and SP approaches, thus advancing multi-omics cancer research and potentially clinical diagnostics in the future.

Detecting immune activity in human tumors with novel high-throughput whole slide imaging approaches using Imaging Mass Cytometry.

Abstract Deciphering the spatial biology of the tumor immune microenvironment (TIME) has potential to inform clinical and translational researchers about the likelihood of immunotherapy success. Imaging Mass Cytometry™ (IMC™) is a spatial biology imaging technique that enables deep characterization of the TIME while fully retaining spatial features of tissues using 40-plus markers simultaneously. We developed two new whole slide imaging methods for IMC. Tissue mode (TM) rapidly acquires an image of the whole tissue that can be used for quantitative analysis of spatial biology. Preview mode (PM) samples the entire tissue at predefined spacing to rapidly capture a low-resolution image of all expressed markers, improving informed identification of areas for high-resolution cell mode (CM) imaging on the same slide. Tumor tissues were stained using a 40-marker panel comprised of the Maxpar™ Human Immuno-Oncology IMC Panel Kit, designed for tumor and immune cell profiling. TM imaging in combination with pixel-based analysis quantified tissue areas containing high or low abundance of myeloid and lymphoid immune, tumor and stromal cell compartments. PM in combination with CM imaging followed by single-cell analysis on selected tumor regions identified cell populations with varying activation states and expression profiles. Overall, we highlight the power of IMC to provide quantitative insights into the spatial biology of the TIME. For Research Use Only. Not for use in diagnostic procedures.

The efficacy of ferroptosis-inducing compounds IKE and RSL3 correlates with the expression of ferroptotic pathway regulators CD71 and SLC7A11 in biliary tract cancer cells.

Biliary tract cancer (BTC) is a lethal disease with a bad overall survivability, partly arising from inadequate therapeutic alternatives, detection at a belated stage, and a resistance to common therapeutic approaches. Ferroptosis is a form of programmed cell death that depends on reactive oxygen species (ROS) and iron, causing excessive peroxidation of polyunsaturated fatty acids (PUFAs). Therefore, the objective of this investigation is, whether ferroptosis can be induced in BTC in vitro and whether this induction is dependent on specific molecular markers. The study conducted resazurin assay and IC25/50 calculation to explore the possible cytotoxic outcomes of different classes of ferroptosis-inducing substances (FINs) on a comprehensive in vitro model of 11 BTC cell lines. Combinatory treatments with different cell death inhibitors were performed to evaluate the magnitude of ferroptosis induction. To ascertain whether ferroptotic cell death occurred, liperfluo and iron assay kits were employed to evaluate lipid ROS and intracellular iron abundance. Potential biomarkers of ferroptosis sensitivity were then assessed via western blot analysis, a rtPCR panel and functional assay kits. The study found that different FINs reduced cell viability in a cell line-dependent manner. In addition, we measured increased lipid ROS and intracellular Fe2+ levels upon exposure to FINs in BTC cells. Combining FINs with inhibitors of ferroptosis, necroptosis or apoptosis suggests the occurrence of ferroptotic events in BTC cell lines CCC-5, HuH-28 and KKU-055. Furthermore, we found that BTC cells display a heterogeneous profile regarding different molecular genes/markers of ferroptosis. Subsequent analysis revealed that sensitivity of BTC cells towards IKE and RSL3 positively correlated with CD71 and SLC7A11 protein expression. Our results demonstrate that induction of ferroptosis is a promising approach to inhibit BTC cell growth and that the sensitivity of BTC cells towards ferroptosis induction might be dependent on molecular markers such as CD71 and SLC7A11.

Abstract LB291: High-plexy intracellular and surface CITE-Seq offers multiomic insights into cell function

Abstract CITE-Seq allows researchers to combine high-plex protein information with whole transcriptome sequencing to gain insights about individual cell states. Due to the harsh fixation and permeabilization required for intracellular (IC) antibody staining, only a few studies have recovered mRNA expression profiles alongside IC protein detection. This experiment utilized a novel IC CITE-Seq assay that enables robust profiling of multiple IC protein targets in combination with mRNA and high-plex surface proteins. In this study, we stimulated human peripheral blood mononuclear cells (PBMCs) alongside a resting control. After stimulation, we stained the surface proteins with a 40-plex BD® AbSeq Panel, including the Immune Discovery Panel and human Single-Cell Multiplexing Kit (SMK). Resting and stimulated cells were pooled, and a portion of the sample pool immediately underwent cell capture as a live control using a BD Rhapsody™ single-cell microwell system. The other portion of the pool was stored in BD® OMICS-Guard Sample Preservation Buffer for either 5 minutes or 24 hours followed by permeabilization, IC staining with a 10-plex IC AbSeq Panel (50-plex total, surface and IC), and cells captured on the the same system. We show IC protein detection for the expected positive targets, including pH2AX, actCaspase-3, and cPARP in the stimulated population, as well as T-Bet, Granzyme B and Helios in the resting lymphocytes. Analysis shows excellent correspondence between IC CITE-Seq protein and transcriptome for T-Bet, Granzyme B and Helios. The gene expression correlation for surface antigen and mRNA between the IC and live control each had R2 values &amp;gt; 0.9. The mRNA sensitivity for the IC sample compared to live control was within whole transcriptome analysis (WTA) assay variation at 86–89% and 95–97% median molecules and genes per cell. Our data showed that the new IC CITE-Seq assay can provide rich single-cell multiomics information including mRNA and surface and intracellular proteins. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD, the BD Logo and BD Rhapsody are trademarks of Becton, Dickinson and Company or itsaffiliates. © 2023 BD. All rights reserved. NPM-2532 (v1.0) 1023 Citation Format: Tracy L. Campbell, Adam Wright, Ying Wah Lee, Anne Tran, Yan Chen, Joe Olives, Hye-Won Song, Larry Wang. High-plexy intracellular and surface CITE-Seq offers multiomic insights into cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB291.

Abstract 585: Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model

Abstract Purpose: In the United States, colorectal cancer (CRC) is the second leading cause of cancer mortality. Metastatic CRC (mCRC) refractory to traditional chemotherapy is managed with regorafenib, a multiple-kinase inhibitor. However, patients show only a modest improvement in overall survival but experience high drug toxicity, adverse side effects, and poor tolerability. Thus, to reduce regorafenib-induced toxicity and to enhance antitumor activity, we combined it with a hybrid/dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition in a single agent. With syngeneic mice, the efficacy and impact on immunomodulation of this drug combination was assessed. Methods: To assess effects of the agents, C57BL/6 immunocompetent mice were injected with MC38 murine CRC cells and treated with regorafenib (6mg/kg body wt) or JAK/HDACi (30mg/kg body wt), and their combination every third day for 21 days. Upon completion of the experiment, the tumors were harvested and processed for RNA and protein expression profiles. The effect of drug treatment on immune response was analyzed by nCounter Gene Expression assays (NanoString Technologies). Cytokines were measured in serum using the MesoScale Discovery mouse V-Plex Proinflammatory Panel kit. For pharmacokinetic studies, plasma samples were assayed from C57BL/6 treated with drugs after 1, 7, 21, and 48 hrs of treatment. Results: The combination treatment significantly reduced tumor growth (volume and weight),relative to the vehicle control or single treatments. Gene expression showed higher CD45 abundance score in the combination treatment. This is an exciting observation, as CD45 has phosphatase activity and dephosphorylates key targets of the drugs used in the study. Additionally, after treatment with the combination, there was higher expression of Gzmb and Gzme and lower expression of Cx3cr1, indicating enhanced immuno modulation relative to regorafenib alone. We confirmed these findings by immunoprofiling of tumors. Higher CD45 staining, noted in tumors of mice treated with the combination, corroborated the gene expression results. Pronounced CD8 T lymphocytes infiltration was observed in combination, compared to regorafenib alone. Reduction of proinflammatory cytokine, TNF-α was noted in the combination group. This is an interesting observation as CD45 negatively regulates TNF, and combination treated mice had highest number of CD45 positive cells. Pharmacokinetic studies showed that the bioavailability of regorafenib was elevated after combination treatment relative to single-agent treatment. Conclusions: Relative to the single agents, the combination of regorafenib with JAK/HDACi was more effective, with an elevated antitumor immune response and with sustained inhibition of tumor growth. A clinical trial to evaluate this combination for the treatment of mCRCs is warranted. Citation Format: Prachi Bajpai, Farrukh Afaq, Sameer Al Diffalha, Sumit Agarwal, Hyung Gyoon Kim, Dennis Otali, Sooryanarayana Varambally, Ashish Manne, Ravi Paluri, Moh’d Khushman, Upender Manne. Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 585.

Abstract 1450: Novel whole slide imaging modes for imaging mass cytometry reveal cellular and structural composition of mouse glioblastoma

Abstract Mouse tumors have become widely used models in translational research of brain malignancies, helping to address the difficulties related to studying the human brain. Mouse brain can be regarded as a miniaturized model of the human brain, permitting visualization of the whole tissue to provide spatial cellular context. Imaging Mass Cytometry™ (IMC™) is a highly relevant tool capable of quantitative evaluation of the multiparametric protein composition in brain tumor microenvironment (TME) without the complications of autofluorescence, tissue degradation, and spectral overlap. The Hyperion XTi™ Imaging System (Standard BioTools™) utilizes IMC technology to simultaneously assess more than 40 individual structural and functional markers in tissue, providing insight into the organization and function of the TME. We demonstrate the whole slide imaging (WSI) application using a 40-marker panel composed of the Maxpar OnDemand™ Mouse Immuno-Oncology IMC Panel Kit and the Maxpar® Neuro Phenotyping IMC Panel Kit on mouse normal and glioblastoma (GBM) brain tissue. The panel was composed of mouse-specific antibodies, which highlight tumor and immune components of the mouse TME. We performed imaging using two new features of Hyperion™ XTi. Ultrafast preview mode (PM) was applied to rapidly screen entire brain sections for marker expression signatures associated with various immuno-oncology processes. This enabled biomarker-guided selection of areas in tumor tissue that were imaged using region of interest-based IMC and analyzed using single-cell analysis (SCA). In parallel, a high-throughput tissue mode (TM) was applied to perform a detailed whole-slide scan of normal and GBM mouse brain tissues that were quantified using pixel-based analysis (PBA) to unravel the composition of the TME. Using TM, we were able to successfully visualize the entire coronal and sagittal sections of normal mouse brain as well as mouse GBM tissue. PBA on TM data provided quantitative spatial expression patterns of structural and immune markers across the whole tissue. In normal tissue, we visualized the highly organized structure of the normal brain and detected neurons, oligodendrocytes, vascular-adjacent astrocytes, and axonal tracks. In GBM tissue, necrotic cores, areas with high immune infiltration, extracellular matrix deposits, and activated tumor cells were detected. SCA of GBM tissue demonstrated expansive vascularization, replicating Olig2+ cells, activation of Ras signaling, and high abundance of infiltrating immune cells. Overall, we demonstrate the successful application of two novel WSI modes and highlight the power of IMC technology to simultaneously explore dozens of relevant biological outputs to better understand the TME of GBM and other tumors. For research use only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Nick Zabinyakov, Thomas D. Pfister, Nikesh Parsotam, David Howell, Liang Lim, Christina Loh. Novel whole slide imaging modes for imaging mass cytometry reveal cellular and structural composition of mouse glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1450.

Abstract 3800: Next generation of spatial biology: High-throughput multiplexed Imaging Mass Cytometry™ with whole slide modes

Abstract Gaining spatial insights into the cellular composition of tumor tissue has tremendous potential to inform clinical and translational researchers about mechanisms behind spatial predictors of immunotherapy success, disease etiology and progression. Imaging Mass Cytometry™ (IMC™) is a high-plex spatial biology imaging technique that enables deep characterization of the diversity and complexity of the tumor microenvironment (TME). IMC supports detailed assessment of cell phenotype and function using 40-plus metal-tagged antibodies simultaneously on a single slide without issues associated with fluorescence-based spectral overlap, tissue autofluorescence or implementation of multiple washing and acquisition cycles. Currently, IMC enables user-defined regions of interest (ROI) in tissues to evaluate cellular and structural composition. To enhance the IMC user experience, we developed two new whole slide imaging (WSI) modes which enable streamlined workflows using ultrafast preview mode (PM) and high-throughput tissue mode (TM). PM samples the entire tissue at predefined spacing to rapidly capture a low-resolution image of all expressed markers in the antibody panel. PM generates an image in minutes to enable informed ROI placements while leaving the stained tissue intact for higher-resolution imaging. PM and TM are designed so acquisitions can easily occur on the same slide without additional processing steps. TM rapidly acquires images of the whole tissue at lower resolution (7 μm pixel size) at a quality that can be used for quantitative analysis of the tissue spatial biology. Specifically designed for high-throughput applications, TM in combination with a newly available 40-slide loader for the Hyperion XTi™ Imaging System permits automated and continuous imaging of more than 40 large tissue samples (400 mm2 per tissue) per week. We showcase the application of WSI modes using the newly developed Maxpar® Human Immuno-Oncology IMC Panel Kit. The 31-marker panel was combined with catalog antibodies to create a 40-plus-marker panel that expands the ability to conduct comprehensive high-plex tumor and immune cell profiling. Tumor tissue microarrays (TMA) and whole tumor tissue sections were stained with the expanded panel. Single-cell analysis of selected ROIs, on whole tumor sections and TMAs, guided by PM data successfully provided quantitative analyses of spatial biology at single-cell resolution. In parallel, TM on whole tumor sections followed by pixel-based analysis provided a spatially resolved quantitative assessment of specific tumor and immune components of the TME. This work demonstrates the expanded capabilities of IMC and establishes it as a reliable high-plex spatial biology imaging platform with high-throughput imaging capabilities ideally suited for translational and clinical applications. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Thomas D. Pfister, Jyh Yun Chwee, Liang Lim, David Howell, Nikesh Parsotam, David King, Christina Loh. Next generation of spatial biology: High-throughput multiplexed Imaging Mass Cytometry™ with whole slide modes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3800.

Abstract 3697: Proof-of-concept study to enumerate large oncosomes using the RareCyte CTC platform

Abstract Introduction: Large oncosomes (LO) are produced by tumor cells and can be detected in blood from cancer patients. The clinical utility of enumerating and characterizing LOs along with CTCs has been demonstrated, especially in settings where CTC counts are low. The RareCyte CTC platform has the potential to capture LOs along with CTCs. We performed a pilot study to evaluate the RareCyte CTC platform’s potential to analyze LOs using established preanalytical procedures. Methods: Metastatic breast cancer patients’ (N=10) blood smears labelled with RarePlex® Enumeration panel kit were re-evaluated to identify objects with LO-like characteristics at 10x magnification, along with negative control slides (N=5). LO-like objects were annotated and re-scanned at 40x magnification using the CyteFinder® imaging instrument. After establishing the criteria for LO identification, LO enumeration was performed on a cohort of MBC patients (N=6). Sets of five blood smear slides stained with the RarePlex® Enumeration panel kit were reviewed and LO counted. Total LO counts were established by extrapolating LO counts to 7.5 mL plasma. Pearson correlation was used to determine the level of correlation between LO counts and previously obtained CTC counts. Results: The established identification criteria enabled the consistent enumeration of LOs at 10X magnification. Total LO count was 9.6 on the negative control slides. In MBC patients the mean total LO count was 306.2 (range 3.2 - 792). There was a significant positive correlation between LO and CTC count (r=0.855, p=0.3). In samples with CTC&amp;gt;0 (N=3), the average total LOs count was 588, about six times higher than CTC count. In CTC=0 patient samples (N=3) the mean total LO count was 24.3 (range 3.2-36). Conclusions: We report a novel protocol to quantify large oncosomes using the RareCyte CTC platform using established preanalytical workflows. The strong CK/EpCam staining in LOs and the strong correlation of LO count with CTC count indicates their tumoral origin. Due to their higher abundance, LOs combined with CTCs offer a more sensitive tool for treatment response monitoring. Due to its broad range, LO counts in patients with CTC=0 may reveal therapy response dynamics undetectable with CTC enumeration alone. Ongoing work focuses on the parallel detection and integrated evaluation of HER2 by immunofluorescent staining of CTCs and LOs, along with digital PCR based HER2 quantification using cfDNA from matched blood plasma samples. Citation Format: Eszter Papp, Jo Vandesompele, Peter B. Vermeulen, Luc Y. Dirix, Mark Kockx. Proof-of-concept study to enumerate large oncosomes using the RareCyte CTC platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3697.

Immunogenicity and Tolerance of BNT162b2 mRNA Vaccine in Allogeneic Hematopoietic Stem Cell Transplant Patients.

Allogeneic hematopoietic stem cell transplantation (ASCT) induces acquired immunodeficiency, potentially altering vaccine response. Herein, we aimed to explore the clinical tolerance and the humoral and cellular immune responses following anti-SARS-CoV-2 vaccination in ASCT recipients. A prospective, non-randomized, controlled study that involved 43 ASCT subjects and 31 healthy controls. Humoral response was investigated using the Elecsys® test anti-SARS-CoV-2. Cellular response was assessed using the QFN® SARS-CoV-2 test. The lymphocyte cytokine profile was tested using the LEGENDplex™ HU Th Cytokine Panel Kit (12-plex). Adverse effects (AE) were observed in 69% of patients, encompassing pain at the injection site, fever, asthenia, or headaches. Controls presented more side effects like pain in the injection site and asthenia with no difference in the overall AE frequency. Both groups exhibited robust humoral and cellular responses. Only the vaccine transplant delay impacted the humoral response alongside a previous SARS-CoV-2 infection. Noteworthily, controls displayed a Th1 cytokine profile, while patients showed a mixed Th1/Th2 profile. Pfizer-BioNTech® anti-SARS-CoV-2 vaccination is well tolerated in ASCT patients, inducing robust humoral and cellular responses. Further exploration is warranted to understand the impact of a mixed cytokine profile in ASCT patients.

Molecular analysis of genetic mutations in non-small cell lung cancer in Morocco.

Non-small cell lung cancer (NSCLC) is a significant global health issue with diverse molecular profiles affecting treatment responses. Yet, NSCLC's molecular epidemiology in Morocco is largely unexplored. This study focuses on NSCLC genetic mutations, specifically in adenocarcinoma, among Moroccan patients to contribute to understanding NSCLC in this population. Ninety-four patients diagnosed with lung adenocarcinoma were analyzed. Formalin-fixed paraffin-embedded tissue samples were processed, and deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) was extracted using standardized protocols. Mutations were detected using the AmoyDx Pan Lung Cancer Polymerase Chain Reaction (PCR) Panel kit, and their frequencies were assessed through statistical analysis. Epidermal Growth Factor Receptor (EGFR) mutations were detected in 22.34% of patients, predominantly exon 19 deletions (66.66%) and exon 21 L858R mutations (23.80%). Anaplastic lymphoma kinase (ALK) gene fusion was observed in 3.19% of patients, and KRAS mutations in 1.06%. No mutations were found in other tested genes. A slightly higher mutation rate was noted in females (54.16%) compared to males (45.84%). The study reveals a distinct mutation profile in Moroccan NSCLC patients, with a notable prevalence of EGFR mutations, albeit lower than in some Asian populations. The significance of EGFR mutations in treatment response aligns with global findings, highlighting the importance of understanding regional molecular variations for personalized therapy. Despite limitations in sample size and clinical data, this study sheds light on the genetic landscape of NSCLC in Morocco. The observed mutation rates, particularly in EGFR, underscore the potential for targeted therapies in Moroccan NSCLC patients, emphasizing the need for further research to refine treatment strategies tailored to this population.

Shisha Consumption and Presence of Cotinine in Saliva Samples among Students in Public Universities in Coastal Kenya.

Despite the well-known adverse health effects of tobacco, shisha use among students in tertiary institutions remains a public health concern. In Kenya, the literature on status of shisha after the 2017 ban is scanty. This study sought to ascertain actual shisha use among university students along the coastal strip. We investigated confirmed and self-reported shisha use. Using proportionate-to-size and snowball sampling methods, 380 respondents were enrolled from three universities. Sociodemographic characteristics and self-reported history of shisha use were documented using a participant-assisted questionnaire. Actual shisha use was determined qualitatively using 6 panel plus alcohol saliva test kit that detected cotinine use among other selected drugs. Of the 380 participants, 278 (73%) were males and their median (IQR) age was 22 (20-23) years. This study reports 29% current use based on testing positive for cotinine. Among those who reported current ever use of shisha, 19% tested positive for cotinine, respectively. In the multivariable analysis, being separated (adjusted risk ratio (aRR): 2.06 (95% CI: 1.45-2.94)) compared to being single and studying for a degree compared to a diploma (aRR: 1.32 (95% CI: 1.10-1.58)) were associated with cotinine positive. The 4th year of study (aRR: 1.68 (95% CI: 1.22-2.33)) compared to the 1st year and reported knowledge of shisha (aRR: 1.84 (95% CI: 1.18-2.87)) were associated with cotinine positive. Nearly one-third of university students along the Kenyan coast are active shisha users. Saliva testing for cotinine is a more reliable method of reporting tobacco use. We recommend upscaling of health education, re-enforcement of the current ban on shisha consumption by concerned authorities, and saliva testing for cotinine while assessing current tobacco use.

Omicron and Delta variant prevalence detection and identification during the fourth COVID-19 wave in Mexico using wastewater-based epidemiology

ObjectivesTo identify the SARS-CoV-2 variants Delta and Omicron during the fourth wave of the COVID-19 pandemic in Mexico using samples taken from 19 locations in 18 out of the 32 states. MethodsThe genetic material concentration was done with PEG/NaCl precipitation, SARS-CoV-2 presence was confirmed by reverse transcriptase-quantitative polymerase chain reaction assay, the variant detection was carried out using a commercial mutation detection panel kit, and variant/mutation confirmation was done by amplicon sequencing of receptor-binding domain target region. The study used 41 samples. ResultsThe Delta variant was confirmed in two samples during August 2021 (Querétaro and CDMX) and in three samples during November 2021 (Aguascalientes, Ciudad Juárez campuses, and Nuevo Leon). In December 2021, another sample with the Delta variant was confirmed in Nuevo Leon. Between January to March 2022 only the presence of Omicron was confirmed, (variant BA.1). Additionally, in this period six samples were identified with the status “Variant Not Determined”. ConclusionTo our knowledge, this study is one of the first to identify Omicron and Delta variants with polymerase chain reaction in Mexico and Latin America and its distribution across the country with 56% Mexican states making it a viable alternative for variant detection without conducting a large quantity of sequencing of clinical tests.

Evaluation of next generation sequencing approaches for SARS-CoV-2

Within public health control strategies for SARS-CoV-2, whole genome sequencing (WGS) is essential for tracking viral spread and monitoring the emergence of variants which may impair the effectiveness of vaccines, diagnostic methods, and therapeutics. In this manuscript different strategies for SARS-CoV-2 WGS including metagenomic shotgun (SG), library enrichment by myBaits® Expert Virus-SARS-CoV-2 (Arbor Biosciences), nCoV-2019 sequencing protocol, ampliseq approach by Swift Amplicon® SARS-CoV-2 Panel kit (Swift Biosciences), and Illumina COVIDSeq Test (Illumina Inc.), were evaluated in order to identify the best approach in terms of results, labour, and costs. The analysis revealed that Illumina COVIDSeq Test (Illumina Inc.) is the best choice for a cost-effective, time-consuming production of consensus sequences.

Preconception care, prenatal care, and postpartum bonding in women with substance use

BackgroundThe status of care in the preconception and pregnancy periods in women who use substances can have an impact on maternal and neonatal health. This study aimed to assess the provision of preconception care, prenatal care, and postnatal mother-to-child bonding among pregnant women who use substances. MethodsAn ambidirectional cohort study was conducted, involving 69 pregnant women who reported substance use and had it confirmed using a ten-parameter panel kit (M10T) manufactured by Hannan Teb Pars Company. These women were selected from a referral maternity hospital between January and December 2020, using a convenience sampling method. Sociodemographic information, obstetric and medical history, and information about preconception and prenatal care were collected. All maternal and neonatal outcomes were recorded from the time of admission to the time of discharge for both the mothers and their neonates. Neonatal abstinence syndrome and mother-to-child bonding were assessed using modified Finnegan's neonatal abstinence tool and a postpartum bonding questionnaire, respectively. The data were analyzed using descriptive and inferential tests using SPSS software version 22. ResultThe mean age of the women was 32.8 ± 5.7 years. The mean duration of substance use was 5.1 ± 3.5 years. 48% of the pregnancies were reported as unwanted. A total of 94.2% and 50.7% of pregnant women did not receive preconception care and prenatal care, respectively. There was no association between pregnancy wantedness and receiving preconception care (P = 0.287), but a significant association was observed for prenatal care (P < 0.001). 31% of the mothers experienced a mother-to-child bonding disorder, with 75% of those who had unwanted pregnancies reporting such a disorder. ConclusionThe findings of this study indicate that the majority of pregnant women who use substances did not receive preconception care, and prenatal care was inadequate with fewer visits than recommended. One-third of the pregnant women who use substances experienced a mother-to-child bonding disorder. It was also observed that women with unwanted pregnancies had poorer perinatal care and mother-to-child bonding.