In-house Analysis Research Articles

An extracellular vesicle delivery platform based on the PTTG1IP protein

Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.

The ribosome profiling landscape of yeast reveals a high diversity in pervasive translation

BackgroundPervasive translation is a widespread phenomenon that plays a critical role in the emergence of novel microproteins, but the diversity of translation patterns contributing to their generation remains unclear. Based on 54 ribosome profiling (Ribo-Seq) datasets, we investigated the yeast Ribo-Seq landscape using a representation framework that allows the comprehensive inventory and classification of the entire diversity of Ribo-Seq signals, including non-canonical ones.ResultsWe show that if coding regions occupy specific areas of the Ribo-Seq landscape, noncoding regions encompass a wide diversity of Ribo-Seq signals and, conversely, populate the entire landscape. Our results show that pervasive translation can, nevertheless, be associated with high specificity, with 1055 noncoding ORFs exhibiting canonical Ribo-Seq signals. Using mass spectrometry under standard conditions or proteasome inhibition with an in-house analysis protocol, we report 239 microproteins originating from noncoding ORFs that display canonical but also non-canonical Ribo-Seq signals. Each condition yields dozens of additional microprotein candidates with comparable translation properties, suggesting a larger population of volatile microproteins that are challenging to detect. Our findings suggest that non-canonical translation signals may harbor valuable information and underscore the significance of considering them in proteogenomic studies. Finally, we show that the translation outcome of a noncoding ORF is primarily determined by the initiating codon and the codon distribution in its two alternative frames, rather than features indicative of functionality.ConclusionOur results enable us to propose a topology of a species’ Ribo-Seq landscape, opening the way to comparative analyses of this translation landscape under different conditions.

Fetal and neonatal echocardiographic analysis of biomechanical alterations for the systemic right ventricle heart

Background The perinatal transition’s impact on systemic right ventricle (SRV) cardiac hemodynamics is not fully understood. Standard clinical image analysis tools fall short of capturing comprehensive diastolic and systolic measures of these hemodynamics. Objectives Compare standard and novel hemodynamic echocardiogram (echo) parameters to quantify perinatal changes in SRV and healthy controls. Methods We performed a retrospective study of 10 SRV patients with echocardiograms at 33-weeks gestation and at day of birth and 12 age-matched controls. We used in-house developed analysis algorithms to quantify ventricular biomechanics from four-chamber B-mode and color Doppler scans. Cardiac morphology, hemodynamics, tissue motion, deformation, and flow parameters were measured. Results Tissue motion, deformation, and index measurements did not reliably capture biomechanical changes. Stroke volume and cardiac output were nearly twice as large for the SRV compared to the control RV and left ventricle (LV) due to RV enlargement. The enlarged RV exhibited disordered flow with higher energy loss (EL) compared to prenatal control LV and postnatal control RV and LV. Furthermore, the enlarged RV demonstrated elevated vortex strength (VS) and kinetic energy (KE) compared to both the control RV and LV, prenatally and postnatally. The SRV showed reduced relaxation with increased early filling velocity (E) compared prenatally to the LV and postnatally to the control RV and LV. Furthermore, increased recovery pressure (ΔP) was observed between the SRV and control RV and LV, prenatally and postnatally. Conclusions The novel hydrodynamic parameters more reliably capture the SRV alterations than traditional parameters.

RNA editing regulates glutamatergic synapses in the frontal cortex of a molecular subtype of Amyotrophic Lateral Sclerosis

BackgroundAmyotrophic Lateral Sclerosis (ALS) is a highly heterogenous neurodegenerative disorder that primarily affects upper and lower motor neurons, affecting additional cell types and brain regions. Underlying molecular mechanisms are still elusive, in part due to disease heterogeneity. Molecular disease subtyping through integrative analyses including RNA editing profiling is a novel approach for identification of molecular networks involved in pathogenesis.MethodsWe aimed to highlight the role of RNA editing in ALS, focusing on the frontal cortex and the prevalent molecular disease subtype (ALS-Ox), previously determined by transcriptomic profile stratification. We established global RNA editing (editome) and gene expression (transcriptome) profiles in control and ALS-Ox cases, utilizing publicly available RNA-seq data (GSE153960) and an in-house analysis pipeline. Functional annotation and pathway analyses identified molecular processes affected by RNA editing alterations. Pearson correlation analyses assessed RNA editing effects on expression. Similar analyses on additional ALS-Ox and control samples (GSE124439) were performed for verification. Targeted re-sequencing and qRT-PCR analysis targeting CACNA1C, were performed using frontal cortex tissue from ALS and control samples (n = 3 samples/group).ResultsWe identified reduced global RNA editing in the frontal cortex of ALS-Ox cases. Differentially edited transcripts are enriched in synapses, particularly in the glutamatergic synapse pathway. Bioinformatic analyses on additional ALS-Ox and control RNA-seq data verified these findings. We identified increased recoding at the Q621R site in the GRIK2 transcript and determined positive correlations between RNA editing and gene expression alterations in ionotropic receptor subunits GRIA2, GRIA3 and the CACNA1C transcript, which encodes the pore forming subunit of a post-synaptic L-type calcium channel. Experimental data verified RNA editing alterations and editing-expression correlation in CACNA1C, highlighting CACNA1C as a target for further study.ConclusionsWe provide evidence on the involvement of RNA editing in the frontal cortex of an ALS molecular subtype, highlighting a modulatory role mediated though recoding and gene expression regulation on glutamatergic synapse related transcripts. We report RNA editing effects in disease-related transcripts and validated editing alterations in CACNA1C. Our study provides targets for further functional studies that could shed light in underlying disease mechanisms enabling novel therapeutic approaches.

Gene-guided identifications of a structure-chimeric cyclotide viphi I from Viola philippica: Potential functions against cadmium and nematodes

The cyclic peptides, cyclotides, are identified mostly with 29–31-aa (amino acid residues) but rarely with ≥ 34-aa in plants. Viola philippica is a well-known medicinal plant but a rare metallophyte with cyclotides. A hypothesis was hence raised that the potential novel 34-aa cyclotide of Viola philippica would clearly broaden the structural and functional diversities of plant cyclotides. After homology-cloning the cyclotide precursor gene of VpCP5, a 34-aa cyclotide (viphi I) was identified to be larger than 22 other known cyclotides in V. philippica. It had a chimeric primary structure, due to its unusual loop structures (8 residues in loop 2 and 6 residues in loop 5) and aa composition (3 E and 5 R), by using phylogenetic analyses and an in-house cyclotide analysis tool, CyExcel_V1. A plasmid pCYC-viphi_I and a lab-used recombinant process were specially constructed for preparing viphi I. Typically, 0.12 or 0.25 mg ml−1 co-exposed viphi I could significantly remain cell activities with elevating Cd2+-exposed doses from 10−8 to 10−6 mol l−1 in MCF7 cells. In the model nematode Caenorhabditis elegans, IC50 values of viphi I to inhibit adult ratios and to induce death ratios, were 184.7 and 585.9 µg ml−1, respectively; the median lifespan of adult worms decreased from 14 to 2 d at viphi I doses ranging from 0.05 to 2 mg ml−1. Taken together, the newly identified viphi I exhibits functional potentials against cadmium and nematodes, providing new insights into structural and functional diversity of chimeric cyclotides in plants.

Identification of Southeast Asian Anopheles mosquito species using MALDI-TOF mass spectrometry.

Malaria elimination in Southeast Asia remains a challenge, underscoring the importance of accurately identifying malaria mosquitoes to understand transmission dynamics and improve vector control. Traditional methods such as morphological identification require extensive training and cannot distinguish between sibling species, while molecular approaches are costly for extensive screening. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and cost-effective tool for Anopheles species identification, yet its current use is limited to few specialized laboratories. This study aimed to develop and validate an online reference database for MALDI-TOF MS identification of Southeast Asian Anopheles species. The database, constructed using the in-house data analysis pipeline MSI2 (Sorbonne University), comprised 2046 head mass spectra from 209 specimens collected at the Thailand-Myanmar border. Molecular identification via COI and ITS2 DNA barcodes enabled the identification of 20 sensu stricto species and 5 sibling species complexes. The high quality of the mass spectra was demonstrated by a MSI2 median score (min-max) of 61.62 (15.94-77.55) for correct answers, using the best result of four technical replicates of a test panel. Applying an identification threshold of 45, 93.9% (201/214) of the specimens were identified, with 98.5% (198/201) consistency with the molecular taxonomic assignment. In conclusion, MALDI-TOF MS holds promise for malaria mosquito identification and can be scaled up for entomological surveillance in Southeast Asia. The free online sharing of our database on the MSI2 platform (https://msi.happy-dev.fr/) represents an important step towards the broader use of MALDI-TOF MS in malaria vector surveillance.

Implementation of an ISO 15189 accredited next generation sequencing service for cell-free total nucleic acid (cfTNA) analysis to facilitate driver mutation reporting in blood: the experience of a clinical diagnostic laboratory

AimsNext generation sequencing (NGS) on tumour tissue is integral to the delivery of personalised medicine and targeted therapy. NGS on liquid biopsy, a much less invasive technology, is an emerging...

Technical Notes: Robustness of three-dimensional treatment and imaging isocenter testing using a new gel dosimeter and kilovoltage CBCT.

Coincidence of the treatment and imaging isocenter coordinates is required to safely perform small-margin treatments, such as stereotactic radiosurgery of multiple brain metastases. A comprehensive and direct methodology for verifying concordance of kilovoltage cone-beam computed tomography (kV-CBCT) and treatment coordinates using an x-ray CT-based polymer gel dosimeter (dGEL) and onboard kV-CBCT was previously reported. Using this methodology, we tested the ability of a new commercially available x-ray CT-based polymer dGEL with a rapid response to provide efficient quality assurance (QA). The aim of this study was to evaluate the robustness of the three-dimensional geometric QA methodology using dGEL. The dGEL were commercially manufactured. The prescribed dose for each field was determined by visually identifying the 5, 10, and 20Gy isodose lines. A linear accelerator was used to irradiate the gels with seven non-coplanar beams. An in-house analysis program was used to identify the beam axes and treatment isocenter in kV-CBCT coordinates by processing the pre- and post-irradiation CBCT images. The impact of the radiation dose on the test reproducibility was examined, and the detectability of an intentional geometric error was assessed. The treatment isocenter was within 0.4mm of the imaging isocenter for all radiation doses. The residual error of the test with the intentional error was within 0.2mm. The analysis and image quality variations for a single dGEL introduced displacement errors less than 0.3mm. The test assessed the coincidence of treatment and kV-CBCT isocenter coordinates and detected errors with high robustness. Even for a 10Gy dose, the test yielded results comparable with those obtained using higher radiation doses owing to the rapid response of the dGEL dosimeter.

Chain-like One-Dimensional Assembly of Mesoporous Silica Nanoparticles: An Approach To Improve Hydrogel Adhesion.

Mesoporous silica nanoparticles (MSNPs) are well known for their adhesive properties with hydrogels and living tissues. However, achieving direct contact between the silica nanoparticle surface and the adherend necessitates the removal of capping agents, which can lead to severe aggregation when exposed to wet surfaces. This aggregation is ineffective for simultaneously bridging the two adherends, resulting in a reduced adhesive strength. In this study, we designed and synthesized mesoporous silica nanochains (MSNCs) to enhance the interactions with hydrogels by promoting the formation of coarser structures with increased nanopore exposure. Chain-like one-dimensional assemblies in the MSNCs were generated by depleting the capping ligand, cetyltrimethylammonium bromide, from the surface of the MSNPs. To quantify the porous areas of the MSNCs, we analyzed scanning electron microscopy (SEM) images using an in-house SEM image analysis algorithm. Additionally, we conducted a comparative assessment of the adhesion energies of MSNCs and MSNPs on a poly(dimethylacrylamide) hydrogel using a universal testing machine. The MSNCs exhibited a maximum adhesion energy of 13.7 ± 0.7 J/m2 at 3 wt %, surpassing that of MSNPs (10.9 ± 0.3 J/m2) at 2 wt %. Moreover, the unique stacking structure of the MSNCs enabled them to maintain an adhesion energy of 13.4 ± 1.0 J/m2 at a high concentration of 9 wt %, whereas the adhesion energy of MSNPs decreased to 8.2 ± 0.4 J/m2. This underscores their potential as superior hydrogel adhesives in challenging wet tissue-like environments.

Global intracranial arterial tortuosity is associated with intracranial atherosclerotic burden

The effect of arterial tortuosity on intracranial atherosclerosis (ICAS) is not well understood. This study aimed to evaluate the effect of global intracranial arterial tortuosity on intracranial atherosclerotic burden in patients with ischemic stroke. We included patients with acute ischemic stroke who underwent magnetic resonance angiography (MRA) and classified them into three groups according to the ICAS burden. Global tortuosity index (GTI) was defined as the standardized mean curvature of the entire intracranial arteries, measured by in-house vessel analysis software. Of the 516 patients included, 274 patients had no ICAS, 140 patients had a low ICAS burden, and 102 patients had a high ICAS burden. GTI increased with higher ICAS burden. After adjustment for age, sex, vascular risk factors, and standardized mean arterial area, GTI was independently associated with ICAS burden (adjusted odds ratio [adjusted OR] 1.33; 95% confidence interval [CI] 1.09–1.62). The degree of association increased when the arterial tortuosity was analyzed limited to the basal arteries (adjusted OR 1.48; 95% CI 1.22–1.81). We demonstrated that GTI is associated with ICAS burden in patients with ischemic stroke, suggesting a role for global arterial tortuosity in ICAS.

Prediction and Analysis of Transient Turbine Tip Clearance Using Long Short-Term Memory Neural Network

Abstract The transient turbine tip clearance (δ) throughout the engine process is crucial to modern high-performance aero engines. However, there is still a lack of efficient and accurate transient prediction models of tip clearances with active thermal control (ATC) system, especially for the tip clearances of the complex turbine structures with various parameters. This study develops a transient prediction model for the tradeoff between computational efficiency and accuracy, which includes an offline dataset generation process and an online δ prediction process. The offline dataset is first generated using an in-house finite element analysis code, which is validated against a transient tip clearance experiment, and data splicing and sensitivity analysis are applied to enrich the sample features and reduce the input parameters' dimensionality. Then, the long short-term memory neural network (LSTM) is employed to learn the transient tip clearances' timing information. The time consumption for the transient prediction model is significantly shorter than that for the tip clearance calculation method by three orders, and the maximum relative error is as low as 3.59%. In addition, the transient characteristics, including the overshoot value (σ) and the response time (ts), are investigated with different jet Reynolds numbers (Rec) and temperatures (Tfc) of ATC cooling flow. The ts decreases with larger Rec and smaller Tfc due to a more significant cooling effect. However, the σ increases with the increase of Rec and Tfc due to the different sensitivity of cooling parameters. This study provides a reference for the transient tip clearance prediction and the adjustments in the cooling strategies.

SKA3 Expression as a Prognostic Factor for Patients with Pancreatic Adenocarcinoma.

The spindle and kinetochore-associated complex subunit 3 (SKA3) is a protein essential for proper chromosome segregation during mitosis and thus responsible for maintaining genome stability. Although its involvement in the pathogenesis of various cancer types has been reported, the potential clinicopathological significance of SKA3 in pancreatic ductal adenocarcinoma (PDAC) has not been fully elucidated. Therefore, this study aimed to assess clinicopathological associations and prognostic value of SKA3 in PDAC. For this purpose, in-house immunohistochemical analysis on tissue macroarrays (TMAs), as well as a bioinformatic examination using publicly available RNA-Seq dataset, were performed. It was demonstrated that SKA3 expression at both mRNA and protein levels was significantly elevated in PDAC compared to control tissues. Upregulated mRNA expression constituted an independent unfavorable prognostic factor for the overall survival of PDAC patients, whereas altered SKA3 protein levels were associated with significantly better clinical outcomes. The last observation was particularly clear in the early-stage tumors. These findings render SKA3 a promising prognostic biomarker for patients with pancreatic ductal adenocarcinoma. However, further studies are needed to confirm this conclusion.

Molecular epidemiology of a Parainfluenza Type 3 virus outbreak: Informing infection control measures on adult hematology wards

ObjectivesParainfluenza virus type 3 (PIV3) outbreaks among hematology patients are associated with high morbidity and mortality. Prompt implementation of infection prevention (IP) measures has proven to be the most efficacious approach for controlling PIV3 outbreaks within this patient population. The most suitable IP measures can vary depending on the mode of virus transmission, which remains unidentified in most outbreaks. We describe the molecular epidemiology of an outbreak of PIV3 among hematology patients and the development of a new method that allows for the differentiation of outbreak and community strains, from which a closed outbreak could be inferred. MethodsPatients were screened for respiratory viruses using multiplex-PCR. PIV3 positive samples with a cycle threshold (Ct)-value of <31 underwent a retrospective characterization via an in-house developed sequence analysis of the hemagglutinin-neuraminidase (HN) gene. ResultsBetween July and September 2022, 31 hematology patients were identified with PIV3. Although infection control measures were implemented, the outbreak persisted for nine weeks. Sequencing the HN gene of 27 PIV3 strains from 27 patients revealed that all outbreak strains formed a distinct cluster separate from the control strains, suggestive of a nosocomial transmission route. ConclusionsSequencing the HN gene of PIV3 strains in an outbreak setting enables outbreak strains to be distinguished from community strains. Early molecular characterization of PIV3 strains during an outbreak can serve as a tool in determining potential transmission routes. This, in turn, enables rapid implementation of targeted infection prevention measures, with the goal of minimizing the outbreak's duration and reducing associated morbidity and mortality.

Resequencing the complete SNCA locus in Indian patients with Parkinson’s disease

The genetic loci implicated in familial Parkinson’s disease (PD) have limited generalizability to the Indian PD population. We tested mutations and the frequency of known mutations in the SNCA gene in a PD cohort from India. We selected 298 PD cases and 301 age-matched controls for targeted resequencing (before QC), along with 363 PD genomes of Indian ancestry and 1029 publicly available whole genomes from India as healthy controls (IndiGenomes), to determine the frequency of monogenic SNCA mutations. The raw sequence reads were analyzed using an in-house analysis pipeline, allowing the detection of small variants and structural variants using Manta. The in-depth analysis of the SNCA locus did not identify missense or structural variants, including previously identified SNCA mutations, in the Indian population. The familial forms of SNCA gene variants do not play a major role in the Indian PD population and this warrants further research in the under-represented population.

Determination of clopidogrel effect in cats using point-of-care Plateletworks ADP and shipped samples for PFA-200 analysis in a clinical practice setting.

Clopidogrel is the recommended first-line antithrombotic in cats for a variety of conditions; however, it is ineffective in 15-20% of cats. The determination of clopidogrel effectiveness with platelet function assays has historically been limited to specialty centers; however, recent work has suggested that in-hospital or shipped analyses of samples may be feasible. The aim of the present study was to investigate the utility of an in-house analysis and shipping of blood samples collected in primary practices for the determination of clopidogrel effectiveness. Citrated blood samples were collected from cats receiving clopidogrel therapy by veterinarians in clinical practices across Canada, a median of 304.4 km from the reference laboratory (range 8-4425). Samples were analyzed in-house using Plateletworks ADP and shipped for remote analysis using PFA-200 P2Y and COL/ADP cartridges. A total of 30 samples were collected from 25 cats. Of these, the percentage of samples analyzable for the presence or absence of the clopidogrel effect was 86% for Plateletworks ADP, 90% for PFA-200 P2Y and 87% for PFA-200 COL/ADP. There was no significant difference in the number of samples unable to be analyzed by each modality (P = 0.689) due to flow obstruction or other sample characteristics. The prevalence of absence of clopidogrel effectiveness on platelet function assays was 8% with the PFA-200 COL/ADP assay, 25% with the PFA-200 P2Y assay and 30% with the Plateletworks ADP assay. The results of this study confirm that samples of feline blood can be collected in clinical practices and shipped to a reference laboratory for PFA-200 analysis with a high rate of success, comparable to point-of-care analysis.

Inhibition of leukotriene-B4 signalling-mediated host response to tuberculosis is a potential mode of adjunctive host-directed therapy.

Treatment of tuberculosis (TB) is faced with several challenges including the long treatment duration, drug toxicity and tissue pathology. Host-directed therapy provides promising avenues to find compounds for adjunctively assisting antimycobacterials in the TB treatment regimen, by promoting pathogen eradication or limiting tissue destruction. Eicosanoids are a class of lipid molecules that are potent mediators of inflammation and have been implicated in aspects of the host response against TB. Here, we have explored the blood transcriptome of pulmonary TB patients to understand the activity of leukotriene B4, a pro-inflammatory eicosanoid. Our study shows a significant upregulation in the leukotriene B4 signalling pathway in active TB patients, which is reversed with TB treatment. We have further utilized our in-house network analysis algorithm, ResponseNet, to identify potential downstream signal effectors of leukotriene B4 in TB patients including STAT1/2 and NADPH oxidase at a systemic as well as local level, followed by experimental validation of the same. Finally, we show the potential of inhibiting leukotriene B4 signalling as a mode of adjunctive host-directed therapy against TB. This study provides a new mode of TB treatment along with mechanistic insights which can be further explored in pre-clinical trials.

Massive Parallel DNA Sequencing of Patients with Inherited Cardiomyopathies in Cyprus and Suggestion of Digenic or Oligogenic Inheritance.

Inherited cardiomyopathies represent a highly heterogeneous group of cardiac diseases. DNA variants in genes expressed in cardiomyocytes cause a diverse spectrum of cardiomyopathies, ultimately leading to heart failure, arrythmias, and sudden cardiac death. We applied massive parallel DNA sequencing using a 72-gene panel for studying inherited cardiomyopathies. We report on variants in 25 families, where pathogenicity was predicted by different computational approaches, databases, and an in-house filtering analysis. All variants were validated using Sanger sequencing. Familial segregation was tested when possible. We identified 41 different variants in 26 genes. Analytically, we identified fifteen variants previously reported in the Human Gene Mutation Database: twelve mentioned as disease-causing mutations (DM) and three as probable disease-causing mutations (DM?). Additionally, we identified 26 novel variants. We classified the forty-one variants as follows: twenty-eight (68.3%) as variants of uncertain significance, eight (19.5%) as likely pathogenic, and five (12.2%) as pathogenic. We genetically characterized families with a cardiac phenotype. The genetic heterogeneity and the multiplicity of candidate variants are making a definite molecular diagnosis challenging, especially when there is a suspicion of incomplete penetrance or digenic-oligogenic inheritance. This is the first systematic study of inherited cardiac conditions in Cyprus, enabling us to develop a genetic baseline and precision cardiology.

Long-term monitoring of SARS-CoV-2 variants in wastewater using a coordinated workflow of droplet digital PCR and nanopore sequencing

Quantitative polymerase chain reaction (PCR) and genome sequencing are important methods for wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse transcription-droplet digital PCR (RT-ddPCR) is a highly sensitive method for quantifying SARS-CoV-2 RNA in wastewater samples to track the trends of viral activity levels but cannot identify new variants. It also takes time to develop new PCR-based assays targeting variants of interest. Whole genome sequencing (WGS) can be used to monitor known and new SARS-CoV-2 variants, but it is generally not quantitative. Several short-read sequencing techniques can be expensive and might experience delayed turnaround times when outsourced due to inadequate in-house resources. Recently, a portable nanopore sequencing system offers an affordable and real-time method for sequencing SARS-CoV-2 variants in wastewater. This technology has the potential to enable swift response to disease outbreaks without relying on clinical sequencing results. In addressing concerns related to rapid turnaround time and accurate variant analysis, both RT-ddPCR and nanopore sequencing methods were employed to monitor the emergence of SARS-CoV-2 variants in wastewater. This surveillance was conducted at 23 sewer maintenance hole sites and five wastewater treatment plants in Michigan from 2020 to 2022. In 2020, the wastewater samples were dominated by the parental variants (20A, 20C and 20 G), followed by 20I (Alpha, B.1.1.7) in early 2021 and the Delta variant of concern (VOC) in late 2021. For the year 2022, Omicron variants dominated. Nanopore sequencing has the potential to validate suspected variant cases that were initially undetermined by RT-ddPCR assays. The concordance rate between nanopore sequencing and RT-ddPCR assays in identifying SARS-CoV-2 variants to the clade-level was 76.9%. Notably, instances of disagreement between the two methods were most prominent in the identification of the parental and Omicron variants. We also showed that sequencing wastewater samples with SARS-CoV-2 N gene concentrations of >104 GC/100 ml as measured by RT-ddPCR improve genome recovery and coverage depth using MinION device. RT-ddPCR was better at detecting key spike protein mutations A67V, del69–70, K417N, L452R, N501Y, N679K, and R408S (p-value <0.05) as compared to nanopore sequencing. It is suggested that RT-ddPCR and nanopore sequencing should be coordinated in wastewater surveillance where RT-ddPCR can be used as a preliminary quantification method and nanopore sequencing as the confirmatory method for the detection of variants or identification of new variants. The RT-ddPCR and nanopore sequencing methods reported here can be adopted as a reliable in-house analysis of SARS-CoV-2 in wastewater for rapid community level surveillance and public health response.

Abstract C006: Combining the methylation and gene expression contiguous signals leads to better pancreatic cancer patient stratification and early detection

Abstract Background Pancreatic cancer (PAC) lacks methods for screening, patient stratification, resulting in late diagnosis and inadequate treatment of patients. Cell-free DNA (cfDNA) methylation patterns are signals representing real-time cancer characteristics such as early presence, tissue, and cell of origin. The aim of this study is to provide a better patient stratification based on differential patterns on the combined methylation and gene expression results in PAC and normal samples and provide proof-of-concept for early detection. Method Differential methylation region (DMR) analysis was performed on 180 TCGA PAAD and normal tissue samples using paired methylation and expression data. Validation was done on 143 Infiltrating duct carcinomas (IDC), (stage I, II, II), and 4 normal samples and the 4 probe clusters (FPC) were defined based on their methylation status: HM = high methylation, IM = intermediate methylation, LM = low methylation and MM = minimum methylation. 3-year and 5-year survival analysis with Kaplan-Meier was done. Gene ontology (GO) and Biological Pathway analyses (PA) were performed on DMRs of FPC. Integration analysis on paired methylation and expression data and the limma analysis was performed for significant genes in 5000 bp vicinities of the selected DMRs. Finally, selected 23 DMRs, were targeted in plasma cfDNA from 9 PAC patients (stage I-III) and 20 matching control samples. Results The survival analysis of FPC, shows the highest probability difference between HM and MM groups with p-values of 0.018 and 0.029 for 3-year and 5-year survival, respectively. Variability analysis shows 5740 significant CpG sites and 1135 significant DMRs comparing IDC tumor samples to controls. GO and PA on DRMs of FPC, indicated the most PAC specific pathways in the group of probes with MM status. Integration analysis on the methylation and mutation data of the selected genes of GO analysis shows potential to discriminate between normal and PAC patients and in between HM and MM groups and showed smaller mutation burden in MM and LM sample groups. Comparing MM to HM groups of patients, shows the highest significance in 5 downregulated genes in 5000 bp vicinity from Hypermethylated probes and 13 upregulated genes and 425 regions in 5000 base pairs vicinity from 170 hypomethylated probes. Survival analysis on the selected genes showed a very high correlation between the expression of the genes like SIX2, DNMT3A, ZFP91 and POR and the survival probability on FPC. Additionally, it has been shown that the selected DMRs, show good individual performance of AUC &amp;gt;0.95 in the in-house analysis of plasma cfDNA targeted data from 9 PAC patients (stage I-III) and 20 matching control samples. Conclusion Methylation markers carry distinct biological signals and combined with gene expression differentiate the PAC patient groups, showing potential of the methylation signals for better patient stratification outcomes for improved survival. High accuracy for cancer detection was observed for early-stage cancers when compared to cfDNA from matching controls. Citation Format: Pardis Saadatmand, Urban Kunej, Kristi Kruusmaa. Combining the methylation and gene expression contiguous signals leads to better pancreatic cancer patient stratification and early detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C006.

Benefits of Implementing Reflex Genomic Analysis for Nonsmall Cell Lung Cancer.

Molecular biomarker analysis is standard of care in advanced nonsmall cell lung cancer (NSCLC). Pathologist-driven reflex testing protocols are one approach to initiating this analysis. Two years after insourcing genomic analysis at our institution, a reflex testing protocol for advanced NSCLC was initiated. A retrospective review of the records of 578 NSCLC biopsies was performed to assess the impact of 3 genomic testing workflows (send-out, in-house clinician-ordered, and in-house reflex) on time to initiation of molecular testing [initiation time (IT)], reporting time (RT), proportion of test failures, and test ordering practices. The proportion of test failures by test methodology was also assessed. IT was lowest for reflex protocol orders (mean weekdays: 30.0 send-out, 27.4 in-house clinician-ordered, 0.95 reflex). Test failure was highest for send-out testing (31.7% vs. 10% each for in-house clinician-ordered and reflex). RT remained consistent across the 3 workflows (mean weekdays: 11.1 send-out, 11.9 in-house clinician-ordered, and 11.4 reflex). Guideline-congruent molecular testing increased upon insourcing genomic analysis and again upon implementing reflex testing with a reduction in nonbiomarker informed care (58.8% send-out, 19.5% in-house clinician-ordered, 11.5% reflex). Implementation of reflex in-house genomic analysis for advanced NSCLC ensured consistency in RT and significantly decreased IT and proportion of test failures. Insourcing genomic analysis and thoughtful care pathway design improve equitable access to molecular biomarker analysis and mitigate nonbiomarker informed cancer care in NSCLC.