V2 Assay Research Articles

B-254 Analytical and Clinical Performance Evaluation of Plasma pTau217 in a Clinical Diagnostic Laboratory

Abstract Background Blood-based biomarkers provide an accessible and cost-effective tool for diagnosing Alzheimer’s disease (AD). Among these biomarkers, phosphorylated tau 217 (pTau217) shows promise for both AD diagnosis and differential diagnosis, as well as for facilitating access to emerging disease-modifying therapies. The ALZpath SIMOA pTau-217 v2 assay is a digital immunoassay designed for quantifying pTau-217 in human EDTA plasma samples. This study assesses the analytical performance of plasma pTau217 assays as a Laboratory Developed Test (LDT) appropriate for diagnostic application. Methods In this analytical study, we evaluate accuracy, precision, detection limits, linearity, and analyte stability. Plasma pTau217 levels were measured using the ALZpath pTau217 v2 assay on a single Quanterix HD-X SIMOA Analyzer platform at BC Neuroimmunology Lab. Inc. Vancouver, British Columbia, Canada. As a clinical testing laboratory, our validation procedures are in accordance with CAP and CLIA requirements, with a primary focus on adhering to CLSI guidelines for both analytical and clinical performance testing. To determine a reference range and cut-off, samples of Amyloid PET-confirmed cases (n=263) and healthy subjects between the ages of 55 and 95, with a roughly equal ratio of males to females (n=219), were tested. Results The ALZpath pTau217 assay demonstrates an analytical measurement range of 0.0046 - 3.4056 ng/L, with a limit of blank (LoB) of 0.0019 ng/L, a limit of detection (LoD) of 0.0046 ng/L, and a limit of quantification (LoQ) of 0.03 ng/L. Linearity spans from 0.06 to 1.75 ng/L, with repeatability showing intra-laboratory precision above LoQ ≤ 20% CV. Stability is maintained at 4⁰C for ≤ 1 week, at room temperature for ≤ 72 hrs, at -20⁰C for ≤ 2 weeks, and at -70⁰C for ≤ 3 weeks, with freeze/thaw cycles tolerated up to 6 times. Moreover, interference is not detected at the maximum concentrations tested for albumin, unconjugated bilirubin, biotin, hemoglobin, creatinine, and heterophilic antibodies. The reference range was determined using the 95% reference interval, resulting in 0.398 ng/L. ROC analysis yielded an AUC of 0.932 with a Youden index of 0.338 ng/L. At the Youden index, sensitivity was 90.0% with a specificity of 85.6%. Specificity reached 95.5% at the upper cut point of 0.471 ng/L, while sensitivity was 94.8% at the lower cut point of 0.242 ng/L. Conclusions The outstanding analytical performance of pTau217 supports its adoption as a reliable screening and diagnostic tool in clinical practice, aiding clinicians in the accurate diagnosis of AD.

Abstract PO5-16-04: Tissue-informed personalized MRD detection assay may outperform tumor-informed fixed panel strategy in Triple Negative Breast Cancer (TNBC)

Abstract Background: Minimal residual disease (MRD) detection by ctDNA analysis enables identifying patients with high recurrence risk and refining risk stratification to guide treatment selection precisely, which may improve patient survival or avoid overtreatment. We previously developed a 1021-gene fixed panel MRD detection assay, MNavigator V1, demonstrating high prognostic evaluation value in various solid tumors, including lung cancer, colorectal cancer, and esophageal carcinoma, and relevant research results have been published. Recently, the assay upgraded to MNavigator V2, a customized patient-specific panel strategy to ultra-deep sequencing, with higher clinical performance and more economical. Here, we head-to-head compared the performance of MNavigator V1 with V2, using the same collection of blood samples from TNBC patients. Methods: The retrospective study recruited 48 patients diagnosed with stage II~III TNBC with adequate samples (NCT04501523/NCT04803539). Five patients in the cohort received neoadjuvant therapy (Neoad-group), and the standard treatment regimen of the remaining 43 patients was adjuvant therapy (ad-group) after surgery. The surgical tumor tissue of all these patients was collected. Peripheral blood samples were collected at various time points, baseline samples before neoadjuvant therapy, 7-day after neoadjuvant therapy, 1-month postoperative, 7-day after adjuvant therapy, and subsequent every 3~6 months follow-up. The personalized, tumor-informed MRD detection approach MNavigator V2 started by identifying somatic mutation of the resected tumor with a 1021-gene panel covering 1.6Mb of the human genome. Then, a customized panel consisting of up to 20 top-ranked patient-specific somatic variants was designed. After that, unique molecular identifiers (UMI) based 100,000X ultra-deep next-generation sequencing on plasma samples was conducted by adopting the mix panel of the patient-specific panel and a 5kb breast cancer universal core panel. MNavigator V1 assay, a fixed panel assay also applied the same off-the-shelf 1021-genes panel with a sequencing depth of 10,000X to detect MRD of blood samples. Results: Until May 2023, the median follow-up time of 660 days, and 8 patients were recurrence confirmed by CT imaging. The median tracked tissue-informed mutations was 7 (2~18). In this study, the screening time point was defined as the combined MRD status of the blood samples collected before and after neoadjuvant therapy, and 1-month post-surgery for the Neoad-group, as for the ad-group, the time point referred to 1-month post-surgery and post-adjuvant therapy. If any one of these time nodes' MRD was positive, the screening period MRD was positive. During the screening period, MNavigator V1 and V2 accurately predicted 6/8 (75.0%) recurrence. MRD-positive status was associated with a worse prognosis, compared to the MRD-negative population, with hazard ratios of 9.0 and 19.9 for V1 and V2 assay, respectively. The longitudinal MRD evaluation yield a higher sensitivity of 7/8 (87.5%) for recurrence prediction with MNavigator V2 at a median lead time of 104 days, whereas the V1 assay predicted 6/8 (75.0%) at 94 days, and the hazard ratio was 40.4 vs 8.5. In one brain-only metastasis patient, MRD was undetectable, which may be due to the limitation of the blood-brain barrier. For recurrence-free patients with 1 year of follow-up, 92.5% (37/40) were MRD-negative with MNavigator V2 assay. These three patients who detected MRD-positive after surgery, ctDNA cleared after adjuvant therapy, and persistently negative during follow-up, may benefit from treatment. Conclusion: Based on the patient-specific tumor mutation profile, the personalized MRD detection assay achieved better clinical performance than the fixed panel strategy. Citation Format: Shunying Li, Wei Gao, Ning Fu, Wuqiang Cao, Xiaoling Zeng, Xinhua Du, Qiang Liu. Tissue-informed personalized MRD detection assay may outperform tumor-informed fixed panel strategy in Triple Negative Breast Cancer (TNBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-04.

Role of the prefrontal cortical protease TACE/ADAM17 in neurobehavioral responses to chronic stress during adolescence.

Chronic adolescent stress profoundly affects prefrontal cortical networks regulating top-down behavior control. However, the neurobiological pathways contributing to stress-induced alterations in the brain and behavior remain largely unknown. Chronic stress influences brain growth factors and immune responses, which may, in turn, disrupt the maturation and function of prefrontal cortical networks. The tumor necrosis factor alpha-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17) is a sheddase with essential functions in brain maturation, behavior, and inflammatory responses. This study aimed to determine the impact of stress on the prefrontal cortex and whether TACE/ADAM17 plays a role in these responses. We used a Lewis rat model that incorporates critical elements of chronic psychosocial stress, such as uncontrollability, unpredictability, lack of social support, and re-experiencing of trauma. Chronic stress during adolescence reduced the acoustic startle reflex and social interactions while increasing extracellular free water content and TACE/ADAM17 mRNA levels in the medial prefrontal cortex. Chronic stress altered various ethological behavioral domains in the observation home cages (decreased ingestive behaviors and increased walking, grooming, and rearing behaviors). A group of rats was injected intracerebrally either with a novel Accell™ SMARTpool TACE/ADAM17 siRNA or a corresponding siRNA vehicle (control). The RNAscope Multiplex Fluorescent v2 Assay was used to visualize mRNA expression. Automated puncta quantification and analyses demonstrated that TACE/ADAM17 siRNA administration reduced TACE/ADAM17 mRNA levels in the medial prefrontal cortex (59% reduction relative to control). We found that the rats that received prefrontal cortical TACE/ADAM17 siRNA administration exhibited altered eating patterns (e.g., increased food intake and time in the feeding zone during the light cycle). This study supports that the prefrontal cortex is sensitive to adolescent chronic stress and suggests that TACE/ADAM17 may be involved in the brain responses to stress.

Transcriptomic and proteomic characterization of the locus coeruleus in a rat model of Alzheimer’s disease

AbstractBackgroundThe noradrenergic locus coeruleus (LC) is the first region to accumulate hyperphosphorylated tau pathology (p‐tau) in Alzheimer’s disease (AD). Using TgF344‐AD transgenic rats, which develop endogenous p‐tau in the LC prior to pathology in forebrain regions, we have recently demonstrated age‐dependent effects of p‐tau on LC firing rates. Specifically, LC neurons exhibit early hyperactivity followed by late hypoactivity. These changes in LC firing rates are coincident with the emergence of behavioral phenotypes that characterize different stages of AD (neuropsychiatric symptoms followed by cognitive deficits). Here, we sought to identify molecular mechanisms that could contribute to these changes using RNAscope and proteomics in TgF344‐AD rats.Methods32 male and female TgF344‐AD rats and wild‐type (WT) littermates aged 6 and 15 months were used for this study. Brains were flash‐frozen in isopentane on dry ice and stored at ‐80°C until being sectioned. At the level of the LC, the left hemisphere LC was collected with a 1 mm tissue punch. Punches of the cerebellum were taken as a control to verify LC enrichment in the brainstem punches. The right LC hemisphere was sectioned at 16 µm for processing with fluorescent in situ hybridization using the RNAscope Multiplex Fluorescent V2 Assay according to the manufacturer’s protocol. Probes included noradrenergic markers (tyrosine hydroxylase, norepinephrine transporter) and those that influence LC activity (α2‐adrenergic receptors, GRIA2, GRIN, ADCYAP1R1). Separately, tissue punches were submitted to the Emory Integrated Proteomics Core for label‐free quantification of proteins.ResultsRNAscope revealed no differences in transcript levels of noradrenergic markers at either age. Similarly, there were no differences in transcript levels of α2‐adrenergic receptors, GRIA, or ADCYAP1R1 at either age, or GRIN in 6‐month animals. We observed an increase of GRIN mRNA in 15‐month TgF344‐AD animals, suggesting an upregulation of excitatory neurotransmission.ConclusionsNoradrenergic neuronal identity remains in‐tact up to 15 months of age, a timepoint when there is no LC cell death in these rats. We have also uncovered a potential compensatory mechanism to maintain LC activity in late stages of disease. Proteomics and RNAscope analysis of other markers that may affect LC activity are ongoing.

Abstract 17051: Oxytocin Receptor in the Spinal Neuraxial Modulation of Ventricular Excitability

Introduction: Cardiac sympathoexcitation is a major contributor to the pathogenesis of ventricular tachyarrhymias (VTs) during ischemia reperfusion (I/R) and chronic myocardial infarction (MI). Afferent network connections in the intermediolateral cell column (IML) and dorsal horn (DH) control sympathetic output to the heart. Our team reported spinal cord stimulation (SCS), decreased cardiac sympathoexcitation and reduced VTs during ischemia. Oxytocin (OXT) is a known neuromodulator; however, whether OXT signaling can modulate the cardiospinal neural network is unknown. Hypothesis: SCS neuromodulation is reducing sympathoexcitation and VTs through OXT signaling pathways. Methods: Two models of cardiac ischemia were used in Yorkshire domestic swine. In acute I/R, a suture was placed around the left anterior descending (LAD) coronary artery, after sternotomy. Chronic infarction (MI) was created by accessing the femoral with fluoroscopy guidance. Polystyrene microspheres (1ml) were injected via a 3mm angioplasty balloon catheter to the LAD. Animals then recovered post-MI for 6-8wk. SCS treatment was delivered through a four-pole lead which was inserted in the epidural space (T1-T4)(50Hz, 0.4ms pulse duration). T2-T4 spinal cord was collected postmortem for molecular analysis. Fluorescence in situ hybridization (FISH) protocol for RNAscope Fluorescent v2 Assay (ACD) was followed. Cell counts represented as 3 puncta or more centered around DAPI. Results: We report that OXT receptor positive cells significantly decreased as a result of I/R and MI (p<0.001, two-way ANOVA n=4-5 animals, average of 2-3 slices per animal). Within the DH the majority of OXT positive cells were inhibitory GABAergic neurons. Within the IML, a majority of the cells regulated by I/R and MI were microglia, and both an inhibitory and excitatory population contributed to the onset of ischemia. SCS treatment after I/R and MI resulted in the recovery of this OXT receptor cell population. Conclusions: By evaluating the role of OXT in reducing cardiac sympathoexcitation at the level of the spinal cord, we provide new insights into optimizing current neuromodulation approaches and developing new pharmacologic therapies.

Molecular profiling of solid tumors by next-generation sequencing: an experience from a clinical laboratory.

Personalized targeted therapies have transformed management of several solid tumors. Timely and accurate detection of clinically relevant genetic variants in tumor is central to the implementation of molecular targeted therapies. To facilitate precise molecular testing in solid tumors, targeted next-generation sequencing (NGS) assays have emerged as a valuable tool. In this study, we provide an overview of the technical validation, diagnostic yields, and spectrum of variants observed in 3,164 solid tumor samples that were tested as part of the standard clinical diagnostic assessment in an academic healthcare institution over a period of 2 years. The Ion Ampliseq™ Cancer Hotspot Panel v2 assay (ThermoFisher) that targets ~2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes was validated, and a total of 3,164 tumor DNA samples were tested in 2 years. A total of 500 tumor samples were tested by the comprehensive panel containing all the 50 genes. Other samples, including 1,375 lung cancer, 692 colon cancer, 462 melanoma, and 135 brain cancer, were tested by tumor-specific targeted subpanels including a few clinically actionable genes. Of 3,164 patient samples, 2,016 (63.7%) tested positive for at least one clinically relevant variant. Of 500 samples tested by a comprehensive panel, 290 had a clinically relevant variant with TP53, KRAS, and PIK3CA being the most frequently mutated genes. The diagnostic yields in major tumor types were as follows: breast (58.4%), colorectal (77.6%), lung (60.4%), pancreatic (84.6%), endometrial (72.4%), ovary (57.1%), and thyroid (73.9%). Tumor-specific targeted subpanels also demonstrated high diagnostic yields: lung (69%), colon (61.2%), melanoma (69.7%), and brain (20.7%). Co-occurrence of mutations in more than one gene was frequently observed. The findings of our study demonstrate the feasibility of integrating an NGS-based gene panel screen as part of a standard diagnostic protocol for solid tumor assessment. High diagnostic rates enable significant clinical impact including improved diagnosis, prognosis, and clinical management in patients with solid tumors.

Towards clinical implementation: Establishing standardized operating procedures to limit the pre‐analytical sample handling effects on the novel Alzheimer’s disease blood‐based biomarkers

AbstractBackgroundClinical implementation of novel blood‐based biomarkers for Alzheimer’s disease (AD) will aid in accurate, less‐invasive diagnosis. However, variation in blood handling may affect the accuracy of biomarker’s measurement. Establishing standardized operating procedures (SOPs) to mitigate these effects is essential for implementing novel blood‐based biomarkers into clinical use. We aim to provide an evidence‐based SOP specific for AD blood‐based biomarkers by studying the effects of multiple pre‐analytical variations in blood samples from AD and healthy controls (HCs). Here, we show the first results obtained, addressing the effect of biomarker’s adsorption to lab plastics, during sample processing.MethodBlood samples are being withdrawn from 150 volunteers (AD and HCs) and treated according to one of ten pre‐analytical experimental protocols. Several aliquots per sample are being obtained, to be shared and analyzed among labs, focusing on labs with novel P‐tau plasma tests. Preliminary results are acquired for the effect of 1x, 2x and 4x tube transfers in fresh polypropylene tubes with fresh pipet tips at every transfer, compared to no tube transfer, as a reference condition. Sample analysis was performed on Simoa using the Simoa neurology 4‐plex E assay (Aβ1‐42, Aβ1‐40, GFAP, NfL), and the P‐tau181 V2 assay. A variation in the median value of one of the measured biomarkers of at least 10% from the reference condition was regarded as a significant change.ResultFormally, medians of all tube transfers for all biomarkers measured (Aβ1‐42, Aβ1‐40, GFAP, NfL and Ptau‐181), stayed within the predefined range, for both AD and HC. However, the levels of Aβ1‐42 and Aβ1‐40 showed an obvious tendency to decrease at the 4th tube transfer. For Aβ1‐40, levels in HCs were more affected compared to those in AD (median 92%; AD: median = 95%). For Aβ1‐42, both AD and HCs showed a similar decrease (median: 91% and 93%). This decrease is not so apparent with the Aβ1‐42/Aβ1‐40 ratio. ConclusionIn line with the results, 4 tube transfers or less should have no influence on the accuracy of the measured biomarkers. For Aβ peptide measurements, using the Aβ1‐42/Aβ1‐40 ratio appears to stabilize the effect of tube transfer.

Multiomic Spatial Phenotyping of the Tumor Immune Microenvironment at Single Cell Resolution

Abstract The Tumor microenvironment (TME) is critical in cancer development, progression, and control. Immunological components within tumors, known as the tumor immune microenvironment (TIME), have been implicated in cancer progression. Effective strategies for cancer immunotherapies will require a deep understanding of factors that shape the TME and TIME. Here, we describe a spatial multiomics assay utilizing RNAscope™ ISH technology paired with high-plex whole-slide spatial phenotyping with the PhenoCycler ®-Fusion platform. This two-step approach is compatible with human FFPE tissues and enables characterization of the TIME more accurately by detecting RNA and protein markers on serial sections. We performed ultrahigh-plex spatial phenotyping on FFPE tumor tissue sections, using an antibody panel designed for immune cell phenotyping, evaluation of immune contexture and proliferation across the TME. On a serial section, we ran the RNAscope HiPlex v2 assay using the PhenoCycler-Fusion system. This assay consisted of a 12-plex immuno-oncology panel of RNA target probes, which detect macrophages, chemokines, and cytokines. In this proof-of-concept study, we demonstrate multiomic spatial phenotyping on the PhenoCycler-Fusion platform. Analysis of the resulting multiplex imaging data revealed structural organization of cells within the TME and activation states of immune cells. Together, this information provides a more complete functional map of immune cells within the TME and TIME and thereby enriches our understanding of tumor biology that may be deterministic of immunotherapy responsiveness.

Abstract 4636: High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with Imaging Mass Cytometry

Abstract Purpose The next breakthroughs in immuno-oncology will be driven by high-plex tools that decipher the spatial arrangement of different cell types within the tumor microenvironment (TME). Imaging Mass Cytometry™ (IMC™) is a proven tool for the study of complex cellular interactions in the TME. It utilizes CyTOF® technology for simultaneous assessment of 40-plus protein markers at subcellular resolution without spectral overlap or background autofluorescence, thus providing unprecedented insight into the organization and function of the TME. Despite this, some protein targets are challenging to include in IMC as they have very few or no commercial antibodies available. Moreover, although cellular identity can easily be deciphered through detection of protein targets, knowledge of the cell’s transcriptome improves understanding of cellular function and activation state. Here, we present a robust and reliable workflow that combines the highly sensitive and specific RNAscope™ technology for RNA detection with the multiplexing capability of IMC to visualize key RNA and protein markers in the same tumor samples. Methods The RNAscope HiPlex v2 assay was combined with protein detection on the Hyperion+™ Imaging System to evaluate expression of both RNA and protein targets in formalin-fixed, paraffin-embedded (FFPE) tumor tissue microarray (TMA). The RNAscope assay was used with 12 target RNA marker probes and associated metal-labeled detection probes, suitable for use in IMC. The RNAscope HiPlex v2 assay workflow was followed as recommended up until applying the fluoros. Instead of using the fluoros, metal-conjugated probes were used for RNA detection. Metal-conjugated antibodies were used for protein detection in the same tissue. Results Target protein markers identified a range of immune cells, tumor cells, stromal cells, endothelial cells, and extracellular matrix. Co-detection of RNA and protein allowed visualization of several cytokines and chemokines, such as CXCL13, CXCL9, CXCL10, IFNγ, IL10, IL8, and IL1B, enabling the identification of the cellular source of these secreted factors. Additionally, immune cell subpopulations and their activation states were visualized using marker-specific antibodies. Immune cell hubs associated with anti-tumor immune responses were detected in tumor niches across the TMA. Conclusions Overall, combining RNAscope with protein co-detection on the IMC platform allowed simultaneous visualization of RNA and protein targets to interrogate the tumor microenvironment. The single-cell resolution offered by the RNAscope assay provides superior sensitivity for RNA detection in addition to the existing protein-detection capability of the IMC platform. This workflow can be applied to FFPE tissues to study therapeutic efficacy, stratify patients based on inflammatory signature, and study cell-cell interactions within the TME. Citation Format: James Pemberton, Smriti Kala, Anushka Dikshit, Clinton Hupple. High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with 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 4636.

Abstract 5626: Multiomic spatial profiling of the tumor immune microenvironment at single cell resolution

Abstract Background: It has been well established that the tumor microenvironment (TME), which comprises cancer cells, stromal cells, and surrounding extracellular matrix, plays a critical role in cancer development, progression, and control. The immunological components within tumors, known as the tumor immune microenvironment (TiME), have also been implicated in tumor development, recurrence, and metastasis. Effective strategies for cancer immunotherapies will require a deep understanding of the factors that shape both the TME and TiME. Here, we describe a spatial multiomics approach that utilizes RNAscope™ ISH technology paired with high-plex whole-slide spatial phenotyping with the PhenoCycler™-Fusion platform. This two-step approach is compatible with human FFPE tissues and enables researchers to characterize the spatial biology of the TiME more accurately by detecting RNA and protein markers on serial sections. The resulting multiomic data more accurately reveal the interplay between TME and TiME by giving insight into cell lineages, surrounding structures, as well as secreted chemokines and cytokines that exist within the TME ecosystem. Methods: We performed ultrahigh-plex spatial phenotyping on the PhenoCycler-Fusion on FFPE tumor tissue sections, using an antibody panel that is designed for immune cell phenotyping, evaluation of immune contexture and proliferation across the TME. Using serial sections from the same tissue blocks, we then ran the RNAscope HiPlex v2 assay automated on the PhenoCycler-Fusion system. This assay consisted of a 12-plex immuno-oncology panel of RNA target probes, which were selected to detect macrophages, chemokines, and cytokines within tumors. We used Phenoplex software to analyze the protein and RNA datasets and to compute cell phenotypes and spatial associations. Results and Conclusions: In this proof-of-concept study, we demonstrate the utility of multiomic spatial profiling on the PhenoCycler-Fusion platform. Analysis of the resulting multiplex imaging data not only revealed the structural organization of cells within the TME, but also activation states of immune cells. Together, this information provides a more complete functional map of immune cells within the TME and TiME and thereby enriches our understanding of tumor biology that may be deterministic of immunotherapy responsiveness. This work paves the way for future research that will rely on deep spatial phenotyping with protein biomarkers coupled with accurate quantification of the expression of regulatory cytokines, chemokines, growth factors, or non-coding RNAs that only RNA probes can detect. Citation Format: Niyati Jhaveri, HaYeun Ji, Anushka Dikshit, Jessica Yuan, Emerald Doolittle, Steve Zhou, Maithreyan Srinivasan, Bassem B. Cheikh, Fabian Schneider, James Mansfield, Julia Kennedy-Darling, Oliver Braubach. Multiomic spatial profiling of the tumor immune microenvironment at single cell resolution. [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 5626.

Paired Comparison of Routine Molecular Screening of Patient Samples with Advanced Non-Small Cell Lung Cancer in Circulating Cell-Free DNA Using Three Targeted Assays.

Progressive advanced non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor targetable activating mutations, such as in-frame deletions in Exon 19 (Ex19del) of EGFR. Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of tyrosine kinase inhibitors. Plasma was collected from patients with NSCLC. We carried out targeted NGS using the Plasma-SeqSensei™ SOLID CANCER IVD kit on cfDNA (circulating free DNA). Clinical concordance for plasma detection of known oncogenic drivers was reported. In a subset of cases, validation was carried out using an orthogonal OncoBEAMTM EGFR V2 assay, as well as with our custom validated NGS assay. Somatic alterations were filtered, removing somatic mutations attributable to clonal hematopoiesis for our custom validated NGS assay. In the plasma samples, driver targetable mutations were studied, with a mutant allele frequency (MAF) ranging from 0.00% (negative detection) to 82.25%, using the targeted next-generation sequencing Plasma-SeqSensei™ SOLID CANCER IVD Kit. In comparison with the OncoBEAMTM EGFR V2 kit, the EGFR concordance is 89.16% (based on the common genomic regions). The sensitivity and specificity rates based on the genomic regions (EGFR exons 18, 19, 20, and 21) were 84.62% and 94.67%. Furthermore, the observed clinical genomic discordances were present in 25% of the samples: 5% in those linked to the lower of coverage of the OncoBEAMTM EGFR V2 kit, 7% in those induced by the sensitivity limit on the EGFR with the Plasma-SeqSensei™ SOLID CANCER IVD Kit, and 13% in the samples linked to the larger KRAS, PIK3CA, BRAF coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit. Most of these somatic alterations were cross validated in our orthogonal custom validated NGS assay, used in the routine management of patients. The concordance is 82.19% in the common genomic regions (EGFR exons 18, 19, 20, 21; KRAS exons 2, 3, 4; BRAF exons 11, 15; and PIK3CA exons 10, 21). The sensitivity and specificity rates were 89.38% and 76.12%, respectively. The 32% of genomic discordances were composed of 5% caused by the limit of coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit, 11% induced by the sensitivity limit of our custom validated NGS assay, and 16% linked to the additional oncodriver analysis, which is only covered by our custom validated NGS assay. The Plasma-SeqSensei™ SOLID CANCER IVD kit resulted in de novo detection of targetable oncogenic drivers and resistance alterations, with a high sensitivity and accuracy for low and high cfDNA inputs. Thus, this assay is a sensitive, robust, and accurate test.

Bio-SCAN V2: A CRISPR/dCas9-based lateral flow assay for rapid detection of theophylline.

Rapid, specific, and robust diagnostic strategies are needed to develop sensitive biosensors for small molecule detection, which could aid in controlling contamination and disease transmission. Recently, the target-induced collateral activity of Cas nucleases [clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases] was exploited to develop high-throughput diagnostic modules for detecting nucleic acids and small molecules. Here, we have expanded the diagnostic ability of the CRISPR-Cas system by developing Bio-SCAN V2, a ligand-responsive CRISPR-Cas platform for detecting non-nucleic acid small molecule targets. The Bio-SCAN V2 consists of an engineered ligand-responsive sgRNA (ligRNA), biotinylated dead Cas9 (dCas9-biotin), 6-carboxyfluorescein (FAM)-labeled amplicons, and lateral flow assay (LFA) strips. LigRNA interacts with dCas9-biotin only in the presence of sgRNA-specific ligand molecules to make a ribonucleoprotein (RNP). Next, the ligand-induced ribonucleoprotein is exposed to FAM-labeled amplicons for binding, and the presence of the ligand (small molecule) is detected as a visual signal [(dCas9-biotin)-ligRNA-FAM labeled DNA-AuNP complex] at the test line of the lateral flow assay strip. With the Bio-SCAN V2 platform, we are able to detect the model molecule theophylline with a limit of detection (LOD) up to 2μM in a short time, requiring only 15min from sample application to visual readout. Taken together, Bio-SCAN V2 assay provides a rapid, specific, and ultrasensitive detection platform for theophylline.

Derivation and evaluation of signatures using plasma β‐amyloid and pTau‐181 for brain amyloid‐β detection

AbstractBackgroundDetection of brain amyloid‐b (Ab) pathology using blood‐based tests would be considerably more convenient for patients, in addition to being a faster and more cost‐effective method for screening and monitoring patients in clinical trials. Using patient screening data from internal clinical trials, we derive signatures for brain Ab detection using plasma Ab42, Ab40, and pTau‐181, and evaluate their performance accuracy in an independent dataset.MethodAb42 and Ab40 were measured using immunoprecipitation coupled with LC‐MS/MS by C2N diagnostics in plasma samples from 513 patients at screening and pTau‐181 was measured in plasma samples from 398 patients (n=273 overlap) using Simoa Advantage V2 assay kit (immunoassay) by Quanterix Corp. Over 90% of these patients had mild cognitive impairment (MCI), and approximately 50% had brain Ab pathology based on Florbetapir‐PET visual read.Signatures using these plasma markers for detecting brain Ab were derived using Bayesian elastic‐net, resampled lasso, and gradient boosting models. Additional signatures were derived for assessing the added value of ApoE4 status, cognitive function scores and brain volumetric measures from magnetic resonance imaging (MRI). Performance of these signatures was assessed via 10‐fold cross‐validation within the training set, and further tested in an independent cohort from ADNI (Alzheimer’s disease neuroimaging initiative).ResultUsing currently available data, signatures derived using plasma Ab42/Ab40 ratio achieved 76% overall accuracy for detecting brain Ab. Similar accuracy was achieved using plasma pTau‐181 alone, which improved to 78% when including Ab42/Ab40, to 80% when adding ApoE4, and to 82% when adding cognitive scores. Inclusion of MRI brain volumetric measures did not improve the accuracy.When testing in the independent ADNI cohort, 75.7% accuracy was achieved by signatures based on plasma Ab42/Ab40 ratio. Signatures using plasma pTau‐181 alone achieved 75% accuracy, which improved to 78% when combined with Ab42 to Ab40 ratio.ConclusionThese results demonstrate the potential of blood‐based amyloid/tau signatures as promising patient screening tools for detecting brain amyloid burden in clinical trials and patient care. Results reported here will be refined further prior to final presentation using impending additional data.

Plasma pTau181 is associated with impaired cognition in the Old Order Amish and adds additional information beyond the known genetic risk factors for AD

AbstractBackgroundAs plasma biomarker assays have become more widely available, they are increasingly being used to characterize AD and related dementias. The Old Order Amish are a cultural and genetic isolate in the US that have participated in genetic studies for decades. This study investigated plasma phosphorylated tau at threonine‐181 (pTau181) in an Old Order Amish cohort to determine if the association with cognitive status is reproducible in this population.MethodOld Order Amish individuals over age 65 (n=467, mean age = 81.3, 60.6% female) in Indiana and Ohio were examined using a modified Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) battery. Each individual was assigned Impaired (n=155, mean age = 82.5, 56.8% female) or Unimpaired (n=312, mean age = 80.8, 62.5% female) cognitive status via consensus review of these examination results. The level of plasma pTau181 was measured using the pTau181 Advantage V2 assay from Quanterix. Genetic Risk Scores (GRS) were calculated using genome‐wide significant variants from Kunkle et al. (2019), weighted by log odds ratio estimates. A t‐test was used to compare plasma pTau181 levels between the Impaired and Unimpaired groups. Logistic regression was then used to model the contribution of age, sex, GRS, and plasma pTau181 on cognitive status.ResultWe found that the average level of plasma pTau181 was significantly higher in the Impaired group (2.46 pg/mL) compared to the Unimpaired group (2.01 pg/mL) at p<0.0001. A multivariable model found increasing age (OR=1.10, p=0.0002), male sex (OR=1.60, p=0.04), increasing GRS (OR=1.97, p<0.0001), and increasing plasma pTau181 (OR=1.46, p=0.001) associated with impaired cognitive status. The effect of GRS is mostly attributable to APOE.ConclusionThe result of these analyses indicates that pTau181 is associated with impaired cognitive status in the Old Order Amish, adding additional information beyond the known genetic risk factors for AD.

Towards a single-assay approach: a combined DNA/RNA sequencing panel eliminates diagnostic redundancy and detects clinically-relevant fusions in neuropathology

Since the introduction of integrated histological and molecular diagnoses by the 2016 World Health Organization (WHO) Classification of Tumors of the Nervous System, an increasing number of molecular markers have been found to have prognostic significance in infiltrating gliomas, many of which have now become incorporated as diagnostic criteria in the 2021 WHO Classification. This has increased the applicability of targeted-next generation sequencing in the diagnostic work-up of neuropathology specimens and in addition, raises the question of whether targeted sequencing can, in practice, reliably replace older, more traditional diagnostic methods such as immunohistochemistry and fluorescence in-situ hybridization. Here, we demonstrate that the Oncomine Cancer Gene Mutation Panel v2 assay targeted-next generation sequencing panel for solid tumors is not only superior to IHC in detecting mutation in IDH1/2 and TP53 but can also predict 1p/19q co-deletion with high sensitivity and specificity relative to fluorescence in-situ hybridization by looking at average copy number of genes sequenced on 1p, 1q, 19p, and 19q. Along with detecting the same molecular data obtained from older methods, targeted-next generation sequencing with an RNA sequencing component provides additional information regarding the presence of RNA based alterations that have diagnostic significance and possible therapeutic implications. From this work, we advocate for expanded use of targeted-next generation sequencing over more traditional methods for the detection of important molecular alterations as a part of the standard diagnostic work up for CNS neoplasms.

Abstract 764: Fully automated sample-to-report NGS workflow for comprehensive genomic profiling for myeloid neoplasms

Abstract Introduction: Myeloid malignancies are associated with a broad and diverse set of genomic alterations, including SNVs, insertions, deletions and gene fusions. Comprehensive characterization of genetic mutations in hematological disorders currently requires a variety of diagnostic tests and takes multiple days to complete. We developed a fully automated NGS Myeloid Assay that offers an easy to use sample-to-report workflow and the capability for processing up to 8 samples per day. Methods: The Genexus System is comprised of two software linked instruments, the Genexus Purification System and the Genexus Integrated Sequencer. The Genexus Purification System was used to isolate the DNA and RNA from blood or bone marrow samples from precharacterized myeloid samples representative of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) and from the blood of healthy donors. The Genexus Integrated Sequencer was used to dilute the nucleic acids to optimal concentration and to sequence the samples in replicates with Oncomine Myeloid Genexus v2 Assay. Six DNA and RNA samples were sequenced per run per day along with commercially available analytical controls and a No Template Control. The report was generated by the Genexus Software analysis pipeline optimized to detect different variant types with high sensitivity and specificity. Results: The purification workflows were tested with blood input of 50-400uL for DNA and 50-150uL for RNA. Genexus Purification System extracted and quantified nucleic acids showed input dependent yields. DNA and RNA yields obtained with 50uL sample inputs consistently met the 27.5ng DNA and 15ng RNA minimum requirements for Genexus Integrated Sequencer workflows. DNA libraries had >97% Uniformity of Amplicon Coverage and >95% Target Base Coverage at 350x. The percentage ratio of Mapped Reads for DNA and RNA libraries was approximately 80:20. Detected genetic variations included key hotspots in CEBPA, FLT3, IDH1/2, NPM1, NRAS, RUNX1, and U2AF1 genes that are prevalent in AML and MDS. Genexus Variant Calling results showed high reproducibility and high concordance to the Ion GeneStudio S5 sequencing platform (>95%). The analytical controls, AcroMetrix Oncology Hotspot Control, Seraseq Myeloid Mutation DNA Mix and Seraseq Myeloid Fusion RNA Mix, were sequenced with Sensitivity and PPV >95%. Conclusion: The Genexus System offers an automated sample-to-report workflow with minimal hands-on-time and run results in 30 hours which allows an easy to use solution for next day turnaround time. When used with the Oncomine Myeloid GX v2 Assay, it provides accurate and comprehensive information on diverse mutations including fusions that are relevant to the study of myeloid cancers. For research use only. Not for use in diagnostic procedures. Citation Format: Marina Sedova, Alexy Ongpin, Jennifer Burke, Collyn Seeger, Sarah Brozio, Janice Au-Young, Jiajie Huang, Thilanka Jayaweera, Iris Casuga, Milton Huynh, Fiona Hyland. Fully automated sample-to-report NGS workflow for comprehensive genomic profiling for myeloid neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 764.

Abstract 1723: Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays

Abstract Interrogating complex tumor microenvironment requires a multi-omics approach that can provide high level of sensitivity and specificity. Identifying immune cell subsets within the tumor can be vital for predicting response and determining therapeutic efficacy. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms that can substantially improve RNA-protein co-detection. We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated RNAscope Multiplex Fluorescent v2 assay, automated RNAscope Chromogenic Duplex assay and manual RNAscope Multiplex Fluorescent v2 assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples. We identified CD4+ helper T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize the expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor cells. This assay is enabled for multiplexing by combining with our fluorescent assay platforms. Similarly, for researchers interested in detecting target expression while retaining morphological context, the codetection assay should be combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization. Citation Format: Anushka Dikshit, Sayantani Basak, Emerald Doolittle, Ilya Kovalenko, Michaeline Bunting. Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1723.

Spatial profiling of immune cell markers in FFPE tumor tissues using the RNAscope&[trade] HiPlex v2 in situ hybridization assay

Abstract The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in FFPE tumor tissues. The RNAscope HiPlex v2 assay iteratively multiplexes up to 12 targets in frozen and FFPE tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. Here we investigate spatial expression of 12 immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokines The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction. We visualized T cell infiltration and activation within tumors using CD3, CD8 and IFNG expression. Subsets of pro- and anti-inflammatory macrophages were detected by CD68 and CD163 expression Hypoxia markers HIF1A and VEGF indicated immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis showed higher copy numbers in the lung tumor as compared to the other tumors. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME. Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, 12 key targets were spatially resolved in four different tumor types. The assay can provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues.

Genetic structure of drug-resistant Mycobacterium tuberculosis strains in Peru based on haplotypes obtained from a line probe assay

To determine the genetic structure of drug-resistant strains of Mycobacterium tuberculosis that circulated throughout Peru during the years 2011-2015, by using haplotypes obtained from a line probe assay. A total of 6589 samples that were admitted to the Instituto Nacional de Salud for routine diagnosis using the GenoType® MTBDRplus v2 assay were analyzed during the study period. Resistant haplotypes were created by concatenating 21 polymorphic sites of the evaluated genes using the line probe assay; and the association analysis was carried out with phenotypes obtained by the 7H10 agar ratio method. The most frequent mutations were: rpoB S531L (55.4%) and rpoB D516V (18.5%) for rifampicin resistance, and katG S315T (59.5%) and inhA c-15t (25.7%) for isoniazid resistance. We obtained 13 representative haplotypes (87.8% of analyzed samples), 6 corresponded to the multidrug-resistant genotype, 4 to the isoniazid mono-resistant genotype and 3 to the rifampicin mono-resistant genotype. Eighteen regions and the province of Callao showed high haplotype diversity; four showed moderate diversity and two showed low diversity. Most regions showed high haplotype diversity; in addition, most drug-resistant strains of Mycobacterium tuberculosis were concentrated in the cities of Lima and Callao. Likewise, drug-resistant Mycobacterium tuberculosis strains circulating in Peru mainly contain the genetic markers with the highest prevalence worldwide, which are associated with resistance to rifampicin and isoniazid.

Evaluation of the Genmark ePlex\xae and QIAstat-Dx\xae respiratory pathogen panels in detecting bacterial targets in lower respiratory tract specimens

BackgroundThe ePlex® and QIAstat-Dx® respiratory pathogen panels detect multiple respiratory pathogens, mainly viruses but also Legionella pneumophila, Mycoplasma pneumoniae and Bordetella pertussis. The assays have been marketed for use in nasopharyngeal swab specimens. For diagnosing bacterial pneumonia, lower respiratory tract (LRT) specimens are indicated. Aim of this study was to evaluate the performance of these syndromic panels for these three bacterial targets in samples from the LRT. Fifty-six specimens were collected from our repositories, five negative samples and fifty-one samples which had been previously tested positive with the routine diagnostic real-time PCR assays for Legionella spp. (N = 20), Bordetella spp. (N = 16) or M. pneumoniae (N = 15).ResultsThe QIAstat-Dx Respiratory Panel V2 (RP) assay detected all of the L. pneumophila and B. pertussis positive samples but only 11/15 (73.3 %) of the M. pneumoniae targets. The ePlex Respiratory Pathogen Panel (RPP) assay detected 10/14 (71.4 %) of the L. pneumophila targets, 8/12 (66.7 %) of the B. pertussis positive samples and 13/15 (86.7 %) of the M. pneumoniae targets.ConclusionsNo false-positive results were reported for all three bacterial pathogens by both assays. The clinical performance of both assays depended highly on the bacterial load in the sample and the type of specimen under investigation.