Molecular Diagnostic Laboratories Research Articles

Diagnostic Performance of a Tumor Marker Gene Test to Personalize Serum CA19-9 Reference Ranges.

CA19-9 synthesis is influenced by common variants in the fucosyltransferase (FUT) enzymes FUT3 and FUT2. We developed a clinical test to detect FUT variants, and evaluated its diagnostic performance for pancreatic ductal adenocarcinoma (PDAC). A representative set of controls from the Cancer of the Pancreas Screening study was identified for each FUT functional group. Diagnostic sensitivity was determined first in a testing set of 234 PDAC cases, followed by a 134-case validation set, all of whom had undergone resection with curative intent without neoadjuvant therapy. Tumor marker gene testing was performed in the Johns Hopkins Molecular Diagnostics Laboratory. CA19-9 levels were measured in the Hopkins Clinical Chemistry lab. Receiver operating characteristic (ROC) curve analysis was used to evaluate the discriminative ability of CA19-9 alone versus with the gene test. Applying the CA19-9 standard cutoff (<36 U/mL) to all 716 subjects yielded a 68.8% sensitivity in the test set of cases, 67.2% in the validation set, at 91.4% specificity. Applying 99th percentile cutoffs according to each individual's FUT group (3, 34.9, 41.8, and 89.2, for the FUT3-null, FUT-low, FUT-intermediate, and FUT-high groups, respectively) yielded a diagnostic sensitivity for CA19-9 in the first set of cases of 66.7%, 65.7% in the validation set, at 98.9% specificity. ROC analysis for CA19-9 alone yielded an AUC of 0.84; with the tumor marker gene test, AUC improved to 0.92 (P < 0.001). Using a tumor marker gene test to personalize an individual's CA19-9 reference range significantly improves diagnostic accuracy.

Genomic Data Heterogeneity across Molecular Diagnostic Laboratories: A Real-World Connect Myeloid Disease Registry Perspective on Variabilities in Genomic Assay Methodology and Reporting

Genomic data variability from laboratory reports can impact clinical decisions and population-level analyses; however, the extent of this variability and the impact on the data's value are not well characterized. This pilot study used anonymized genetic and genomic test reports from the Connect Myeloid Disease Registry (NCT01688011), a multicenter, prospective, observational cohort study of patients with newly diagnosed myelodysplastic syndromes, acute myeloid leukemia, or idiopathic cytopenia of undetermined significance, to analyze laboratory test variabilities and limitations. Results for 56 randomly selected patients enrolled in the Registry were independently extracted and evaluated (data cutoff, January 2020). Ninety-five reports describing 113 assay results from these 56 patients were analyzed for discrepancies. Almost all assay results [101 (89%)] identified the sequencing technology applied, and 94 (83%) described the test limitations; 95 (84%) described the limits of detection, but none described the limit of blank for detecting false positives. RNA transcript identifiers were not provided for 20 (43%) variants analyzed by next-generation sequencing and reported by the same laboratory. Of 42 variants with variant allele frequencies ≥30%, 16 (38%) of the variants did not have report text indicating that the variants might be germline. Variabilities and lack of standardization present challenges for incorporating this information into clinical care and render data collation ineffective and unreliable for large-scale use in centralized databases for therapeutic discovery.

Çanakkale (Türkiye) balık pazarından alınan üç ekonomik balık türünden izole edilen nematodların moleküler incelenmesi

This study was based on the molecular identification of endoparasites sampled from three economically valuable fish species caught from the waters of the Canakkale (Türkiye). Subjected samples in the study were from chub mackerel (Scomber japonicus Houttuyn, 1782), anchovy (Engraulis encrasicolus Linnaeus, 1758), and bogue (Boops boops Linnaeus, 1758) without gender discrimination. The nematode parasites obtained from the samples were sent to molecular diagnostic laboratories in alcohol and the results were interpreted. As a result of the study, Anisakis typica (Diesing, 1860) Baylis, 1920 and Anisakis pegreffii Campana-Rouget and Biocca, 1955 were isolated from the chub mackerel fish and Anisakis pegreffii species were also isolated from anchovy and bogue. This parasite was the most isolated nematode parasite among all nematodes.

Discovery of gene fusions in driver-negative NCI-MATCH screening samples.

3112 Background: NCI-MATCH aimed to determine the treatment efficacy of identifying actionable genetic alterations, irrespective of cancer histology, and to tailor treatment to the putative molecular drivers of tumors. Leveraging next-generation sequencing (NGS) and ThermoFisher’s Oncomine assay, this phase II trial genotyped 5,540 patient samples during active screening. Reviewing the genotyping statistics, we noted that NCI-MATCH identified fusions in 164 tumors, only 2.96%. This result is substantially lower than expected, given that clinical assays used at MGH’s and Yale’s diagnostic laboratories identified fusions in 8.5% of the 7,500 sequenced cases, 2.5% of which were novel. Notably, the Oncomine assay, based on multiplexed PCR, is unable to detect driver fusion events when either of the gene partners are unknown, or a novel breakpoint is involved. Cross-referencing identified fusions from MGH’s cohort with Oncomine’s primers, 5-30% of these fusions were potentially missed, suggesting that we could detect up to 30% more fusions than originally identified. Developed at MGH’s Molecular Diagnostics Laboratory, Anchored Multiplex PCR (AMP) interrogates 138 known fusion partners, enabling unbiased fusion detection when only one partner is known. With a high likelihood of identifying novel fusion drivers using AMP technology, we sequenced 663 cases that had no identified mutations of interest (MOI) from the NCI-MATCH cohort. Methods: A total of 200ng of RNA per sample was shipped from ECOG-ACRIN’s repository at MD Anderson Cancer Center to MGH (n = 319) and Yale (n = 349). Samples were processed with ArcherDX’s FusionPlex Pan Solid Tumor v2 kit and sequenced on the NextSeq500 with 150 base pair (bp) Paired-End (PE) kits. Data analysis was completed with ArcherDX’s analytics platform in conjunction with custom Python and R code. Reported fusions were manually verified on a case-by-case basis. All statistical tests were conducted in R. Results: Of the cases sequenced, most (663/693, 95.7%) were successfully processed with AMP. We identified 142 cases with at least one gene fusion out of 663 MOI-Negative cases (21%; 95% CI [18%, 25%]), with 90 as unique fusions, significantly more than the 8.5% observed at MGH (P &lt; .001). Of the 142 fusion-positive cases, 36 have potentially actionable driver events, 27 contain novel fusions not previously described, and in 63 cases no drugs are available to target either component of the fusion. Additionally, we identified fusions in (12/34, 35%) Cholangiocarcinoma and (38/101, 37.6%) Sarcoma cases, 9 and 3 of which were potentially actionable respectively. Conclusions: As hypothesized, we identified fusions that the NCI-MATCH screening had missed, detailing sequencing technology improvement that could be broadly applicable to many clinical laboratories sequencing tumors. Clinical trial information: NCT02465060 .

Plasma-based methylated SEPTIN9 circulating tumor DNA to predict progression-free and overall survival in metastatic colorectal cancer.

e15615 Background: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths, as about half of cases develop incurable metastatic disease. Recent studies have shown circulating tumor DNA (ctDNA) markers reflect disease burden and responses to neoadjuvant therapy in metastatic CRC more accurately than carcinoembryonic antigen (CEA). We aimed to determine if a semi-quantitative evaluation of the methylated SEPTIN9 (mSEPT9) ctDNA marker, used in a CRC screening test, is capable of predicting progression free survival (PFS) and overall survival (OS) in a longitudinal cohort of metastatic CRC cases. Methods: A longitudinal cohort of 70 CRC cases who presented with stage IV disease, or experienced recurrent disease, was prospectively assembled from patients undergoing treatment at Cedars-Sinai Medical Center. Blood samples were drawn either prior to, or within 2 weeks following, treatment initiation, and 1-2 monthly thereafter for two years or until death. mSEPT9 ctDNA levels were measured in 1 mL plasma using a modified Epi-proColon V2.0 (Epigenomics) protocol. Univariate and multivariate analyses of clinical endpoints, PFS and OS, were conducted using a Cox regression model with CEA and mSEPT9 ctDNA as time-dependent covariates, with and without adjustments for multiple confounding factors (sex, age, race, organ of metastasis, neo- and adjuvant chemotherapy, immunotherapy, surgery, mismatch repair, KRAS and BRAF mutation status). Results: In univariate analysis, higher level of ctDNA, peritoneal metastasis, gynecologic metastasis, and additional liver metastasis, were associated with poor PFS. Elevated CEA was not associated with PFS either as a continuous variable or as a categorical variable (threshold 10ng/mL). In multivariable analysis, higher level of ctDNA (HR: 1.41; 95% CI: 1.17-1.71; P&lt; 0.001) remained associated with poor PFS after adjusting for adjuvant chemotherapy and peritoneal metastasis. CEA remained not associated with PFS. In univariate analyses, high level of CEA, high level of mSEPT9 ctDNA, neoadjuvant chemotherapy, male sex, and peritoneal metastasis were associated with poor OS. In multivariable analysis, higher level of CEA (HR: 1.63; 95% CI: 1.12-2.36; P= 0.011) and mSEPT9 ctDNA (HR: 1.50; 95% CI: 1.22-1.84; P&lt; 0.001) remained associated with poor OS after adjusting for sex and peritoneal metastasis. Conclusions: Semi-quantification of the single ctDNA marker, mSEPT9, a prevalent biomarker of CRC, was an independent predictor of PFS whereas CEA was not. High levels of both CEA and mSEPT9 ctDNA were independent predictors of OS, showing that mSEPT9 ctDNA added value to CEA in evaluation of OS. The mSEPT9 ctDNA test is rapid turn-around and easy to perform in molecular diagnostic pathology laboratories, thus may serve as a valuable adjunct to CEA for frequent testing in the setting of metastatic CRC.

Abstract A41: Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia

Abstract TP53 mutations in acute myeloid leukemia (AML) are associated with copy number abnormalities (CNA), structural variants and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018; Bernard et al. 2020). In spite of relatively high remission rates obtained by targeted therapies, TP53 mutant (TP53-mut) clones persist, invariably resulting in relapse (Short et al., 2021; Takahashi et al., 2016). Delineating the clonal architecture and the immunophenotypes of TP53-mut clones during AML therapy may provide a better understanding of the role of TP53-mutations in AML biology. Recent progress in sequencing technologies allows the integration of genotyping and phenotyping at the single cell level. Here, we took advantage of MissionBio Tapestri’s newest platform: single cell DNA + protein for simultaneous genotyping and phenotyping with 45 surface oligo-conjugated antibodies in 10 paired samples from 5 patients with TP53-mut AML before and after therapy. Samples with at least 70% viability were stained with the TotalSeq™-D Human Heme Oncology Cocktail, V1.0. Following surface marker staining, single-cell suspension, encapsulation, and barcoding were performed according to manufacturer’s instruction. For scDNA library preparation, we utilized a validated custom panel (Morita et al. 2020) consisting of 279 amplicons covering recurrent mutations in 37 genes in AML. We sequenced a total of 44,550 cells from 10 samples. We confirmed mutations reported by MD Anderson molecular diagnostic laboratory in the genes covered by the scDNA custom panel. The clonal architecture analysis distinguished between TP53-mut clones with or without loss of heterozygosity (LOH) of the normal TP53 allele. Our data show a primitive immunophenotype in TP53-mut with LOH (LOH+) clones in comparison to TP53-mut LOH- clones. We see 2.4 LOG2FC increase in CD34 and 1.8 LOG2FC increase in CD117 (p&amp;lt;0.001) in TP53-mut LOH+ clones in comparison to TP53-mut LOH- clones. Clonal evolution analysis shows that TP53-mut LOH+ clones are significantly more resistant to therapy than TP53-mut LOH-, consistent with previous publications. On the other hand, TP53-mut LOH- clones showed significantly higher levels of CD2, CD16, CD5, CD3, and CD8 among other lineage markers (LOG2FC= 1.1, 1.2, 1.4, 1.1, and 1.1 respectively; p.value &amp;lt;0.0001) compared to TP53-mut LOH+. This data indicates that TP53-mut LOH- cells can express lymphoid phenotypic markers. Single cell cytokine analysis (IsoPlexis) reveals profound lack of secreted cytokines in T-cells from TP53-mut AML. Further data from bulk-RNA sequencing, ddPCR, and CyTOF will be presented that validates a lymphoid phenotype of TP53-mut LOH-. In summary, we utilize a scDNA+protein multiomic approach to dissect clonal architecture and provide a link between genotype-phenotype in TP53-mut AML. We show that while TP53-mut LOH+ clones are exclusively primitive, TP53-mut LOH- clones retain the capacity to exist outside primitive immunophenotype and might lack differentiation block. Citation Format: Edward Ayoub, Vakul Mohanty, Yuki Nishida, Tallie Patsilevas, Mahesh Basyal, Russell Pourebrahim, Muharrem Muftuoglu, Ken Chen, Ghayas C. Issa, Michael Andreeff. Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A41.

MOLECULAR DIAGNOSTIC LABORATORY SETUP AND MAINTENANCE FOR SARS-COV-2

Abstract&#x0D; Aim: Importance of laboratory diagnosis has come to the spotlight once again with the Covid-19 pandemic caused by Sars-CoV-2 and significant changes have taken place in terms of laboratory operation. A global effort has emerged when all healthcare professionals faced a biological threat. Interlaboratory collaboration and multidisciplinary approach contributed to this effort. This study aims to explain step-by-step establishment of a fully capable laboratory for Sars-CoV-2 diagnosis to support local and global fight for the COVID-19 pandemic.&#x0D; Methods: Several precautions were taken, and disaster plans were updated because of the changes in employee health and workload distribution. Some of these are setting up a laboratory from scratch for microorganism diagnostic tests performed in pandemic cases, measures for healthcare workers, personnel assignment planning, changes in the variety and number of tests, innovations in quality standards and the contribution of laboratories to scientific studies.&#x0D; Results: XXXXX Molecular Diagnostic Laboratory has become one of the laboratories in Türkiye where Sars-Cov-2 and its mutations have been studied the most with 1,710,856 samples between 01 October 2020 and 01 May 2022 since its establishment and it has become the laboratory with the highest number of equipment and technical personnel in the capital.&#x0D; Conclusion: This study summarizes all the phases of XXXXX Molecular Diagnostic Laboratory beginning with its establishment from the scratch and covers all the steps to render this facility fully operational.&#x0D; &#x0D; Keywords: COVID-19, laboratory disaster planning, laboratory staff, personal protective equipment, Sars-CoV-2

Evaluating the Transition from Targeted to Exome Sequencing: A Guide for Clinical Laboratories.

The transition from targeted to exome or genome sequencing in clinical contexts requires quality standards, such as targeted sequencing, in order to be fully adopted. However, no clear recommendations or methodology have emerged for evaluating this technological evolution. We developed a structured method based on four run-specific sequencing metrics and seven sample-specific sequencing metrics for evaluating the performance of exome sequencing strategies to replace targeted strategies. The indicators include quality metrics and coverage performance on gene panels and OMIM morbid genes. We applied this general strategy to three different exome kits and compared them with a myopathy-targeted sequencing method. After having achieved 80 million reads, all-tested exome kits generated data suitable for clinical diagnosis. However, significant differences in the coverage and PCR duplicates were observed between the kits. These are two main criteria to consider for the initial implementation with high-quality assurance. This study aims to assist molecular diagnostic laboratories in adopting and evaluating exome sequencing kits in a diagnostic context compared to the strategy used previously. A similar strategy could be used to implement whole-genome sequencing for diagnostic purposes.

The COVID-19 laboratory response in Timor-Leste; a story of collaboration.

Timor-Leste is a small nation of 1.3 million people which shares a land border with Indonesia and is 550km from Darwin, Australia. It is one of the poorest nations in Asia. The National Health Laboratory (NHL) and its network of smaller laboratories in Timor-Leste had limited capacity to perform molecular diagnostic testing before the coronavirus disease 2019 (COVID-19) pandemic began. With the support of international development partners, the NHL rapidly expanded its molecular testing service. From March 2020 to February 2022, over 200,000 molecular tests were performed; COVID-19 testing sites were established in hospital and community health center laboratories and all 13 municipalities, and the number of scientists and technicians at the molecular diagnostic laboratory at the NHL increased from five to 28 between 2019 and2022. Molecular diagnostic testing for COVID-19 was successfully established at the NHL and in the municipalities. The molecular diagnostic laboratory at NHL is now equipped to respond to not only large-scale COVID-19 testing but also laboratory detection of other infectious diseases, preparing Timor-Leste for future outbreaks or pandemics.

Impact of Molecular Testing Using Next-Generation Sequencing in the Clinical Management of Patients with Non-Small Cell Lung Cancer in a Public Healthcare Hospital

Next-generation sequencing (NGS) is a molecular approach able to provide a comprehensive molecular profile of non-small cell lung cancer (NSCLC). The broad spectrum of biomarker-guided therapies has positioned molecular diagnostic laboratories as a central component of patient clinical management. Here, we show the results of an UNE-EN ISO 15189:2022 NGS-accredited assay in a cohort of 350 patients. TP53 (51.0%), KRAS (26.6%) and EGFR (12.9%) were the most frequently mutated genes. Furthermore, we detected co-occurring and mutually exclusive alterations, as well as distinct molecular profiles according to sex and smoking habits. Actionable genetic alterations were significantly more frequent in female patients (80.5%, p < 0.001) and in never-smoker patients (87.7%, p < 0.001). When NGS was established as the main molecular testing strategy, 36.4% of patients received at least one line of targeted treatment. Among 200 patients with stage IV NSCLC, first-line treatment with targeted therapies was associated with a longer progression-free survival (PFS) (13.4 months (95% CI, 10.2-16.6) (p = 0.001)). Similarly, the overall survival (OS) of patients receiving at least one targeted drug was significantly longer (26.2 months (95% CI, 11.8-40.5) (p < 0.001)). Our results show that the implementation of NGS in the public healthcare system has provided a broader application of precision medicine.

Evaluation of the aggressive variant prostate cancer molecular profile (AVPCm) in CLIA environments.

250 Background: 20-30% of prostate cancers respond poorly to androgen receptor (AR)-directed therapies and have an atypical, virulent course. The aggressive variant prostate cancers ( AVPC) encompass virulent androgen indifferent tumors and are characterized by combined defects in ≥ 2/3 of TP53, RB1, and PTEN (AVPC-m). Post-hoc analysis in a phase I/II trial indicated a benefit of carboplatin+cabazitaxel in men with AVPCm+ metastatic castrate resistant prostate cancer (mCRPC). We assess the feasibility of detecting the AVPCm in CLIA-certified environments. Methods: Men with progression of mCRPC undergoing biopsies for standard molecular profiling at MD Anderson Cancer Center (MDACC) were eligible. TP53, RB1, and PTEN expression was assessed by immunohistochemistry (IHC) and read by 2 pathologists independently. Next generation sequencing of solid tumor DNA (stDNA) and circulating tumor DNA (ctDNA) was performed on the MDACC Molecular Diagnostics Laboratory and the University of Washington OncoPlex ctDNA platforms respectively . Results: Of 64 eligible patients, 49 (77%), 37 (58%), and 54 (84%) had results reported for IHC, stDNA, and ctDNA respectively. Concordance of IHC reads between pathologists was variable: Kappa 0.78, 0.67, and 0.91 for TP53, RB1, and PTEN respectively. 36 (66.7%) ctDNA samples had low tumor content, such that false negatives could not be excluded. AVPC-m was detected in 14 (29%) of patients by IHC per the original reading pathologist, 2 (5%) by stDNA NGS, and 7 (39%) by ctDNA NGS. Median turnaround times for IHC, stDNA and ctDNA were 12 (2-35), 28 (12-56) and 16 (11-56) days respectively. Conclusions: We show the operational characteristics of the AVPCm detection using CLIA certified assays. IHC detected a greater proportion of patients with AVPCm+ tumors than stDNA, likely due, partly, to the challenges of copy number loss detection. ctDNA detected the greatest proportion of AVPCm+ tumors but was not evaluable in two thirds of patients. These data serve to inform the design of clinical trials using AVPCm as a criterion for patient selection or stratification. [Table: see text]

PipeIT2: A tumor-only somatic variant calling workflow for molecular diagnostic Ion Torrent sequencing data

Precision oncology relies on the accurate identification of somatic mutations in cancer patients. While the sequencing of the tumoral tissue is frequently part of routine clinical care, the healthy counterparts are rarely sequenced. We previously published PipeIT, a somatic variant calling workflow specific for Ion Torrent sequencing data enclosed in a Singularity container. PipeIT combines user-friendly execution, reproducibility and reliable mutation identification, but relies on matched germline sequencing data to exclude germline variants. Expanding on the original PipeIT, here we describe PipeIT2 to address the clinical need to define somatic mutations in the absence of germline control. We show that PipeIT2 achieves a > 95% recall for variants with variant allele fraction >10%, reliably detects driver and actionable mutations and filters out most of the germline mutations and sequencing artifacts. With its performance, reproducibility, and ease of execution, PipeIT2 is a valuable addition to molecular diagnostics laboratories.

Accreditation of a molecular HIV diagnostic laboratory following the Strengthening Laboratory Management Towards Accreditation (SLMTA)-Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) approach in Kenya: an implementation science study.

accreditation is the most effective approach to ensure the quality of services. Laboratory performance can be evaluated using the World Health Organization (WHO)-SLIPTA checklist, which checks a laboratory´s compliance with ISO 15189 on a five-star score scale and improved using the SLMTA approach. Compliance is assessed by an external body and can result in accreditation. In this paper, we describe the steps taken by the Kenya Medical Research Institute (KEMRI) HIV Laboratory, Alupe, a resource-limited public entity, towards accreditation, and discuss the lessons learned. the laboratory adopted a SLMTA-SLIPTA approach that included targeted mentorship, on-site workshops, and training. Mentorship-based interventions were used to establish a robust quality management system. Targeted mentorship, on-site workshops, and training were conducted between September 2015 and July 2016. Audits used the SLIPTA checklist to detect gaps in 12 quality system essentials. Performance indicators including turnaround time, external quality assurance, sample rejection rates, and corrective actions were tracked. An external assessment by the national accreditation body was conducted between September 2016 and November 2016. training and mentorship-based interventions were successfully conducted. Quality management systems aligned with ISO 15189 were established. Baseline, midterm, and exit audits yielded scores of 47%, 75%, and 94% respectively. Early infant diagnosis external quality assurance scores were 100% in 2014-2016, while average viral load scores were at 60%, 70% and 90% during the same period. Turnaround time from September 2015 surpassed the 80% target. Accreditation was awarded in March 2017. the SLMTA-SLIPTA approach is suitable for quality improvement in resource-limited laboratories.

Nucleic Acid-Based Detection of Pythium insidiosum: A Systematic Review.

Pythiosis, a life-threatening infectious condition caused by Pythium insidiosum, has been increasingly reported in humans and animals worldwide. Antifungal drugs usually fail to control the pathogen. The surgical removal of an infected organ is the treatment of choice. Many affected patients die due to advanced infection. A timely and accurate diagnosis could lead to a better prognosis in pythiosis patients and save their lives. Although a standard culture method is available in microbiological laboratories, it is time-consuming, laborious, and insensitive for P. insidiosum identification. Immunological assays have been developed to improve the diagnosis of pythiosis. However, immunological methods are commercially unavailable and primarily detect anti-P. insidiosum antibodies, which constitute indirect evidence of pythiosis, making it challenging to differentiate a past from a recent infection. Moreover, such immunological tests cannot diagnose patients with a local infection, such as in the eye. Nucleic acid-based tests (NATs) are efficient for the direct and rapid detection of P. insidiosum DNA in trace-amount or culture-negative specimens. The reagents and equipment required for NATs are usually available in molecular diagnostic laboratories. Herein, we provide a systematic review to comprehensively present the principal and clinical usages, advantages, and limitations of such NATs in the detection of P. insidiosum. Various NATs have been established to detect P. insidiosum, which can be classified into amplification-based (i.e., PCR assays, isothermal tests, and next-generation sequencing methods) and non-amplification-based (i.e., DNA hybridization) techniques. This concise review on NATs constitutes an up-to-date reference with which healthcare professionals can learn about and decide upon which detection method is suitable for their respective laboratory environments.

Pethema NGS-AML Project. Final Analysis and Clinical Validation of New Genomic Classifications

Introduction Next-Generation Sequencing (NGS) implementation to perform accurate diagnosis in acute myeloid leukemia (AML) represents a major challenge for molecular diagnostic laboratories in terms of the specialization, cost and logistical support. The PETHEMA group established a diagnostic network of 7 reference laboratories to provide standardized NGS studies for AML patients, performing cross-validation (CV) rounds to ensure the quality of the studies and regular updating of gene panels. We report the CV rounds of the diagnostic platform and the clinical validation of the new genomic classification which proposes unified framework for disease classification and risk-stratification in AML based on cytogenetic and 32 genes (Tazi et al. 2022). Methods CV rounds were performed by exchanging control samples among reference laboratories every 9-12 months. In the last CV round, four control samples harboring 34 variants were distributed (21 variants with VAF>5% and 13 with VAF<5%). NGS analyses were performed in 1631 samples collected from 1471 patients enrolled in NGS-AML project (NCT03311815). Genomic classification and risk stratification was validated in 954 patients treated with intensive (n=568) or non-intensive therapy (n=386) with available clinical, cytogenetic and molecular data. An additional cohort of 1225 samples was included for molecular analysis. Results The error rate (ER) in variants with VAF>5% was decreasing in the successive CV rounds (1st: 39%, 2nd: 14.4% and 3rd: 4.76%). However, in variants with VAF <5%, ER was raised (2nd: 28.6% and 3st: 59.62%). Consequently, the diagnostic platform decided to maintain the cut-off of VAF>5% to report clinically relevant variants. In the global cohort, 7768 variants were detected. 96.5% of samples showed at least one mutation, with a mean of 3.1 mutations/sample (range 0-13). FLT3 (24.6%), DNMT3A (24.3%), NPM1 (22.4%) and TET2 (21.6%) were the most frequently mutated genes. Our series also reflected different mutational profile according to disease status (DNMT3A, IDH1, KRAS, NPM1, NRAS, RUNX1 and TP53), age (ASXL1, DNMT3A, EZH2, FLT3, IDH2, NPM1, RUNX1, SF3B1, SRSF2, TP53 and U2AF1) and sex (ASXL1, DNMT3A, EZH2, FLT3, NPM1, RUNX1, SRSF2, and U2AF1); and revealed co-occurring mutations and exclusivity gene patterns (Table 1). According to genomic classification, the PETHEMA cohort was categorized in 16 molecular classes which encompass established WHO2016 entities (33.5%) [inv(16), t(8;21), NPM1, CEBPAbi, t(11;x), t(6;9) and inv(3)] and novel entities: "sAML1" (8.7%) [Single mutation in: SRSF2, SF3B1, U2AF1, ASXL1, EZH2, RUNX1 and SETBP1], "TP53/complex karyotype" (19.2%), "sAML2" (25.4%) [More than one mutated sAML1 gene], "driver mutations but no defining class" (8.5%), "DNMT3A/IDH1-2" (1.8%), "WT1" (1.4%), trisomies (0.73%) and "No events" (0.94%). These molecular subgroups have also been associated with different prognostic values: "TP53/Complex karyotype" [4.2 months (95%CI 2.9-5.5)], inv(3) [4.9 months (95%CI 0.8-9.1)] and "sAML2" [11.87 months (95%CI 9.7-14.0)] showed the lower values of OS, while patients classified in the "No events" and "inv(16)" (Median OS not reached at 33 and 42 months) and "NPM1" [28.97 months (95%CI 19.9-38.0)] had the best outcomes. Figure 1. Integrated risk score based on cytogenetic and 32 genes was assessed in the PETHEMA "real-life" cohort: 23.5% of patients were classified in the favorable risk group [OS 29.23 months (95%CI 18.40-40.06)], 27.5% in the intermediate [18.77 months (95%CI 14.62-22.92)] and 49.1% in the adverse risk group [9.37 months (95%CI 7.76-10.98)]; (P<0.001). HR are shown in figure 1. Finally, we identified a dismal prognosis and higher risk of death in older patients compared to younger ones for all risk groups: Favorable: 4.5 (95%CI 2.68-7.56; P<0.001), intermediate: 1.93 (95%CI 1.33-2.79; P<0.01) and adverse: 2.63 (95%CI 2.01-3.46; P<0.001). Conclusions We show the experience of the PETHEMA diagnostic network in standardized NGS studies in terms of quality metrics and variant reporting criteria. Based on the current landscape of AML, these strategies ensure technical quality and equity in access to NGS studies. Furthermore, this cooperative work allows the assessment of new molecular subgroups in "real-life" cohorts as well as the application of the novel risk classifications. Partially funded by BMS/Celgene. PI18/01340, PI19/00730, FI19/00059 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Thermal Shock Viral RNA Release in COVID-19 Testing

Abstract Introduction/Objective The public health emergency of the COVID-19 pandemic emphasized the crucial role of medical laboratory professionals and scientists in molecular diagnostics laboratories to ensure success in infection control strategies. The demand for laboratory testing using nucleic acid amplification tests to detect SARS-CoV-2 RNA imposed strains in laboratory supplies. Here, we explored an alternative cost-effective solution that will simplify the pre-PCR steps by using a simple heating method to release viral RNA. Methods/Case Report Samples tested using the reference automated extraction method were used:100 samples identified as positive for SARS-CoV-2 RNA and 500 samples tested negative for SARS-CoV-2 RNA were used for the study and sorted with equal distribution according to Ct values of (a) &amp;lt;20, (b) 20–30, and (c) &amp;gt;30.100 ul from swab preserved in Universal Transport Medium was treated with 30 μg of proteinase K, and another set was tested without proteinase K pre-treatment. All samples with or without proteinase K were diluted to minimize PCR inhibitors. The thermal shock protocol was set at (98°C, 5 minutes; 4°C, 2 minutes) and screened for purity. Performance and method verification studies were performed. Internal extraction, positive template, and no template controls were markers used for testing quality. The experimental study was performed by qualified testing personnel and all under the same experimental conditions. Results (if a Case Study enter NA) The Ct values from the thermal shock RNA release were compared to the automated extraction method and statistically analyzed.The criteria for acceptability for validation of this new RNA extraction proceeding were set to 100% concordance compared to the commercial kit using an automated extraction. PCR efficiency was at 98% and a slope of -3.3. Within run precision of 2% and limits of detection from 200 to 20,000 copies/uL The method 100% (50/50) concordance on samples previously identified as negative by automated methods and identified 86% (86/100) with a mean difference of 3 Ct. Conclusion Our findings suggest that the thermal shock treatment of nasopharyngeal swabs in viral transport media can successfully extract viral nucleic acid for nucleic acid amplification and is a reasonable alternative for chemical extraction methods when molecular diagnostic laboratories persistently encounter supply chain issues.

Usage of DNA Fingerprinting Technology to Check Sample Error and Contamination in Molecular Laboratories.

Sample identification error is a severe medical error in clinical molecular diagnostic laboratories, which can lead to reporting the wrong results for the patient involved. Sample contamination can also lead to incorrect test reports. Avoiding sample identification error and sample contamination could be life-saving. Sample switch and sample contamination could happen on laboratory bench works, especially when pipetting into multi-well plates. It is difficult to realize such errors during laboratory bench work. Laboratory staff may not be aware of such an error when it happens. DNA fingerprinting technology can be used to determine sample identity and subsequently identify sample switch and sample contamination in the laboratory. Our laboratory has explored the usage of this technology in our quality control process and successfully established that DNA fingerprinting can be used to monitor sample switch and sample contamination in next-generation sequencing and BCR/ABL1 real-time PCR bench work.

The Relationship Between Population-Level SARS-CoV-2 Cycle Threshold Values and Trend of COVID-19 Infection: Longitudinal Study.

The distribution of population-level real-time reverse transcription-polymerase chain reaction (RT-PCR) cycle threshold (Ct) values as a proxy of viral load may be a useful indicator for predicting COVID-19 dynamics. The aim of this study was to determine the relationship between the daily trend of average Ct values and COVID-19 dynamics, calculated as the daily number of hospitalized patients with COVID-19, daily number of new positive tests, daily number of COVID-19 deaths, and number of hospitalized patients with COVID-19 by age. We further sought to determine the lag between these data series. The samples included in this study were collected from March 21, 2021, to December 1, 2021. Daily Ct values of all patients who were referred to the Molecular Diagnostic Laboratory of Iran University of Medical Sciences in Tehran, Iran, for RT-PCR tests were recorded. The daily number of positive tests and the number of hospitalized patients by age group were extracted from the COVID-19 patient information registration system in Tehran province, Iran. An autoregressive integrated moving average (ARIMA) model was constructed for the time series of variables. Cross-correlation analysis was then performed to determine the best lag and correlations between the average daily Ct value and other COVID-19 dynamics-related variables. Finally, the best-selected lag of Ct identified through cross-correlation was incorporated as a covariate into the autoregressive integrated moving average with exogenous variables (ARIMAX) model to calculate the coefficients. Daily average Ct values showed a significant negative correlation (23-day time delay) with the daily number of newly hospitalized patients (P=.02), 30-day time delay with the daily number of new positive tests (P=.02), and daily number of COVID-19 deaths (P=.02). The daily average Ct value with a 30-day delay could impact the daily number of positive tests for COVID-19 (β=-16.87, P<.001) and the daily number of deaths from COVID-19 (β=-1.52, P=.03). There was a significant association between Ct lag (23 days) and the number of COVID-19 hospitalizations (β=-24.12, P=.005). Cross-correlation analysis showed significant time delays in the average Ct values and daily hospitalized patients between 18-59 years (23-day time delay, P=.02) and in patients over 60 years old (23-day time delay, P<.001). No statistically significant relation was detected in the number of daily hospitalized patients under 5 years old (9-day time delay, P=.27) and aged 5-17 years (13-day time delay, P=.39). It is important for surveillance of COVID-19 to find a good indicator that can predict epidemic surges in the community. Our results suggest that the average daily Ct value with a 30-day delay can predict increases in the number of positive confirmed COVID-19 cases, which may be a useful indicator for the health system.

27. Review and comparison of the Oncomine Myeloid Assay GX v2 on Genexus System

Rapid molecular results for FLT3, IDH1/2 and NPM1 are necessary for treatment decisions of newly diagnosed acute leukemia patients. However, as treatment protocols and clinical trials request more gene variants with a faster turnaround time, single gene STAT testing using polymerase chain reaction-based testing becomes more cumbersome for the molecular diagnostic laboratory. Therefore, a rapid next generation sequencing panel for acute leukemias is necessary for laboratory operations and, more importantly, clinical utility. Cedars-Sinai molecular pathology laboratory validated the automated Oncomine? Myeloid Assay GX v2 panel using the Ion Torrent? Genexus? Integrated Sequencer (v.6.6) for a 24-hour turnaround time from DNA and RNA purification to variant reporting. We compared the results from Oncomine? Myeloid Assay GX v2 panel to Archer® VariantPlex® Myeloid panel on Illumina-based platforms. The differences between the two systems include technician intervention, turnaround time, variant accuracy, and the usability of the software package. While Archer® VariantPlex® Myeloid panel demonstrates unrivaled accuracy and reliability, the ThermoFisher Myeloid Assay GX v2 using the Genexus (IonTorrent sequencing) offers quick results and fusion detection. Therefore, we have chosen to implement both systems as standard clinical diagnostics for new acute leukemia patients.

Nationwide molecular survey of Dirofilaria immitis and Dirofilaria repens in companion dogs and cats, United States of America

BackgroundHeartworms, Dirofilaria immitis, are known to be widespread in dogs and cats in the USA, but there have been no country-wide prevalence studies performed to date. There have also been no large-scale studies to determine whether the closely related species, Dirofilaria repens, occurs in the USA.MethodsTo provide this large-scale data, we examined whole blood samples (n = 2334) submitted from around the USA to the Molecular Diagnostic Laboratory at Auburn University between 2016 and 2022. Quantitative PCRs for D. immitis (targeting 16S rRNA) and D. repens (targeting cytochrome c oxidase subunit 1 gene) were performed to determine the presence of Dirofilaria DNA. DNA sequencing was performed to confirm the results.ResultsDirofilaria immitis DNA was found in 6.3% (68/1080) of the dogs from 17/39 states, and 0.3% (4/1254) of the cats from 4/42 states. None of the dogs or cats were positive for D. repens. The average 16S rRNA copy number of D. immitis in the dogs was 1,809,604 in 200 µl whole blood, while only a single copy was found in each of the four D. immitis-positive cats. The prevalence of D. immitis in dogs of different ages, sexes, and breeds did not differ significantly, but the prevalence in Southern states (7.5%, 60/803) was significantly higher than in the Western (1.7%, 1/58), Midwest (3.3%, 4/120), and Northeastern states (3.1%, 3/98) (P < 0.05). Dogs positive for D. immitis were identified in each study year (2016: 4.2%, 2/48; 2017: 9.8%, 4/41; 2018: 5.1%, 8/156; 2019: 4.9%, 15/306; 2020: 9.8%, 26/265; 2021: 4.9%, 13/264). Interestingly, dogs infected with Hepatozoon spp. (11.8%, 37/313) were significantly more likely to also be positive for D. immitis than dogs without evidence of Hepatozoon infection (3.9%, 30/760) (P < 0.0001).ConclusionsTo our knowledge, this is the first nationwide molecular survey of Dirofilaria spp. in dogs and cats in the USA, and the largest molecular survey of canine and feline dirofilariosis worldwide. Further studies are warranted to combine PCR with standard heartworm diagnostics to better understand the prevalence of Dirofilaria spp. and aid in determining the risks posed to dogs and cats in the USA.Graphical