Use Of Diagnostic Techniques Research Articles

Identifying spatial immuno-oncological parameters of mouse tumors using Imaging Mass Cytometry

Abstract Identification of immuno-oncological (IO) processes that dictate tumor growth, metastasis, and immune response is essential for selecting promising drug candidates in preclinical models. Imaging Mass Cytometry™ (IMC™) is a proven high-plex imaging technology that enables deep characterization of the complexity of tumor tissue without disrupting spatial context while eliminating artifacts caused by tissue autofluorescence. Here, we showcase the Maxpar® OnDemand™ Mouse Immuno-Oncology IMC Panel Kit for application on mouse tumor tissues. We compiled a 33-plex antibody panel to quantitatively assess IO-related processes and applied it to a tissue microarray containing a large variety of mouse tumors. We digitized high-plex data from mouse tissues using the Hyperion™ Imaging System and generated images demonstrating the detailed layout of the tumor microenvironment (TME). We further conducted single-cell analysis to identify specific populations of tumor and immune cells in the TME. The Mouse IO IMC Panel Kit successfully identified pathophysiological processes such as immune cell infiltration and activation, signaling pathway activation, biomarkers of epithelial-to-mesenchymal transition (EMT), metabolic activity, growth, and the TME tissue architecture. Single-cell analysis of several highly relevant tumor types separated distinct cellular clusters representing tumor, immune, stromal, and vascular cells. Our quantitative analysis of tumor composition revealed critical insights regarding prognostic parameters such as metastatic and growth potential of tumor cells and activity of immune cell infiltrates. For Research Use Only. Not for use in diagnostic procedures.

A 47-marker immune profiling flow cytometry assay to enable comprehensive antigen-specific immune analysis

Abstract The complex nature of the immune system requires deep interrogation at the single-cell level. Flow cytometry using CyTOF® technology employs antibodies tagged with isotopically enriched metals instead of fluorophores, resulting in discrete signals that facilitate highly multiparametric characterization on a single-cell level. CyTOF technology enables analysis of over 50 parameters simultaneously without the impediments of spectral overlap or autofluorescence, allowing for a reliable evaluation of immune responses. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, and validated 30-marker antibody cocktail for single-tube staining of human whole blood or PBMC and acquisition on CyTOF systems. Using the assay and Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations. In this study, we highlight the customizability of the Maxpar® Direct panel by adding an array of 17 surface and intracellular parameters with the Maxpar Direct™ T Cell Activation Expansion Panel and live-cell barcoding. The simultaneous detection of key surface and intracellular markers in this expanded panel enabled comprehensive analysis of immune cell activation and antigen-specific recall responses to PBMC stimulated with a pool of immunodominant microbial peptides. Taken together, this 47-marker panel and automated analysis unlocked deeper immunophenotyping and functional profiling of activated immune cells and provided insight into the cell-mediated adaptive immune response to foreign targets. Such phenomena are hallmarks for research on infection, vaccine development, and immunotherapy. For Research Use Only. Not for use in diagnostic procedures.

Abstract LB004: Ultra-sensitive targeted DNA panel for very low-frequency mutation detection in circulating cell-free DNA

Abstract Due to its noninvasive nature and being readily available in peripheral blood and other bodily fluids, analysis of cell-free DNA (cfDNA) has become a promising application in cancer diagnosis, prognosis and treatment monitoring. However, disease-relevant cfDNA is present in a very limited amount in the huge background of normal cfDNA. This remains a challenge for any detection technology. Many currently available target enrichment and library preparation methods use regular DNA polymerase and amplification processes that introduce substantial bias and artifacts. This results in artifactual errors that greatly limit the detection of true low-frequency variants below 0.5% in heterogeneous samples, such as cfDNA. Here, we present a new targeted cfDNA workflow that overcomes challenges such as biases and artifacts. The workflow uses a highly optimized, high-fidelity reaction chemistry and incorporates UMIs into a single gene-specific, primer-based targeted enrichment process. Compared to regular DNA polymerase, this high-fidelity chemistry resulted in a five- to ten-fold decrease in base substitution error. An individual cancer panel was designed to specifically cover cancer-relevant hotspots and copy number genes with a dense primer design to accommodate the short length of cfDNA. Due to its high-fidelity chemistry and optimal panel design, our streamlined workflow can be completed in a single day. We also report consistent panel performance across different samples. Detection sensitivity and specificity were evaluated on a reference cfDNA sample and a simulated cfDNA sample by mixing enzyme-digested Genome in a Bottle samples, NA12878 and NA24385. We achieved close to 90% detection sensitivity and above 99.9% specificity for 0.1% variant. In addition, copy number variation could be successfully detected with a 1.5-fold difference over normal control samples. These results demonstrate an efficient targeted cfDNA panel workflow that enables cancer-relevant mutation detection with high sensitivity and accuracy. The applications presented here are for research use only. Not for use in diagnostic procedures. Citation Format: Michelle Baird, Mariam Ashraf, Emil Chistensen, Christa Haldrup, Zhong Wu, Eric Lader. Ultra-sensitive targeted DNA panel for very low-frequency mutation detection in circulating cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB004.

Abstract 6530: Preferential low frequency allele enrichment with lower sequencing depth requirement using KAPA HyperPETE specialized primer designs

Abstract Background: The presence of circulating tumor DNA (ctDNA) can be used for minimal residual disease (MRD) monitoring and guide adjuvant therapy decisions. However, even when present, total ctDNA could be less than 0.01% of cell free DNA (cfDNA). Sequencing such low mutation frequency can be costly due to high sequencing read depth and accuracy requirements. The ability to enrich for low-abundance variants could improve detection performance and greatly lower sequencing cost by reducing the sequencing depth required. Purpose: The KAPA HyperPETE (Primer Extension Target Enrichment) workflow employs primer extension reactions to specifically capture and release target library molecules for sequencing, detects all major somatic variant types (SNV, InDel, CNV, MSI status, and novel fusion transcript partners) and could be leveraged to specifically target certain alleles. In this study, we assessed the variant enrichment capabilities of the KAPA HyperPETE workflow. Methods: Variant specific primers were designed to 8 low allele frequency single nucleotide variants (SNVs) in mixed cell line samples (NA12878 and NA24143, two Epstein-Barr virus transformed B cell female cell lines, with variant allele frequency (VAF) 1%, 0.5%, and 0.1%). Variant enrichment primers were used alongside a 37kb panel in the KAPA HyperPETE workflow. Additional optimizations and a study enriching all low allele frequency SNVs* in SeraCare ctDNA Mutation Mix v2 reference samples (VAF 1%, 0.5%, and 0.125%) are planned. Results: The preliminary results exhibited a median of 249✕, 159✕, and 86.5✕ enrichment of the targeted variants compared to the expected VAFs of 0.1%, 0.5%, and 1%, respectively. All 8 targeted variants were detected at 100K, 150K, and 550K read pairs per variant for VAF 1%, 0.5%, and 0.1%, respectively. Results from further optimizations and the study using SeraCare ctDNA samples will be shared at the AACR meeting. Conclusion: Preferential enrichment of specific variants down to 0.1% can be achieved using the KAPA HyperPETE Workflow. Utilizing primers designed for variant enrichment, we simultaneously enriched multiple variants and demonstrated the potential to minimize required sequencing. The KAPA HyperPETE workflow can be a powerful, cost-effective tool for detecting low frequency mutations in MRD research applications. KAPA HYPERPETE is For Research Use Only. Not for use in diagnostic procedures. KAPA HYPERPETE is a trademark of Roche. *Except GNA11 (COSM52969) due to stretches of polybase. Seraseq ctDNA Mutation Mix v2 gene list available on SeraCare website. Citation Format: Florence K. Crary-Dooley, Liu Xi, Ruben van der Merwe, Nitya M. Furtado, Jingchuan Li, Junyan Lin, Manuel Ochoa, Brian Godwin. Preferential low frequency allele enrichment with lower sequencing depth requirement using KAPA HyperPETE specialized primer designs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6530.

Abstract 1033: Evaluation of HER2 status in circulating tumor cells isolated using the Parsortix® system

Abstract Background: Evaluation of HER2 status in tissue from breast cancer patients is a standard test for clinical diagnosis and treatment. However, tissue biopsy is invasive and repeated testing is generally not possible. Liquid biopsy allows minimally invasive, repeat sample collection for regular monitoring. This study combined ANGLE’s Parsortix® PR1 Research System*, an epitope-independent microfluidic device that isolates and harvests rare cells from blood based on size and deformability, with two downstream assays: an Immunofluorescence (IF) assay for HER2 protein detection and an IF assay combined with FISH targeting HER2 gene amplification, to determine HER2 status on circulating tumor cells (CTCs). Methods: Blood was collected from healthy volunteers into Streck Cell-Free DNA tubes. Two aliquots of blood from each donor were spiked with cultured SKBR3 (HER2+) and Hs 578T (HER2-) breast cancer cells and then processed using Parsortix® PR1 instruments. Captured cells were harvested and spun onto positively charged slides. One slide per donor was stained using ANGLE’s IF panel for CTC detection, consisting of a nuclear dye, antibodies targeting epithelial and mesenchymal markers, and antibodies targeting blood cell markers. After imaging, slides were de-stained and processed using a commercially available HER2 FISH kit. Cancer cells identified by the IF panel were re-imaged to examine FISH signal. Cells were considered HER2+ if the ratio of HER2 foci to CEP17 foci was ≥2. The second slide was stained using the same IF CTC panel in combination with an antibody targeting HER2 protein. HER2 expression was analyzed based on intensity of fluorescence signal. Same workflow was implemented in a small cohort of breast cancer patients with blood processed 72-144 hours post-draw using Parsortix® PC1 Clinical System. Results: The numbers of epithelial and mesenchymal CTCs detected on each slide were comparable in both assays. In HER2 FISH-stained samples, 97.3% of the SKBR3 cells and 13.3% of the Hs 578T cells harvested showed HER2 amplification. This data was consistent with the literature (~20% of Hs 578T cells tend to form isochromosomes, altering the HER2/CEP17 ratio). In IF-only stained samples, 96.8% of the SKBR3 cells and 0% of the Hs578T cells harvested overexpressed HER2 protein. In patient samples, CTCs were identified in 80% of the donors (mean: 21; median: 6; range: 0-200), with 17% of the CTC-positive donors having ≥1 CTC overexpressing HER2 protein. Conclusions: This study demonstrates the feasibility of HER2 evaluation using IF for protein expression and HER2 FISH for gene amplification on CTCs enriched from blood using the Parsortix® systems. It also demonstrates that blood collection in Streck tubes is suitable for both assays, with the advantage of allowing longer blood sample storage and facilitating batch shipping and processing of clinical samples. *Research Use Only. Not for use in diagnostic procedures. Citation Format: Nerea Borreguero-Munoz, Alex Young, Jessica Kimber, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Evaluation of HER2 status in circulating tumor cells isolated using the Parsortix® system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1033.

Abstract 780: Digital PCR-based EGFR T790M mutation detection from human blood samples collected in PAXgene Blood ccfDNA Tubes

Abstract Introduction: Circulating cell-free DNA (ccfDNA) is used as an analyte in liquid biopsy-based cancer research with promising applications in the fields of cancer screening, therapeutic decision making and monitoring and minimal residual disease control. However, despite the multitude of future applications, challenges regarding the sensitivity of analytical tests remain, thereby limiting the routine use of ccfDNA within the above mentioned fields. In this study, we investigated the compatibility of the PAXgene® Blood ccfDNA Tube and the QIAcuity® Digital PCR System as an optimal “sample to insight” workflow with regard to the detection of EGFR T790M mutations spiked into apparently healthy human donor blood specimens. Methods: Blood from 22 apparently healthy consented donors was collected into PAXgene Blood ccfDNA Tubes* (PreAnalytiX). Within 2 h of blood draw, blood samples were either spiked with an EGFR-Multiplex 5% AF cfDNA standard (SensID) containing an EGFR T790M mutation or left unspiked as a control. Tubes were incubated for 3 d at room temperature to simulate transport over a weekend and plasma was generated by double centrifugation. Plasma pools were generated from spiked and unspiked samples and ccfDNA was isolated with the QIAamp® Circulating Nucleic Acid Kit (QIAGEN). CcfDNA quantity was measured by Qubit® dsDNA HS Assay (Thermo Fisher Scientific) and fragment size distribution was determined by TapeStation® Cell-free DNA ScreenTape® (Agilent Technologies). EGFR T790M mutation detection was performed using the dPCR Mutation Assay EGFR 6240 Human (QIAGEN) with a 26K 24-well Nanoplate (QIAGEN) on the QIAcuity Digital PCR System (QIAGEN). Data analysis was conducted using the QIAcuity Software Suite (QIsAGEN). Results: CcfDNA isolated from PAXgene Blood ccfDNA Tubes had a fragment size distribution profile with distinct mononucleosomal peaks at ~180 bps without high-molecular weight DNA, indicating successful nucleated cell stabilization over the simulated transport duration and extraction of high-quality ccfDNA. Digital PCR analysis of extracted ccfDNA revealed positive detection of EGFR T790M mutations in spiked blood samples at a relative mutation fraction of approximately 2% in the investigated samples. Conclusion: PAXgene Blood ccfDNA Tubes enabled high-quality ccfDNA isolation by stabilizing nucleated cells during simulated transport condition. Spiked EGFR T790M mutations were successfully detected by dPCR following sample storage. This proof of principle study shows the combined usability of both systems for mutation detection in human blood specimens, highlighting the potential of both technologies for a broader application in the liquid biopsy field to detect oncogenic driver as well as tumor suppressor gene mutations in a multitude of tumor types. *The PAXgene Blood ccfDNA Tube is for Research Use Only in the US. Not for use in diagnostic procedures. Citation Format: Christian Neander, Andrea Huxhold, Maike Schoenborn, Thorsten Voss. Digital PCR-based EGFR T790M mutation detection from human blood samples collected in PAXgene Blood ccfDNA Tubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 780.

Abstract 6009: An end-to-end workflow for improved methylation detection

Abstract Introduction: DNA methylation at CpG nucleotide sites in eukaryotes is a key epigenetic mark that can help regulate gene expression. Specific changes in CpG methylation occur in many human cancers, making them a promising biomarker for early cancer detection. However, existing assays can be costly, lack specificity to regions of interest and often provide only semi-quantitative estimates of the methylation fraction. Here, we present a targeted methylome panel to decrease costs associated with next generation sequencing (NGS), methylation controls to calibrate quantitative assays and UMIs for accurate deduplication in low-diversity samples. Experimental Procedures: Genomic DNA (gDNA) was prepared for sequencing using the Twist Methylation Detection System consisting of enzymatic methylation conversion and hybrid capture using the Twist Human Methylome Panel. Twist’s synthetic CpG methylation level controls were spiked-in to gDNA and taken through the Twist Methylation Detection System to demonstrate their utility in calibrating methylation assays. Additionally, libraries were generated using cell free DNA (cfDNA) and either conventional or UMI-containing adapters to investigate the impact on quantitative detection and total unique coverage. Results: Using the Twist Human Methylome Panel at 150x raw coverage achieves uniform coverage with low off-bait Picard metrics. 6.59 million CpG sites were detected using a minimum depth of 10X. Target capture with the Human Methylome Panel allows for informative CpG calling of up to 82 samples on a single Illumina Novaseq S4 flowcell, compared to 3 samples per flowcell with a tradition whole-genome bisulfite sequencing (WGBS) assay. The Twist CpG methylation specific controls are constructed of 48 unique contrived sequences that contain a total of 8 different levels of methylation, ranging from 100% to 0%. Including these controls allows for quantitation of methylation levels in the experimental samples and qualification of the enzymatic conversion process. Conclusions: Our study leverages the Twist methylation detection portfolio to interrogate genome-wide methylation patterns for various applications. In combination, these tools can be used to design cost effective end-to-end assays. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Lydia Bonar, Kristin Butcher, Michael Bocek, Holly Corbitt, Bryan Hoglund, Cibelle Nassif, Patrick Cherry, Derek Murphy, Jean Challacombe, Esteban Toro. An end-to-end workflow for improved methylation detection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6009.

Abstract 615: Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways

Abstract Spatial distribution of T cells is key in understanding the escape of tumors from immune surveillance via the adaptive immune response, including interactions between immune cells and the surrounding tumor microenvironment. T cells are critical to the adaptive immune response to pathogens and cancers, mediating an antigen-specific response through both specificity and diversity of T cell receptor (TCR) clonotypes. Many methods exist to determine specific clonotypes and overall TCR diversity present from bulk tissues or sorted cell populations; however, nearly all fail to capture spatial orientation and arrangement of T cells engaging with their microenvironment, and most require large amounts of starting material from precious samples. Here, we present a TCR expression profiling panel for the GeoMx® Digital Spatial Profiler that can be combined with the GeoMx Cancer Transcriptome Atlas (CTA) or Human Whole Transcriptome Atlas (WTA) on archival formalin-fixed paraffin embedded (FFPE) tissue specimens. This represents the first commercial spatial expression profiling assay for the simultaneous quantification of TCR constant, variable, and joining segments in situ. We show reliable sensitivity and specificity (>90%) with respect to orthogonal sequencing and robust detection of TCR chains with evidence of clonal expansion and CD8 infiltration across tumor regions in colorectal cancer tissue. These events also corresponded to increased signatures of exhaustion from the T cells and suggest that the T cells resident in or near the tumor are tumor-specific and poised for activation via checkpoint blockade. Signaling pathways and tumor-specific signatures were also evaluated to look for mechanisms through which tumor cells respond to T cell infiltration. We further validated the performance of the TCR probe pool in cell pellet arrays with orthogonal TCR sequencing, tonsil and colorectal cancer tissues. Together, the combination of our TCR add-on panel with the CTA or WTA illuminates T cell phenotypes, signaling pathways, population dynamics, and transcriptomic changes, yielding an unparalleled view of the T cell response in any context. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Katrina van Raay, Michelle Kriner, Jason Reeves, Erin Piazza, Hargita Kaplan, John Vivian, Francis Fernandez, Margaret Hoang, Joseph Beechem. Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 615.

Abstract 4461: Optimized human immunophenotyping panels enhance the flexibility for high-dimensional flow cytometry analysis with CyTOF

Abstract The immune system plays a pivotal role in the pathogenesis of cancer. Beyond its importance in leukemia and lymphoma, scientists now understand that the immune system is involved in virtually all malignancies. Heterogeneity of cell phenotypes within a tumor microenvironment, including blood cell phenotypes, is a hallmark of cancer that is poorly understood. Resolution of these phenotypes is essential to direct therapeutic development. CyTOF® technology has pioneered the field of high-dimensional flow cytometry through the use of isotopically pure metal-tagged antibodies and a highly sensitive mass cytometer to enable 50-plus-parameter analysis. Easy panel design without the need for compensation controls or issues of autofluorescence allows for comprehensive single-cell analysis in complex biological samples and is particularly well suited for revealing the intricacies of oncogenesis. Maxpar® OnDemand reagents were recently introduced for CyTOF to increase flexibility and facilitate larger panel design in a short period of time. To this end, lineage markers that have strong, reliable expression were reassigned to optimal metal isotopes to reserve the high-sensitivity lanthanide channels for more difficult-to-detect markers with low endogenous expression, common in immuno-oncology studies. In this study, we aimed to test the newly released Maxpar OnDemand™ Antibodies to create an optimized immune phenotyping panel and maximize further customization for CyTOF. Individual antibody performance was compared between the new and existing antibodies with different metal tags. Human peripheral blood mononuclear cells (PBMC) and fresh whole blood (WB) were stained to confirm equivalent capabilities for population gating. Importantly, we demonstrated the power of these antibodies to generate reproducible, impactful data by creating functional human immunophenotyping panels. A 20-marker human phenotyping panel was tested in PBMC and WB. We identified major immune cell populations including T cells, B cells, granulocytes, natural killer (NK) cells, and monocytes. Furthermore, the addition of CD45RO to the panel enhanced the delineation of naive, memory and effector T cell subsets. Additional myeloid markers were included to resolve the complexity within this compartment. With the updated placement of key lineage markers, the optimized phenotyping panel is ideal for future immuno-oncology studies as it can be easily expanded to interrogate cell cycle proteins, cytokines, cell signaling proteins, and oncogenic transcription factors. This work demonstrates the capability of CyTOF for robust, high-parameter immunophenotyping and a high degree of flexibility to expand our understanding of the complex processes underlying carcinogenesis and response to therapy. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Lauren J. Tracey, Michael Cohen, Christina Loh. Optimized human immunophenotyping panels enhance the flexibility for high-dimensional flow cytometry analysis with CyTOF. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4461.

Abstract 5640: Identifying pathophysiological features of mouse tumors using imaging mass cytometry

Abstract Mouse tumor models are widely utilized for cancer studies and preclinical drug development. An obstacle in predicting therapeutic drug efficacy is the ability to quantitatively evaluate the multi-parametric post-treatment response in the tumor microenvironment (TME). Identification of immuno-oncological processes that dictate tumor growth, metastasis, and immune response is essential for selecting promising drug candidates for further clinical evaluation. Imaging Mass Cytometry™ (IMC™) is a vital and proven high-plex imaging technology that enables deep characterization of the complexity and diversity of tumor tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC technology to simultaneously assess 40-plus individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of the TME. We have previously demonstrated the application of IMC in combination with Maxpar® and Maxpar OnDemand™ antibodies to highlight cellular composition of normal mouse tissues. Here, we showcase the Maxpar OnDemand Mouse Immuno-Oncology IMC Panel Kit for application on a variety of mouse tumor tissues. We compiled the antibody panel to quantitatively assess IO-related processes and applied it to a tissue microarray (TMA) containing a large variety of mouse tumors. Antibodies in panel kits were selected from the Maxpar and Maxpar OnDemand catalogs. We digitized high-plex data from mouse tissues using the Hyperion Imaging System and generated images demonstrating the detailed layout of the TME. We further conducted single-cell analysis to identify specific populations of tumor and immune cells in the TME. The Mouse Immuno-Oncology IMC Panel Kit successfully identified pathophysiological processes such as immune cell infiltration and activation, signaling pathway activation, biomarkers of epithelial-to-mesenchymal transition (EMT), metabolic activity, growth, and the tissue architecture of the TME. Single-cell analysis of several highly relevant tumor types separated distinct cellular clusters representing tumor, immune, stromal, and vascular cells. Activation of cellular processes associated with signaling, growth, and metastasis were identified in tumor cells. In addition, cytotoxic and inflammatory activation in lymphoid and myeloid immune cell subtypes were detected. Application of IMC based multiparametric analysis successfully identified the spatial landscape of the TME at single-cell resolution. Quantitative analysis of tumor composition revealed critical insights regarding prognostic parameters such as metastatic and growth potential of tumors, and identification and activation of immune cell infiltrates. Overall, we demonstrate the power of IMC and provide evidence of its successful application in mouse tumor models. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Liang Lim, Thomas D. Pfister, Nick Zabinyakov, Christina Loh. Identifying pathophysiological features of mouse tumors using imaging mass cytometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5640.

Abstract 247: Twist pan-cancer synthetic RNA fusion control for assay development

Abstract Fusion genes are structural variants that arise when the coding region of two previously independent genes become joined together; fusion genes can be a major driving cause of cancer development. Many gene fusion events have been classified as clinically-relevant and are targets for both diagnostic applications, such as TMPRSS2-ERG in prostate cancer, and therapeutic applications, such as CD74-ROS1 and EML4-ALK in non-small cell lung cancer. Given the potential clinical benefits of early detection, there is significant interest in developing highly sensitive and accurate diagnostic assays to detect cancer using these gene fusions as biomarkers. A significant barrier to the development of these diagnostic assays is the lack of a reliable and renewable sample source harboring gene fusions of interest for use in assay development and analytical validation. Here, we describe the development and performance of a new fusion reference material: the Twist Pan-cancer RNA Fusion Control, a highly-multiplexed positive control designed to be spiked into a selected RNA background or as a stand-alone positive control. The Twist Pan-cancer RNA Fusion Control provides a wide selection of 80+ common and curated cancer targets for analytical validation and assay development. The synthetic RNAs are designed centered around the known and documented fusion sites with 750 nt of RNA 5′- of and 3′- of the fusion junction, suitable for short read sequencing and qPCR experiments. Pooled synthetic fusion RNAs are quantified at known molar ratios, NGS QC’d for uniformity, and ddPCR’d to establish a highly precise pool concentration. Finally, in fusion detection NGS experiments, we observe over a 95% recall rate of fusion events. The Pan-cancer RNA Fusion Control is designed to serve as a spike-in or stand-alone positive control and specifically pairs well with a clinically-relevant target enrichment (TE) panel within the Twist Targeted Enrichment for Gene Expression solution. This control sample positive for RNA fusions can be applied in both qPCR and NGS workflows to validate panel/probe sets, establish limits of detection, and monitor ongoing assay performance. The Twist Pan-cancer RNA Fusion Control provides a valuable one-stop solution to assay developers seeking reference materials for a wide array of cancer-associated fusion transcripts. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Patrick Daniel Cherry, Jason Corwin, Kit Fuhrman, Jean Challacombe, Derek Murphy, Esteban Toro. Twist pan-cancer synthetic RNA fusion control for assay development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 247.

Abstract 232: Fully automated comprehensive genomic profiling for detection of cancer variants, gene fusions, and complex oncology endpoints

Abstract Introduction: We present the comprehensive genomic profiling performance of the Ion Torrent Genexus system using the Oncomine Comprehensive Assay Plus (OCA Plus), a 500+ gene targeted AmpliSeq-based oncology research panel that evaluates DNA variants (including copy number alterations), RNA fusions, and key oncology research endpoints including tumor mutational burden (TMB), microsatellite instability (MSI), and homologous recombination repair deficiency (HRD) via characterization of genomic instability by the newly introduced Genomic Instability Metric (GIM). Methods: The Ion Torrent Genexus System provides comprehensive genomic profiling via automated sample-to-report workflow with next day results. The Genexus System supports oncology research panels such as OCA Plus, which is comprised of over 13,000 amplicons, and enables low input requirements of just 20ng of FFPE DNA and RNA. This study utilized cell lines, reference controls, and orthogonally tested FFPE research samples to evaluate detection of DNA variants, copy number alterations, RNA fusions, and key research endpoints, including MSI, TMB, and HRD. The OCA Plus panel was also evaluated for the ability to detect arm-level copy number changes in orthogonally validated FFPE samples. Results: Commercial reference controls and FFPE research samples were sequenced using OCA Plus on the Genexus System to an average depth of ≥24 million reads per sample, with four DNA and RNA samples supported per run. SNV and MNV calling performance was assessed using the AcroMetrix Oncology Hotspot Control which has 377 variants covered by OCA Plus and delivered SNV sensitivity and PPV >99% and MNV sensitivity of >99% and PPV >95%. MSI status was assessed using orthogonally tested FFPE samples from various tumor tissues (stomach, endometrial, colorectal) and returned status concordance of 99.4% with sensitivity and PPV >99%. The TMB endpoint was tested using commercial controls and FFPE samples with a correlation of r2 > 0.90 to orthogonal measurements. RNA Fusion reference controls showed 100% positive correlation. Copy number gain detection shows sensitivity of 99% and PPV >95%, while homologous copy loss gives 100% PPV and sensitivity of >90%. We also demonstrate high concordance to orthogonal methods in detection of HER2 amplifications, and the ability to detect arm-level copy number alterations such as 1p/19q co-deletions in IDH1 positive glioma samples. Conclusion: The Genexus System combines minimal touch points and a rapid turnaround time to enable comprehensive genomic profiling for research assays such as OCA Plus for detection of rare variants and low-level fusion transcripts. Further, by providing accurate characterization of key oncology research endpoints, the Genexus System can accelerate research in oncology. For research use only. Not for use in diagnostic procedures. Citation Format: Geoffrey Marc Lowman, Dinesh Cyanam, Emily Norris, Michelle Toro, Coleen Nemes, Tanaya Puranik, Yan Zhu, Alex Phan, Derek Wong, Portia Bernado, Anelia Kralcheva, Srinivas Nallandhighal, Loni Pickle, April Bigley, Mohit Gupta, Ying Jin, Sameh El-Difrawy, Amir Marcovitz, Fatima Zare, Charles Scafe, Yu-Ting Tseng, Jianjun Guo, Vinay Mittal, Scott Myrand, Santhoshi Bandla, Paul Williams, Eugene Ingerman, Elaine Wong-Ho, Seth Sadis, Mark Andersen, Rob Bennett. Fully automated comprehensive genomic profiling for detection of cancer variants, gene fusions, and complex oncology endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 232.

Abstract 3656: A multi-scale approach to quantitatively evaluate the SMAD signaling pathway

Abstract Angiogenesis and cell proliferation are fundamental biological processes whose pathologic states represent two hallmarks of cancer. Central to controlling both processes is the TGF-β/BMP signaling pathway, for which the SMAD proteins serve as major downstream effector molecules. Upon ligand-binding, TGF-β or BMP type I/II receptor heterotetramers phosphorylate receptor-regulated SMADs (R-SMADs). TGF-β-receptor binding leads to SMAD2/3 phosphorylation, whereas BMP-receptor binding induces phosphorylation of SMADs 1/5/8. Phosphorylated SMADs (pSMADs) then form a complex with the co-SMAD SMAD4 at which point they undergo nuclear translocation, where they function as transcriptional modulators of target genes. The magnitude of SMAD phosphorylation and functional output (e.g., stimulated angiogenesis or attenuated cell proliferation) depends on both the ligand concentration, as well as the duration of exposure. In this study, we quantitatively evaluate the phosphorylation states of R-SMADs, as well as their downstream biological effects at three different biological scales: 1) biochemical, 2) cellular, and 3) tissue/3D models. At the biochemical level, the temporal and dose-dependent phosphorylation of R-SMADs in response to growth factor stimulation can be quantified using a quantitative ELISA approach. Growth factor stimulation can be quantified at the cellular level by high content imaging of SMAD phosphorylation and nuclear translocation, while simultaneously monitoring cell proliferation rates. Lastly, high-resolution confocal imaging can be employed to observe developmental consequences of phospho-SMAD signaling using a HUVEC-based 3D angiogenesis model system. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Ernest Heimsath, Lisa Arvidson, Reginaldo Prioli, Antony Wood. A multi-scale approach to quantitatively evaluate the SMAD signaling pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3656.

Abstract 4617: A complete pipeline for high-plex spatial proteomic profiling and analysis on the cosmxtm spatial molecular imager and atomtm spatial informatics platform

Abstract Detecting and analyzing large numbers of proteins using whole-slide imaging is critical for a comprehensive picture of immune response to cancer. Many existing approaches for high-plex proteomics face issues around simplicity, speed, scalability, and big data analysis. Here, we present an integrated workflow from sample preparation through downstream analysis that addresses many key concerns around high plex proteomics. The CosMx Spatial Molecular Imager (SMI) and AtoMx Spatial Informatics Platform (SIP) comprise of a turnkey, end-to-end workflow that efficiently handles highly multiplex protein analysis at plex sizes exceeding 110 targets. We demonstrate an extension of our commercially available 64-plex human immuno-oncology panel to higher numbers of targets and show how the cloud computing-enabled AtoMx SIP allows flexible construction of analytic pipelines for cell typing and spatial analyses. The CosMx protein assay uses antibodies conjugated with oligonucleotides, which are detected using universal, multi-analyte CosMx readout reagents. The CosMx Human Immuno-oncology panel was optimized to comprehensively profile lymphoid and stromal lineages within the tumor microenvironment as well as markers of cancer signaling and progression. Each CosMx SMI antibody was validated on multi-organ FFPE tissue microarrays covering prevalent solid tumor types with matched controls, and 52 human FFPE cell lines, including overexpression lines for key targets such as GITR, CD278, PD-L1, and PD-1. CosMx SMI uses a deep learning algorithm to segment whole cells and a semi-supervised algorithm to classify cell types. The AtoMx SIP provides full analysis support, including a whole-slide image viewer, and methods for performing built-in or fully customizable analyses for cell typing, ligand-receptor analysis, neighborhood analysis and spatial differential expression. Within the cancer sample profiled, we performed in-depth single-cell proteomic profiling across different cell populations. We detected TLS, characterized TLS maturation, and identified immune interactions with the tumor microenvironment. The CosMx SMI assay profiled the composition and spatial organization of infiltrating immune cells within and around the tumor microenvironment. We found that markers of T cell activation and exhaustion varied across the tumor landscape. CosMx SMI is a high-plex spatial multi-omics platform that enables detection of more than 110 proteins at subcellular resolution in real-world FFPE tissues. The extensibility of the CosMx protein assay to large numbers of protein targets and our flexible, scalable bioinformatic platform provides a straightforward and robust solution for comprehensive immune phenotyping with full spatial context. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Tien Phan-Everson, Zachary Lewis, Giang Ong, Yan Liang, Emily Brown, Liuliu Pan, Aster Wardhani, Mithra Korukonda, Carl Brown, Dwayne Dunaway, Edward Zhao, Dan McGuire, Sangsoon Woo, Alyssa Rosenbloom, Brian Filanoski, Rhonda Meredith, Kan Chantranuvatana, Brian Birditt, Hye Son Yi, Erin Piazza, Jason Reeves, John Lyssand, Vik Devgan, Michael Rhodes, Gary Geiss, Joseph Beechem. A complete pipeline for high-plex spatial proteomic profiling and analysis on the cosmxtm spatial molecular imager and atomtm spatial informatics platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4617.

Abstract 5631: Expanding tools for multiplex mRNA imaging in spatialomics through the ViewRNA tissue assay kits

Abstract Spatialomics is a rapidly growing field as it allows researchers to gain a deeper understanding of transcriptomes and corresponding protein expression profiles in cells within complex tissue microenvironments. One critical technology for spatialomics is in-situ hybridization (ISH) technology, which enables direct visualization and quantitation of nucleic acid in cells with single molecule resolution. The Invitrogen ViewRNA ISH assays incorporate branched DNA (bDNA) technology provides tools for interrogating multiple RNA transcripts at the same time, paving the way for improved spatialomics research. This technology is a powerful tool for spatialomics, giving insight into important mechanisms within cells and tissue. With researchers increasingly using spatialomic data in fields like neuroscience, immuno-oncology & single-cell analysis, these scientists will need the rapid and efficient detection of mRNA co-expression profiles that ViewRNA portfolio provides. In our newest offering, ViewRNA fluorescence tissue kits we extended our branched DNA technology, proprietary probe set design and signal amplification technology with Alexa Fluor probes offering a unique, robust, and sensitive in situ hybridization assay for RNA localization in fixed tissues. With fluorescence detection using Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, and Alexa Fluor 750, these kits allow multiplexed detection and imaging paving the way for improved spatialomics. This technology lays the foundation for expanded fluorescent profiles to provide scientists additional tools to probe multiple transcripts per sample, allow broader spatialomics research. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Nancie Mooney. Expanding tools for multiplex mRNA imaging in spatialomics through the ViewRNA tissue assay kits. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5631.

Abstract 2250: Immuno-oncology study to profile the tumor microenvironment in multiple human cancers using high-plex imaging mass cytometry

Abstract Immune profiling of tumor tissues has become a key tool in understanding the complexity of the tumor microenvironment (TME), for predictive biomarker discovery, and in cancer treatment. The presence of tumor-infiltrating lymphocytes has been associated with benefit from therapy. Furthermore, the TME also contains immunosuppressive elements that can impede immune response. Imaging Mass Cytometry™ (IMC™) enables detailed assessment of cell phenotype and function using 40-plus markers simultaneously at subcellular resolution on a single slide without spectral overlap or background autofluorescence. High-plex IMC has enabled us to evaluate the TME in different cancer histologies including in highly autofluorescent tissue types like lung, hepatocellular carcinoma, and skin melanoma. The Maxpar® Human Immuno-Oncology IMC Panel Kit (201508) was customized using antibodies from the Standard BioTools™ catalog to create panels for tissue-based immuno-oncology research. Data acquisition was performed using a Hyperion™ Imaging System. To facilitate cell segmentation, an IMC Cell Segmentation Kit (TIS-00001) was applied to enhance cell membrane boundaries. We applied a pixel classification approach and CellProfiler™ for single-cell segmentation. We used histoCAT™ for single-cell analysis to visualize protein expression in various cancer types via PhenoGraph clustering and t-SNE maps. Our panels were applied to normal and cancer human tissue microarrays (TMAs) to phenotype and analyze cell populations in these tissues. We provide detailed analysis of the TME by classifying activation state of immune cell populations, epithelial-to-mesenchymal transition (EMT) progression, and composition of the extracellular matrix. In-depth single-cell analysis quantitatively evaluated the cellular makeup and immune cell component in the TME of cancer tissues and identified major tumor, immune, and stromal cell phenotypes. This work demonstrates the capability of IMC for quantitative and spatial identification of multiple immune parameters in the TME on a single slide of cancer patient samples (e.g. tumor microarray). For Research Use Only. Not for use in diagnostic procedures. Citation Format: Thomas D. Pfister, Liang Lim, Shaida Ouladan, Nick Zabinyakov, Qanber Raza, Christina Loh. Immuno-oncology study to profile the tumor microenvironment in multiple human cancers using high-plex imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2250.

Abstract 764: Automated walk away NGS sample preparation for constructing in-line molecular barcoded RNA libraries from Fresh Frozen and FFPE samples

Abstract RNA-Seq is a revolutionary technology for transcriptome analysis, which has been used to quantify transcript levels, confirm gene annotation, identify novel transcripts, splice variants, and fusion detection. Traditionally, the usefulness of RNA-Seq has been limited by pre-analytical (sample availability, low integrity, tumor heterogeneity, complex protocols) and analytical factors (small mRNA fraction, high read duplicate rate), which can lead to higher costs, longer turnaround times, and false negative results. We have developed and automated a streamlined workflow for construction of highly complex RNA Libraries from fresh frozen and FFPE samples that addresses these concerns. We use target enrichment by bait selection with FFPE-derived samples, where rRNA depletion is found to be inconsistent and poly(A) enrichment cannot be used due to degradation of the source material. A fully automated protocol using pre-aliquoted reagents, has been optimized on the Magnis NGS Prep System for a wide range of RNA inputs (10-200 ng), various sample types (Intact and FFPE total RNA) and equipped with 192 Unique Dual sample Indices (UDI) to minimize the effects of possible index hopping and contamination. Addition of in-line molecular barcodes incorporated at the ligation step helps differentiate fragmentation from PCR duplicates, which increases the total number of reads available for further analysis (gene expression and fusion detection). With minimal risk of contamination, this automated protocol delivers up to eight target-enriched NGS Illumina sequencing-ready cDNA libraries from total RNA input per run in about 12 hours without the need of any user intervention. Both catalog and custom probes can be utilized, and PCR cycles can easily be adjusted from the touchscreen to optimize the library yields. For Research Use Only. Not for use in Diagnostic procedures. Citation Format: Saharnaz Bigdeli, Bahram Arezi, Jan Godoski, Denise Rhodes, Ryan Aguilar, Karen William, Bernd Buehler, Ji Zhu, Ronda Allen. Automated walk away NGS sample preparation for constructing in-line molecular barcoded RNA libraries from Fresh Frozen and FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 764.

Abstract 6671: Simplifying high-parameter phenotypic and functional characterization of cancer immune cells

Abstract Interrogating immune cell composition and function in patients with cancer is critical for making disease prognoses, monitoring clinical efficacy of tumor immunotherapies, identifying novel therapeutic targets, and discovering predictive biomarkers of disease. Both the adaptive and innate arms of the immune system play important roles in generating pro- or anti-tumor milieus. In particular, natural killer (NK) cells are gaining increasing attention as potential cell-based therapies in immuno-oncology, including in adoptive transfer of chimeric antigen receptor (CAR) and iPSC-derived NK cells or off-the-shelf NK cell lines to target multiple myeloma. Since NK cells can also indirectly impact CAR T cell or antibody-based immunotherapies, characterizing these cells using optimized and reproducible assays is critical. CyTOF® is a high-plex flow cytometry technology that exploits metal-isotope-tagged antibodies to probe cellular phenotypes and functions. In contrast to fluorescence-based conventional and spectral flow cytometry, CyTOF experimental workflows are streamlined as autofluorescence is not an issue and signal spillover is minimal, allowing rapid design and application of 40-plus-marker panels. To expand on the increasing clinical and preclinical utility of the 30-marker Maxpar® Direct™ Immune Profiling Assay™ (Maxpar Direct Assay), we developed nine add-on Expansion Panels for deeper phenotyping of specific cell types and activation states, including panels designed to characterize ex vivo and activated myeloid cells, T cells, and NK cells. Peripheral blood mononuclear cells from healthy donors and donors with multiple myeloma were stimulated in vitro, then stained in the 30-antibody Maxpar Direct Assay tube with the NK Cell Expansion Panel (CD181, NKp30, NKp46, PD-1, NKG2A, ICOS, and TIGIT) or the T Cell Panel 3 (OX40, TIGIT, CD69, PD-1, Tim-3, ICOS, and 4-1BB) as drop-in antibodies. Surface staining was followed by intracellular staining with the Basic Activation Expansion Panel antibodies (IL-2, TNFα, IFNγ, perforin, granzyme B) after cell fixation and permeabilization. Anti-CD107a was added during stimulation to measure degranulation. Samples were acquired on a CyTOF XT™ instrument in automated batch acquisition mode. Automated analysis was performed with Maxpar Pathsetter™ software to enumerate immune cell types and quantify marker expression. In addition to the 37 populations identified by Pathsetter with the base Maxpar Direct Assay, the new Expansion Panels allowed deeper profiling of NK and T cells, while the Basic Activation Panel revealed their cytokine responsiveness and cytotoxic potential. Thus, the optimized single-tube Maxpar Direct Assay is a powerful tool that can be further expanded and customized with predefined panels or antibodies to comprehensively study immune cells in health and disease. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Deeqa Mahamed, Michael Cohen, Stephen Li, Lauren Tracey, Huihui Yao, Christina Loh, Leslie Fung. Simplifying high-parameter phenotypic and functional characterization of cancer immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6671.

Dojrzałość szkolna/ gotowość szkolna – fenomen wciąż kontrowersyjny

Following the indication formulated by Barbara Wilgocka-Okoń, who passed from measuring a child’s school maturity to postulating school maturity, the article is about the discussion of the concept “school maturity of a child” and the effects of its use in diagnostic procedures. As a construct that is non-obvious as far as a child’s development is considered but used as an important criterion for assessing it from the perspective of an institution such as school, it is a specific psychological-administrative phenomenon. Its use has many proponents and opponents, and each of them evokes different arguments derived from paradigmatically different theoretical and methodological rationalities. In this text, I will indicate some limitations and controversies related to the concept of “school maturity of a child” and the tests derived from it, trying to establish them theoretically, paradigmatically and pointing some potential dangers associated with their use.

Detection and Diagnosis of Cardiac Amyloidosis in Egypt.

Cardiac amyloidosis is a life-threatening disease that occurs when amyloid proteins, most commonly immunoglobulin light chain or transthyretin, mutate or become unstable, misfold, deposit as amyloid fibrils, and accumulate in the myocardium. Early diagnosis of cardiac amyloidosis is hindered by insufficient awareness, specifically regarding clinical red flags and diagnostic pathways. Cardiac amyloidosis diagnosis comprises two important phases, clinical suspicion (phase one) followed by definitive diagnosis (phase two). Each phase is associated with specific clinical techniques. For example, clinical features, electrocardiography, echocardiography, and cardiac magnetic resonance imaging serve to raise suspicion of cardiac amyloidosis and facilitate early diagnosis, whereas laboratory tests (i.e., blood or urine electrophoresis with immunofixation), biopsy, scintigraphy-based nuclear imaging, and genetic testing provide a definitive diagnosis of cardiac amyloidosis. In Egypt, both the lack of cardiac amyloidosis awareness amongst healthcare providers and the unavailability of clinical expertise for the use of diagnostic techniques must be overcome to improve the prognosis of cardiac amyloidosis in the region. Previously published diagnostic algorithms for cardiac amyloidosis have amalgamated techniques that can raise clinical suspicions of cardiac amyloidosis with those that definitively diagnose cardiac amyloidosis. Though such algorithms have been successful in developed countries, diagnostic tools like echocardiography, scintigraphy, and cardiac magnetic resonance imaging are not ubiquitously available across Egyptian facilities. This review presents the current state of knowledge regarding cardiac amyloidosis in Egypt and outlines a new diagnostic algorithm which leverages regional nuclear imaging expertise. Importantly, the proposed diagnostic algorithm guides accurate amyloid-typing to mitigate misdiagnosis and erroneous treatment selection and improve the cardiac amyloidosis diagnostic accuracy in Egypt.