Clinical Molecular Diagnostics Research Articles

Abstract 1771: NYU Langone Genome PACT - Genome Profiling of Actionable Cancer Targets (LG-PACT) for clinical patient molecular diagnostics and treatment

Abstract Next-generation sequencing (NGS) for the detection of somatic variants has become the tool of choice in a variety of molecular oncology fields and in the clinic. Its use ranges from sequencing entire tumor genomes and transcriptomes to targeted clinical diagnostic gene panels. The NYU Langone Genome PACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients to detect gene alterations in a 607-gene panel. Indications for testing are cancer (solid tumors or hematological malignancies) where a mutational profile from multiple genes would be informative for disease stratification, prognosis, or treatment options including targeted therapies and eligibility for clinical trials. The test is intended to provide information on somatic mutations including point mutations, copy number aberrations, and small insertions/deletions (indels) for diagnostic and treatment decisions. LG-PACT is a United States Food and Drug Administration (FDA) approved diagnostic test. The clinical interpretation of sequencing data of molecular tumor markers from NGS encompasses automated variant calling tools with human interpretation. The final mostly manual review of data is intensive, involving highly trained scientists, encompassing literature review, interpretation and tier classification by pathologists, who then provide a complete molecular diagnostic report to the treating oncologists. Since January 2022 to present (October 2023) we have provided clinical genomic reports for 1029 oncological cases from 124 different cancers and their subtypes, including Brain (489 cases incl. meningioma, glioma and glioblastoma), Gastrointestinal (123), Lung (114), among others. We first present the technical challenges of validating an NGS oncological diagnostic targeted assay for appropriate clinical grade accuracy and sensitivity acceptable for patient care. We show how copy number alterations provide a more comprehensive description of the tumors genomic profile. We then outline the actionability of targeted panel sequencing for our current patient cohort. Where analysis of variant detection has led to 47% (490) of our clinical tumor samples containing known tier 1 therapeutic variants. We conclude with presenting case studies that identify both the clinical utility and informatic challenges of variant calling specific to gene panel sequencing e.g., i) the capture of potential targetable rare and novel indels and multivariant mutations in exons 19 and 20 of EGFR, and ii) a unique KIT tandem duplication event in a gastrointestinal stromal tumor (GIST). We demonstrate the value in precision oncology for multiple cancer types and how the capture of unique variants can provide better targeted treatment options to cancer patients. Citation Format: Varshini Vasudevaraja, Yiying Yang, Jonathan Serrano, Kazimierz Wrzeszczynski, Matija Snuderl. NYU Langone Genome PACT - Genome Profiling of Actionable Cancer Targets (LG-PACT) for clinical patient molecular diagnostics and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1771.

CRISPR/Cas13a catalyzed self-assembly of quantum dot-DNA hydrogel for microRNA assay

Taking the advantages of outstanding precision in target recognition and trans-cleavage ability, the RNA-guided CRISPR-Cas systems have shown great promise in cancer diagnostics. Here, we developed a novel detection system based on CRISPR-Cas-catalyzed formation of quantum dot-DNA (QD-DNA) hydrogel for sensitive miRNA assay. After target miRNA recognition and Cas-mediated cleavage of the uracil ribonucleotide-embedded hairpin DNA (rU-HD), the cleaved rU-HD released primer DNA, which triggered toehold-mediated strand displacement (TMSD) amplification to form trefoil DNA quenchers. The trefoil DNA quenchers hybridized with DNA functionalized quantum dots (DNA-QDs) to construct self-assembled QD-DNA hydrogel and the fluorescence of the QDs could be efficiently quenched by the modified black hole quencher at the 3′ terminal of the trefoil DNA quenchers. This assay, termed CRISPR-Cas-catalyzed TMSD based QD-DNA hydrogel (Cas-TMSD-QDH), was applied for the sensitively detection of miR-17 and the limit of detection was as low as 182aM. The Cas-TMSD-QDH assay showed an accurate detection of miR-17 levels in different cell lines and the detection accuracy is confirmed by qRT-PCR. Besides, the assay was able to assess miR-17 levels in serum sample, revealing great potential for miRNAs assay in clinical molecular diagnostics.

Target Recognition Initiated Self-Assembly-Based Signal Amplification Strategy for Sensitive and Colorimetric Staphylococcus aureus Detection and Diagnosis of Skin Infection.

Staphylococcus aureus (S. aureus), as a Gram-positive bacterium, is commonly encountered in various infectious diseases, such as acute skin and soft tissue infections. Despite that many efforts have been made, sensitive and reliable quantitative determination of S. aureus remains a huge challenge. Here, we depict a novel colorimetric approach for sensitive and accurate detection by combining allosteric probe-based target recognition and chain extension-based dual signal recycling. The single-strand DNA (ssDNA) products generated by the chain extension process lead to the liberation of G-quadruplex sequences, which can fold into active DNAzyme under the assistance of hemin. The active DNAzyme can work as peroxidase mimics to catalyze the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS2-)-H2O2 system, causing the color change of the system. Eventually, the method exhibits a wide detection range from 103cfu/mL to 106cfu/mL. The limit of detection of the approach was determined 232cfu/mL. Considering the robust capability of the approach in S. aureus detection, we believe that it will be a potential alternative tool for biomedical research and clinical molecular diagnostics.

DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging.

Sensitive quantification of multiple analytes of interest is of great significance for clinical diagnosis. CRISPR Cas platforms offer a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostics, while their multiplex analysis capability is still limited and challenging. Herein, we develop a novel DNA Tetrahedron (DTN)-supported biosensor based on the spatially separated CRISPR Cas self-amplification strategy and multiple-metal-nanoparticle tagging coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection to improve the sensitivity and feasibility of the platform for multiplex detection of HPV-DNA (HPV-16, HPV-18 and HPV-52). Given target DNA induces robust trans-cleavage activity of the Cas12a/crRNA duplex, and the surrounding corresponding single-stranded DNA (ssDNA) linker are cleaved into short fragments that are unable to bond metal-nanoparticle probes (197Au, 107Ag, 195Pt) onto DTN modified magnetic beads probe (MBs-DTN), resulting in obvious ICP-MS signal change. Of note, compared with ssDNA functionalized MBs, a higher Signal-to-Noise Ratio was obtained by using MBs-DTN in our system, further amplifying the signal by regulating probes on the surface of MBs. As expected, the HPV-DNA could be detected with detection limits as low as 218fM and be multiplexed assayed at one test with high accuracy and specificity by this proposed strategy. Furthermore, we demonstrated that the HPV-DNA in cervical swab samples could be detected, showing high consistency with DNA sequencing results. We believe that this work provides a promising option in designing CRISPR based multiplex detection system for high sensitivity, good specificity, and clinical molecular diagnostics.

Copy number variation (CNV) calling of MET, MYC, and ERBB2 using PiVAT bioinformatic platform.

e15145 Background: CNV in tumor-associated genes such as MET, MYC and ERBB2 amplification can lead to cancer progression and poor survival in multiple cancers, and is associated with treatment regimen decision. Whole genome sequencing (WGS) and comprehensive genomic profiling (CGP) panels covering hundreds of genes are currently used to detect CNV in hospitals and CLIA-accredited laboratories. However, cost and complex workflow of WGS/CGP restrict their application in clinical molecular diagnostics. CGP panel covering dozens of genes based on targeted amplicon sequencing (TAS) method is preferred, but it is not ideal to detect CNV accurately due to unique methodology. Here we established a CNV calling toolkit in PiVAT bioinformatic platform to solve CNV calling problems in small-size TAS panel. Methods: 46 CNV positive (CNV+) and 14 CNV negative (CNV-) samples were sequenced using MGISEQ-2000 with a paired end, 2x100 read length protocol and adapter sequences were trimmed from 3’ ends of each read. PiVAT’s CNV calling method is based on double coverage normalization, including one per-sample normalization and one per-amplicon normalization. CNV- samples were used as normalization references. MET, MYC and ERBB2 copy number was investigated to determine sensitivity, specificity, precision, and accuracy of the overall test. Copy number deletion is defined as ratio < 0.8 and amplification is defined as ratio > 1.2. PiVAT CNV output calls were compared against excepted copy number to calculate and plot the concordance. CNVkit and Control-FREEC, 2 other CNV calling tools, were chosen as comparators to compare ERBB2, MET and MYC copy number levels with PiVAT’s CNV caller. Results: Sequencing results from all 46 CNV+ samples showed high coverage uniformity, with > 90% of sites having base coverage depth > 20% of mean coverage for all samples. Average mapping rate and on-target rate of sequencing run, analyzed by PiVAT, were 98.98±0.41% and 98.39±0.45%. Results from PiVAT showed a sensitivity of 97.83% and a specificity of 100% for MET, MYC, ERBB2 amplifications. PiVAT estimated copy number more accurately compared with CNVkit and Control-FREEC. PiVAT’s R2, measuring degree of difference from expected copy number, was greater than CNVkit and Control-FREEC for MET, MYC and ERBB2, with values at 0.78, 0.69 and 0.96, respectively. In contrast, CNVkit and Control-FREEC were on average 77.38% and 164.04% worse. For CNVkit and Control-FREEC, many of MET, MYC and ERBB2 CNV calls were seen at a much lower magnitude than expected. Conclusions: Our studies have shown that PiVAT is a reliable tool for calling CNVs. PiVAT’s sensitivity and accuracy were calculated to be > 97%. And PiVAT outperformed other CNV calling tools, CNVkit and Control-FREEC. This demonstrates that PiVAT’s CNV caller can be used to provide CNV results from TAS panel, and guide relevant cancer diagnosis and clinical decision more accurately.

Overview of Molecular Detection Technologies for MET in Lung Cancer.

MET tyrosine kinase receptor pathway activation has become an important actionable target in solid tumors. Aberrations in the MET proto-oncogene, including MET overexpression, the activation of MET mutations, MET mutations that lead to MET exon 14 skipping, MET gene amplifications, and MET fusions, are known to be primary and secondary oncogenic drivers in cancer; these aberrations have evolved as predictive biomarkers in clinical diagnostics. Thus, the detection of all known MET aberrations in daily clinical care is essential. In this review, current molecular technologies for the detection of the different MET aberrations are highlighted, including the benefits and drawbacks. In the future, another focus will be on the standardization of detection technologies for the delivery of reliable, quick, and affordable tests in clinical molecular diagnostics.

Abstract 2115: Zero-waste molecular diagnostics from biopsy needle wash water

Abstract Background: Over the past decade, the WHO classification of brain tumors has increasingly integrated molecular diagnostics into official classification criteria. However, biopsy tissue is precious due to high morbidity associated with repeat biopsies, and usually earmarked for histology, leaving little or no tissue for clinical molecular diagnostics. We have identified that the fluid used to wash biopsy needles after cores contains excess tumor DNA and cells that can be diagnostically useful. Additional research of tumor DNA and tumor tissue recovered from biopsy needle washes provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients, while utilizing material that is otherwise thrown away. Methods: In two cases, surgeons were instructed to save 5-15ml of fluid used to wash biopsy needles. Isothermal targeted amplification of H3.3 and H3.1 histone genes was performed on raw wash fluid. Resulting amplicons were sequenced using an Oxford Nanopore MinION. DNA extracted from wash fluid was subjected to NGS sequencing via the Illumina TruSight Oncology 500 panel and compared to a CLIA certified NGS sequencing results from clinically indicated biopsy tissue. Results: For case 1, 5ul of raw wash fluid was subjected to rapid Loop-mediated Isothermal Amplification (LAMP) targeting the H3.1 and H3.3 K27M histone mutations and immediately sequenced using a MinION sequencer via rapid library preparation. Sequencing revealed a H3.3 K27M histone mutation present in wash fluid at 12% variant allele fraction (VAF). The variant call (99.99% CI) was available 1hr5mins after acquisition of material. For case 2, 5ml of wash fluid was processed using a cell-free DNA extraction kit resulting in ~250ng of recovered DNA. A LAMP assay targeting the H3.3 K27M mutation was performed on extracted DNA from wash fluid. Sequencing revealed an H3.3 K27M histone mutation present at 55% VAF, which was later corroborated using digital droplet PCR. Clinically indicated tissue from both biopsies was submitted for cancer panel sequencing in a CLIA certified setting. DNA extracted from wash fluid from both cases was subjected to Illumina TruSight Oncology 500 panel sequencing. In both cases, the panel was able to corroborate all SNV mutations reported by clinical sequencing that were also covered by the panel. Accrual of needle wash specimens is ongoing, with multiple monthly procedures. Conclusions: Large amounts of diagnostically useful material is present in biopsy needle wash fluid, which is usually discarded. Isothermal assays coupled with MinION sequencing can rapidly identify clinically relevant point mutations directly from wash fluid (e.g. IDH1), and enough DNA was recovered from wash fluid from both case-studies to submit for targeted cancer panel sequencing. These results suggest biopsy needle wash fluid should be considered a first-class diagnostic resource in any cancer. Citation Format: Jack Wadden, Vishal John, Seongbae Kong, Kait Verbal, Amy K. Bruzek, Wajd Al-Holou, Jason A. Heth, Hugh Garton, Carl Koschmann. Zero-waste molecular diagnostics from biopsy needle wash water [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 2115.

Engineering m6A demethylation-activated DNAzyme for visually and sensitively sensing fat mass and obesity-associated protein

Fat mass and obesity-associated protein (FTO) regulating the N6-methyladenine (m6A, the most pervasive epigenetic modification) levels within the nucleus has been identified as a potential biomarker for cancer diagnosis and prognosis. However, current methods for FTO detection are complicated or/and not sensitive enough for practical application. Herein, we propose a colorimetric biosensor for detecting FTO based on a delicate design of m6A demethylation-activated DNAzyme. Specifically, an m6A-blocked DNAzyme is constructed as a switch of the biosensor that can be turned on by target FTO. The decreased thermal stability resulting from substrate cleavage leads to a DNAzyme recycling to produce multiple primers. Then the rolling circle amplification (RCA) reactions can be initiated to generate G-quadruplex-DNAzymes catalyzing 2,2-azino-bis-(3-ethylben-zthiazoline-6-sulfonic acid (ABTS) oxidation which can be readily observed by the naked eye. Quantitative detection can also be achieved with a limit of detection (LOD) down to 69.9 fM, exhibiting higher sensitivity than previous reports. Therefore, this biosensor opens a simple and sensitive way to achieve visual assay of FTO via triple signal amplification. In addition, our biosensor has been successfully applied to FTO detection in clinical samples, which shows great potential in clinical molecular diagnostics.

A color-based FHGC approach facilities DNA-based clinical molecular diagnostics

A color-based FHGC approach facilities DNA-based clinical molecular diagnostics

Development of a Biosensor for fast point-of-care Blood Analysis of Troponin.

We present the development of novel tetrapolar EIS biosensor for the detect of troponin. Troponin has considerable diagnostic power and provide invaluable prognostic information for risk stratification. of acute coronary syndromes. Clinical Relevance- A feasibility study was undertaken to assess the diagnostic performance of serial cardiac troponin measurements which is excellent as these structural proteins are unique to the heart and thus sensitive and specific of damage to the myocardium. clinical molecular diagnostics and home healthcare. Troponin's biosensors would provide point-of-care and rapid decision making for the early detection of CS. Clinically relevant window of cTnI testing, concentrations from 10pM to 0.1μM were achieved.

Combining CRISPR/Cas12a with isothermal exponential amplification as an ultrasensitive sensing platform for microRNA detection

MicroRNA (miRNA) is considered as a potential biomarker in the diagnosis of cancer. Thus, a new diagnostic technique with high sensitivity and selectivity is crucial for miRNA detection in clinical testing. Herein, we presented a sensing platform based on CRISPR/Cas12a and isothermal exponential amplification reaction (EXPAR) to realize ultrasensitive analysis of miRNA. MiRNA-27a (miR-27a) was chosen as a model target. In this sensing platform, a miR-27a-triggered cascade EXPAR process was introduced to achieve a mass of activators. The resultant activators could hybrid specifically with crRNA of CRISPR/Cas12a system, triggering the multiple-turnover trans-cleavage activity of Cas12a for achieving signal amplification. The sensing platform rendered a wide detection (ranging from 100 aM to 10 nM) and a low limit of detection (90 aM). In particular, this strategy achieved ultrasensitive detection of the other microRNA biomarkers such as let-7a and miR-141 by the flexible design of the region of template, which demonstrated the universal detection ability. Furthermore, an excellent selectivity feature was obtained by the discrimination of microRNA family members and base mismatch analysis. The proposed sensing platform also was able to assess miR-27a levels in breast cancer cell and serum sample, revealing a promising application in clinical molecular diagnostics.

Tissue-specific genotype-phenotype correlations among USH2A-related disorders in the RUSH2A study.

We assessed genotype-phenotype correlations among the visual, auditory, and olfactory phenotypes of 127 participants with Usher syndrome (USH2) (n =80) or nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) (n = 47) due to USH2A variants, using clinical data and molecular diagnostics from the Rate of Progression in USH2A Related Retinal Degeneration (RUSH2A) study. USH2A truncating alleles were associated with USH2 and had a dose-dependent effect on hearing loss severity with no effect on visual loss severity within the USH2 subgroup. A group of missense alleles in an interfibronectin domain appeared to be hypomorphic in ARRP. These alleles were associated with later age of onset, larger visual field area, better sensitivity thresholds, and better electroretinographic responses. No effect of genotype on the severity of olfactory deficits was observed. This study unveils a unique, tissue-specific USH2A allelic hierarchy with important prognostic implications for patient counseling and treatment trial endpoints. These findings may inform clinical care or research approaches in others with allelic disorders or pleiotropic phenotypes.

Next-generation sequencing of cerebrospinal fluid for clinical molecular diagnostics in pediatric, adolescent and young adult brain tumor patients.

Safe sampling of central nervous system tumor tissue for diagnostic purposes may be difficult if not impossible, especially in pediatric patients, and an unmet need exists to develop less invasive diagnostic tests. We report our clinical experience with minimally invasive molecular diagnostics using a clinically validated assay for sequencing of cerebrospinal fluid (CSF) cell-free DNA (cfDNA). All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families. We analyzed 64 CSF samples from 45 pediatric, adolescent and young adult (AYA) patients (pediatric = 25; AYA = 20) with primary and recurrent brain tumors across 12 histopathological subtypes including high-grade glioma (n = 10), medulloblastoma (n = 10), pineoblastoma (n = 5), low-grade glioma (n = 4), diffuse leptomeningeal glioneuronal tumor (DLGNT) (n = 4), retinoblastoma (n = 4), ependymoma (n = 3), and other (n = 5). Somatic alterations were detected in 30/64 samples (46.9%) and in at least one sample per unique patient in 21/45 patients (46.6%). CSF cfDNA positivity was strongly associated with the presence of disseminated disease at the time of collection (81.5% of samples from patients with disseminated disease were positive). No association was seen between CSF cfDNA positivity and the timing of CSF collection during the patient's disease course. We identified three general categories where CSF cfDNA testing provided additional relevant diagnostic, prognostic, and/or therapeutic information, impacting clinical assessment and decision making: (1) diagnosis and/or identification of actionable alterations; (2) monitor response to therapy; and (3) tracking tumor evolution. Our findings support broader implementation of clinical CSF cfDNA testing in this population to improve care.

Multiplex digital PCR with digital melting curve analysis on a self-partitioning SlipChip.

Digital polymerase chain reaction (digital PCR) can provide absolute quantification of target nucleic acids with high sensitivity, excellent precision, and superior resolution. Digital PCR has broad applications in both life science research and clinical molecular diagnostics. However, limited by current fluorescence imaging methods, parallel quantification of multiple target molecules in a single digital PCR remains challenging. Here, we present a multiplex digital PCR method using digital melting curve analysis (digital MCA) with a SlipChip microfluidic system. The self-partitioning SlipChip (sp-SlipChip) can generate an array of nanoliter microdroplets with trackable physical positions using a simple loading-and-slipping operation. A fluorescence imaging adaptor and an in situ thermal cycler can be used to perform digital PCR and digital MCA on the sp-SlipChip. The unique signature melting temperature (Tm) designed for amplification products can be used as a fingerprint to further classify the positive amplification partitions into different subgroups. Amplicons with Tm differences as low as 1.5 degrees celsius were clearly separated, and multiple amplicons in the same partition could also be distinguished by digital MCA. We further demonstrated this digital MCA method with simultaneous digital quantification of five common respiratory pathogens, including Staphylococcus aureus, Acinetobacter baumannii, Streptococcus pneumoniae, Hemophilus influenzae, and Klebsiella pneumoniae. Since digital MCA only requires an intercalation dye instead of sequence-specific hydrolysis probes to perform multiplex digital PCR analysis, it can be less expensive and not limited to the number of fluorescence channels.

1029. Torus Synestia Nucleic Acid Analysis Platform for Fast, High Multiplex Analysis of Nucleic Acids With Single-Nucleotide Discrimination Level

BackgroundNucleic acid amplification testing (NAAT) is an essential tool both for biomedical research and for clinical molecular diagnostics. Currently, there are multiple NAAT platforms available, each offering certain performance and utility advantages and disadvantages as compared to each other. Next generation NAAT platforms aim to deliver increased target detection sensitivity and specificity, low limits of target detection, quantitative high multiplex target capacity, rapid time to results, and simple sample-to-answer workflow.MethodsHere we describe the Torus Synestia System, a NAAT platform capable of rapid, highly multiplexed amplification and detection of both DNA and RNA targets. The platform comprises a small, portable (~ 2kg) amplification and detection device and a disposable single-use cartridge housing a PCR amplification chamber with an integrated label-free microarray for real-time data acquisition and interpretation. The platform offers a 30-min turnaround time with a detection limit of 10 DNA/RNA molecules per assay and single nucleotide discrimination. ResultsWe demonstrate the Synestia System performance and utility with three distinct molecular applications: 1) detection of 20 genetic loci and 30 single nucleotide polymorphisms in human genomic DNA; 2) detection and genotyping of 43 unique bacterial species associated with human urinary tract infections; and 3) detection and profiling human respiratory viral pathogens including SARS-CoV-1/2, seasonal coronaviruses, Influenza A/B, and human respiratory syncytial viruses. In addition, the single-nucleotide specificity of our label-free microarray probes allowed for robust identification and discrimination of newly emerging SARS-CoV-2 lineages, such as B.1.1.7 (a.k.a. UK), B.1.351 (a.k.a. South African), P.1 (a.k.a. Brazilian), and B.1.617 (a.k.a. Indian).ConclusionThe Torus Synestia System has broad applicability in both clinical and research environments. We are confident that the Torus Synestia System will revolutionize syndromic diagnostics at the point of care (PoC) and lead to improved response times during future epidemic and pandemic pathogen outbreaks.Disclosures All Authors: No reported disclosures

Target invasion-triggered signal amplification based on duplex-specific nuclease for selective and sensitive detection of miRNAs

Dysregulation of MicroRNAs (miRNAs) cause various diseases in humans, and developing reliable methods to detect miRNAs is critical for molecular diagnostics and personalized medicine. This study developed a toehold-mediated target invasion combined with duplex-specificity nuclease (DSN)-assisted cyclic signal amplification fluorescent sensor. Herein, we take advantage of toehold-mediated target invasion process to ensure the high selectivity of miRNA determination, coupled with the unique cleavage properties of DSN to improve the sensitivity of the strategy significantly. Throughout the assay, the whole procedure of detection the target let-7a has a limit of detection (LOD) as low as 9.00 fM and an excellent linear range from 1 pM to 100 nM for no more than 60 min. The assay shows reasonable specificity in detecting mismatched miRNAs and can realize single-base discrimination in the let-7 families. Finally, the developed method was applied to detect the miRNAs extracted from human serum. The results were consistent with those based on the quantitative reverse transcription-polymerase chain reaction(qRT-PCR) method, which shows great potential application value in clinical molecular diagnostics and biological research.

P70.14 PRKDC Mutations Recurrently Found in Non-Small Cell Lung Cancer in East Asian Patients

Protein kinase, DNA-activated, catalytic polypeptide (PRKDC) encodes a 465 kDa catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) that plays a pivotal role in the maintenance of genomic stability and is a critical component of DNA double-strand break repair and recombination. Because of the rarity of those mutations, associated clinical features and prognostic significance have not been thoroughly described so far. The aim of this study is to investigate mutations and prognosis of NSCLC harboring PRKDC mutations. A total of 328 patients with non-small-cell lung cancer were recruited between July 2012 and December 2018. The status of PRKDC mutations and other genes were detected by next generation sequencing. PRKDC gene mutation rate was 1.22% (4/328) in non-small cell lung cancer, including D3411N(1 patient), D2724H(1 patient), M4002L(1 patient) and A1634T(1 patient), and median overall survival (OS) for these patients was 14.0 months. Among them, all patients were PRKDC gene with co-occurring mutations. Briefly, patients with (n=2) or without (n=2) co-occurring BCOR mutations had a median OS of 15.5 months and 22.0 months respectively (P=0.64); patients with (n=2) or without (n=2) co-occurring TP53 mutations had a median OS of 12.5 months and 19.0 months respectively (P=0.23); patients with (n=2) or without (n=2) co-occurring ARID1A mutations had a median OS of 17.5 months and 14.0 months respectively (P=0.81); patients with (n=2) or without (n=2) co-occurring KRAS mutations had a median OS of 12.5 months and 19.0 months respectively (P=0.23). BCOR, TP53, ARID1A, KRAS gene accompanied may have less correlation with PRKDC mutation in NSCLC patients. Next generation sequencing provides a simplified strategy and reasonably high detection rate for PRKDC mutation, which suggested application of the strategies into clinical molecular diagnostics.

Clinical Discernment, Bone Marrow, and Molecular Diagnostics Are Equally Important to Solve the Phenotypic Mimicry among Subtypes of Myeloproliferative Neoplasms

The 2016 WHO classification integrates clinical, bone marrow (BM)-morphology, and molecular features to define disease entities. This together with the advancements in molecular detection and standardization of BM features enable an accurate diagnosis of myeloproliferative neoplasms (MPN) in the majority of patients. Diagnostic challenges remain due to phenotypic mimicry of MPN, failing specificity of BM-morphology, and the fact that phenotype-driver mutations, such as JAK2V617F, are not exclusive to a particular MPN, and their absence does not preclude any of these. We present a series of cases to illustrate themes to be considered in complex cases of MPN, such as triple-negative (TN)-MPN or MPN-unclassifiable (MPN-U). Eleven patients labelled as TN-MPN or MPN-U were included. Serum tryptase and NGS were part of a systematic/sequential multidisciplinary evaluation. Results were clustered into four categories based on diagnostic entities and/or how these diagnoses were made: (A) With expanding molecular techniques, BCR-ABL1 and karyotyping should not be missed; (B) systemic mastocytosis is underdiagnosed and often missed; (C) benign non-clonal disorders could mimic MPN; and (D) NGS could prove clonality in some “TN”-MPN cases. The prognostic/therapeutic consequences of an accurate diagnosis are immense. In TN-MPN or MPN-U cases, a multidisciplinary re-evaluation integrating molecular results, BM-morphology, and clinical judgment is crucial.

Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients.

Genetic screening of somatic mutations in circulating free DNA (cfDNA) opens up new opportunities for personalized medicine. In this study, we aim to illustrate the implementation of NGS-based liquid biopsy in clinical practice for the detection of somatic alterations in selected genes. Our work is particularly relevant for the diagnosis and treatment of NSCLC. Beginning in 2020, we implemented the use of Roche’s Avenio ctDNA expanded panel in our diagnostic routine. In this study, we retrospectively review NGS-based clinical genetic tests performed in our laboratory, focusing on key analytical parameters. Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at >0.5% variant allele frequency (VAF), and high consistency in reproducibility. Since 2020, we performed cfDNA genotyping test in 86 NSCLC patients, and we successfully sequenced 96.5% (83/86) of samples. We observed consistency in sequencing performance based upon sequencing depth and on-target rate. At least one gene variant was identified in 52 samples (63%), and one or more actionable variants were detected in 21 out of 83 (25%) of analysed patients. We demonstrated the feasibility of implementing an NGS-based liquid biopsy assay for routine genetic characterization of metastatic NSCLC patients.

SARS-CoV-2 N gene mutations impact detection by clinical molecular diagnostics: reports in two cities in the United States

Nasal and nasopharyngeal swab specimens tested by the Cepheid Xpert Xpress SARS-CoV-2 were analyzed by whole-genome sequencing based on impaired detection of the N2 target. Each viral genome had at least one mutation in the N gene, which likely arose independently in the New York City and Pittsburgh study sites.