Molecular Diagnostic Laboratories Research Articles

Role of Quality Management System in Setting up and Sustaining a Molecular Diagnostic Laboratory during COVID-19 Pandemic

Introduction: Coronavirus Disease-2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV-2) catapulted the need to build, upgrade and expand the number of diagnostic laboratories having molecular capacity. Setting up and sustaining a molecular laboratory especially in the backdrop of a lockdown presented many challenges. The Department of Microbiology, in a tertiary level hospital in Mumbai was one of the first to start the molecular testing laboratory. All other tests performed in the department are accredited as per International Organisation for Standardisation (ISO) 15189:2012 since 2015. However, starting a molecular diagnostic facility for COVID-19 testing presented a unique set of challenges as the organism in question belonged to risk category 2 and had the potential for airborne transmission. Aim: To determine the challenges faced and activities undertaken especially with regards to the role of Quality Management System (QMS) in setting up and sustaining a molecular diagnostic facility during COVID-19 pandemic. Materials and Methods: A retrospective analysis was carried out of experiences and data generated from March 2020 to April 2021 at the Microbiology Department of a tertiary level medical college and hospital in Mumbai, Maharashtra, India. The article included the processes which required and data generated during setting up and sustaining a new molecular testing facility as per the QMS with special reference to the 12 Quality System Essentials (QSE). Quality Indicators (QI) were identified, objectives defined and monitored over the period of the study. It was a descriptive study and statistical analysis was not indicated. Results: All the objectives of the QI were met with. Only 4% staff needed corrective training. Specimen rejection rate pretest and post-test was 0.26% and 0.56%, respectively. Quality control failure was seen in 0.16% runs and Turnaround Time (TAT) deviated beyond 12 hours in 0.52% samples. The run contamination, equipment problems and laboratory associated infections were 0.08%, 0.56% and 0%, respectively. There were no External Quality Assessment (EQAS) failure and negative feedback. Laboratory contamination rate was 1.02%. Definite improvement was observed over time in all identified parameters. Conclusion: Implementation of QMS with specific reference to strengthening QSE is a necessary requirement for achieving quality standards.

PipeIT2: Somatic Variant Calling Workflow for Ion Torrent Sequencing Data.

The detection of somatic genetic variants forms the cornerstone of precision oncology. Molecular diagnostics laboratories require economical and rapid protocols to generate accurate variant calls for clinical purposes. The Ion Torrent sequencing platform is frequently used but lacks robust analysis workflows tailored to the needs of custom sequencing panels. We present PipeIT2, a somatic variant calling workflow for both matched tumor-germline and tumor-only analyses. PipeIT2 is enclosed in a Singularity container to ensure reproducibility and ease of deployment.

Immunoglobulin/T Cell Receptor Capture Strategy for Comprehensive Immunogenetics.

In the era of genomic medicine, targeted next generation sequencing strategies (NGS)are becoming increasingly adopted by clinical molecular diagnostic laboratories to identify genetic diagnostic and prognostic biomarkers in hemato-oncology. We describe the EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay, which is designed to simultaneously detect B and T cell clonal rearrangements, translocations, copy number alterations, and sequence variants. The accompanying validated bioinformatics pipeline enables production of an integrated report. The combination of the laboratory protocol and bioinformatics pipeline in the EuroClonality-NDC minimizes the potential for human error, reduces economic costs compared to current molecular testing strategies, and should improve diagnostic outcomes.

A method for campus-wide SARS-CoV-2 surveillance at a large public university.

The systematic screening of asymptomatic and pre-symptomatic individuals is a powerful tool for controlling community transmission of infectious disease on college campuses. Faced with a paucity of testing in the beginning of the COVID-19 pandemic, many universities developed molecular diagnostic laboratories focused on SARS-CoV-2 diagnostic testing on campus and in their broader communities. We established the UC Santa Cruz Molecular Diagnostic Lab in early April 2020 and began testing clinical samples just five weeks later. Using a clinically-validated laboratory developed test (LDT) that avoided supply chain constraints, an automated sample pooling and processing workflow, and a custom laboratory information management system (LIMS), we expanded testing from a handful of clinical samples per day to thousands per day with the testing capacity to screen our entire campus population twice per week. In this report we describe the technical, logistical, and regulatory processes that enabled our pop-up lab to scale testing and reporting capacity to thousands of tests per day.

A clinical laboratory–developed LSC17 stemness score assay for rapid risk assessment of patients with acute myeloid leukemia

AbstractLeukemia stem cells (LSCs) are linked to relapse in acute myeloid leukemia (AML). The LSC17 gene expression score robustly captures LSC stemness properties in AML and can be used to predict survival outcomes and response to therapy, enabling risk-adapted, upfront treatment approaches. The LSC17 score was developed and validated in a research setting. To enable widespread use of the LSC17 score in clinical decision making, we established a laboratory-developed test (LDT) for the LSC17 score that can be deployed broadly in clinical molecular diagnostic laboratories. We extensively validated the LSC17 LDT in a College of American Pathologists/Clinical Laboratory Improvements Act (CAP/CLIA)-certified laboratory, determining specimen requirements, a synthetic control, and performance parameters for the assay. Importantly, we correlated values from the LSC17 LDT to clinical outcome in a reference cohort of patients with AML, establishing a median assay value that can be used for clinical risk stratification of individual patients with newly diagnosed AML. The assay was established in a second independent CAP/CLIA-certified laboratory, and its technical performance was validated using an independent cohort of patient samples, demonstrating that the LSC17 LDT can be readily implemented in other settings. This study enables the clinical use of the LSC17 score for upfront risk-adapted management of patients with AML.

Effects of acute severe acute respiratory syndrome coronavirus 2 infection on male hormone profile, ACE2 and TMPRSS2 expression, and potential for transmission of severe acute respiratory syndrome coronavirus 2 in semen of Asian men

ObjectiveTo confirm if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen of men with acute coronavirus disease 2019 and if their male hormone profile (testosterone, follicle-stimulating hormone, luteinizing hormone, sex hormone binding globulin, and free androgen index) is adversely affected during the acute phase of infection and any relation to the ACE2 and/or TMPRSS2 expression in human semen.DesignClinical study.SettingNational University Hospital, Singapore.Patient(s)Asian men aged 21–55 years who were admitted to National University Hospital, Singapore, with a laboratory-confirmed diagnosis of SARS-CoV-2 infection via nasopharyngeal swab in the acute phase of the infection, within 2–14 days of the development of symptoms or contact history, were recruited for the study.Intervention(s)Blood was collected in the morning to assess the male hormone profile. Human semen were obtained by masturbation and sent to the molecular diagnostic laboratories to detect the presence of SARS-CoV-2 RNA and assess the ACE2 and TMPRSS2 expression.Main Outcome Measure(s)Male hormone profile level and expression of SARS-CoV-2 RNA, ACE2, and TMPRSS2 in human semen.Result(s)A total of 63 men of Asian ethnicities agreed to participate in the study. Subsequently, 65% of recruited men had completely normal levels of male hormone profile. Moreover, 27% were noted to have higher luteinizing hormone levels between 6.6 and 16.1 IU/L (normal range, 0.8–6.1 IU/L), and 10% had higher follicle-stimulating hormone levels between 13.6 and 41.6 IU/L (normal range, 1.5–12.4 IU/L); all had normal testosterone levels. No SARS-CoV-2 RNAs were detected in all human semen. The ACE2 and TMPRSS2 expression was undetectable in 26 samples, whereas 23 samples only had a detectable TMPRSS2 expression and 4 only had an ACE2 expression. The remaining 3 expressed both ACE2 and TMPRSS2.Conclusion(s)Severe acute respiratory syndrome coronavirus 2 could not be found in the semen of a cohort of young to middle-aged Asian men with mild acute SARS-CoV-2 infection. However, there was a detectable expression of ACE2 and TMPRSS2 in semen, although not causal, and it may be correlated to changes in male hormone profiles and male age.

Genetic Landscape of Myeloproliferative Neoplasms with an Emphasis on Molecular Diagnostic Laboratory Testing.

Chronic myeloproliferative neoplasms (MPNs) are hematopoietic stem cell neoplasms with driver events including the BCR-ABL1 translocation leading to a diagnosis of chronic myeloid leukemia (CML), or somatic mutations in JAK2, CALR, or MPL resulting in Philadelphia-chromosome-negative MPNs with constitutive activation of the JAK-STAT signaling pathway. In the Philadelphia-chromosome-negative MPNs, modern sequencing panels have identified a vast molecular landscape including additional mutations in genes involved in splicing, signal transduction, DNA methylation, and chromatin modification such as ASXL1, SF3B1, SRSF2, and U2AF1. These additional mutations often influence prognosis in MPNs and therefore are increasingly important for risk stratification. This review focuses on the molecular alterations within the WHO classification of MPNs and laboratory testing used for diagnosis.

Development and Implementation of In-House Pharmacogenomic Testing Program at a Major Academic Health System.

Pharmacogenomics (PGx) studies how a person’s genes affect the response to medications and is quickly becoming a significant part of precision medicine. The clinical application of PGx principles has consistently been cited as a major opportunity for improving therapeutic outcomes. Several recent studies have demonstrated that most individuals (> 90%) harbor PGx variants that would be clinically actionable if prescribed a medication relevant to that gene. In multiple well-conducted studies, the results of PGx testing have been shown to guide therapy choice and dosing modifications which improve treatment efficacy and reduce the incidence of adverse drug reactions (ADRs). Although the value of PGx testing is evident, its successful implementation in a clinical setting presents a number of challenges to molecular diagnostic laboratories, healthcare systems, providers and patients. Different molecular methods can be applied to identify PGx variants and the design of the assay is therefore extremely important. Once the genotyping results are available the biggest technical challenge lies in turning this complex genetic information into phenotypes and actionable recommendations that a busy clinician can effectively utilize to provide better medical care, in a cost-effective, efficient and reliable manner. In this paper we describe a successful and highly collaborative implementation of the PGx testing program at the University of Minnesota and MHealth Fairview Molecular Diagnostic Laboratory and selected Pharmacies and Clinics. We offer detailed descriptions of the necessary components of the pharmacogenomic testing implementation, the development and technical validation of the in-house SNP based multiplex PCR based assay targeting 20 genes and 48 SNPs as well as a separate CYP2D6 copy number assay along with the process of PGx report design, results of the provider and pharmacists usability studies, and the development of the software tool for genotype-phenotype translation and gene-phenotype-drug CPIC-based recommendations. Finally, we outline the process of developing the clinical workflow that connects the providers with the PGx experts within the Molecular Diagnostic Laboratory and the Pharmacy.

Biallelic variants in SLC38A3 encoding a glutamine transporter cause epileptic encephalopathy.

The solute carrier (SLC) superfamily encompasses >400 transmembrane transporters involved in the exchange of amino acids, nutrients, ions, metals, neurotransmitters and metabolites across biological membranes. SLCs are highly expressed in the mammalian brain; defects in nearly 100 unique SLC-encoding genes (OMIM: https://www.omim.org) are associated with rare Mendelian disorders including developmental and epileptic encephalopathy and severe neurodevelopmental disorders. Exome sequencing and family-based rare variant analyses on a cohort with neurodevelopmental disorders identified two siblings with developmental and epileptic encephalopathy and a shared deleterious homozygous splicing variant in SLC38A3. The gene encodes SNAT3, a sodium-coupled neutral amino acid transporter and a principal transporter of the amino acids asparagine, histidine, and glutamine, the latter being the precursor for the neurotransmitters GABA and glutamate. Additional subjects with a similar developmental and epileptic encephalopathy phenotype and biallelic predicted-damaging SLC38A3 variants were ascertained through GeneMatcher and collaborations with research and clinical molecular diagnostic laboratories. Untargeted metabolomic analysis was performed to identify novel metabolic biomarkers. Ten individuals from seven unrelated families from six different countries with deleterious biallelic variants in SLC38A3 were identified. Global developmental delay, intellectual disability, hypotonia, and absent speech were common features while microcephaly, epilepsy, and visual impairment were present in the majority. Epilepsy was drug-resistant in half. Metabolomic analysis revealed perturbations of glutamate, histidine, and nitrogen metabolism in plasma, urine, and CSF of selected subjects, potentially representing biomarkers of disease. Our data support the contention that SLC38A3 is a novel disease gene for developmental and epileptic encephalopathy and illuminate the likely pathophysiology of the disease as perturbations in glutamine homeostasis.

Molecular Diagnosis of Pompe Disease in the Genomic Era: Correlation with Acid Alpha-Glucosidase Activity in Dried Blood Spots.

Measurement of alpha-glucosidase activity on dried blood spots has been the main method to screen for Pompe disease, but a paradigm shift has been observed in recent years with the incorporation of gene panels and exome sequencing in molecular diagnostic laboratories. An 89-gene panel has been available to Canadian physicians since 2017 and was analyzed in 2030 patients with a suspected muscle disease. Acid alpha-glucosidase activity was measured in parallel in dried blood spots from 1430 patients. Pompe disease was diagnosed in 14 patients, representing 0.69% of our cohort. In 7 other patients, low enzyme activities overlapping those of Pompe disease cases were attributable to the presence of pseudodeficiency alleles. Only two other patients had enzymatic activity in the Pompe disease range, and a single heterozygous pathogenic variant was identified. It is possible that a second variant could have been missed; we suggest that RNA analysis should be considered in such cases. With gene panel testing increasingly being performed as a first-tier analysis of patients with suspected muscle disorders, our study supports the relevance of performing reflex enzymatic activity assay in selected patients, such as those with a single GAA variant identified and those in whom the observed genotype is of uncertain clinical significance.

Time scale performance of rapid antigen testing for SARS-CoV-2: Evaluation of 10 rapid antigen assays.

There is a great demand for more rapid tests for SARS‐CoV‐2 detection to reduce waiting time, boost public health strategies for combating disease, decrease costs, and prevent overwhelming laboratory capacities. This study was conducted to assess the performance of 10 lateral flow device viral antigen immunoassays for the detection of SARS‐CoV‐2 in nasopharyngeal swab specimens. We analyzed 231 nasopharyngeal samples collected from October 2020 to December 2020, from suspected COVID‐19 cases and contacts of positive cases at Biotechnology Research Center laboratories, Tripoli, Libya. The performance of 10 COVID‐19 Antigen (Ag) rapid test devices for the detection of SARS‐CoV‐2 antigen was compared to a quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR). In this study, 161 cases had symptoms consistent with COVID‐19. The mean duration from symptom onset was 6.6 ± 4.3 days. The median cycle threshold (C t) of positive samples was 25. Among the 108 positive samples detected by RT‐qPCR, the COVID‐19 antigen (Ag) tests detected 83 cases correctly. All rapid Ag test devices used in this study showed 100% specificity. While tests from six manufacturers had an overall sensitivity range from 75% to 100%, the remaining four tests had a sensitivity of 50%–71.43%. Sensitivity during the first 6 days of symptoms and in samples with high viral loads (C t < 25), was 100% in all but two of the test platforms. False‐negative samples had a median C t of 34 and an average duration of onset of symptoms of 11.3 days (range = 5–20 days). Antigen test diagnosis has high sensitivity and specificity in early disease when patients present less than 7 days of symptom onset. Patients are encouraged to test as soon as they get COVID‐19‐related symptoms within 1 week and to seek medical advice within 24 h if they develop disturbed smell/taste. The use of rapid antigen tests is important for controlling the COVID‐19 pandemic and reducing the burden on molecular diagnostic laboratories.

New Approach for Detection of Normal Alternative Splicing Events and Aberrant Spliceogenic Transcripts with Long-Range PCR and Deep RNA Sequencing.

Simple SummaryRNA splicing defects, caused by genetic variants, are a common molecular mechanism of disease. To detect variants that cause splicing impairment, mRNA-based studies must be performed. Classical mRNA assays are time-consuming, which is why we have validated a new reliable straightforward approach to detect normal alternative splicing events and also splicing aberrations. Using our approach, we were able to reclassify three variants of uncertain significance in NBN and STK11 genes, which is of great importance for a proper clinical management of the patients.RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

Whole-genome sequencing of SARS-CoV-2 in Uganda: implementation of the low-cost ARTIC protocol in resource-limited settings.

Background: In January 2020, a previously unknown coronavirus strain was identified as the cause of a severe acute respiratory syndrome (SARS-CoV-2). The first viral whole-genome was sequenced using high-throughput sequencing from a sample collected in Wuhan, China. Whole-genome sequencing (WGS) is imperative in investigating disease outbreak transmission dynamics and guiding decision-making in public health. Methods: We retrieved archived SARS-CoV-2 samples at the Integrated Biorepository of H3Africa Uganda, Makerere University (IBRH3AU). These samples were collected previously from individuals diagnosed with coronavirus disease 2019 (COVID-19) using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). 30 samples with cycle thresholds(Cts) values <25 were selected for WGS using SARS-CoV-2 ARTIC protocol at Makerere University Molecular Diagnostics Laboratory. Results: 28 out of 30 (93.3%) samples generated analyzable genomic sequence reads. We detected SARS-CoV-2 and lineages A (22/28) and B (6/28) from the samples. We further show phylogenetic relatedness of these isolates alongside other 328 Uganda (lineage A = 222, lineage B = 106) SARS-CoV-2 genomes available in GISAID by April 22, 2021 and submitted by the Uganda Virus Research Institute. Conclusions: Our study demonstrated adoption and optimization of the low-cost ARTIC SARS-CoV-2 WGS protocol in a resource limited laboratory setting. This work has set a foundation to enable rapid expansion of SARS-CoV-2 WGS in Uganda as part of the Presidential Scientific Initiative on Epidemics (PRESIDE) CoV-bank project and IBRH3AU.

Abstract 249: Comparison of variant classification between molecular diagnostics experts and decision support software

Abstract INTRODUCTION: Assessing the actionability of variants identified in next-generation sequencing (NGS) of solid tumors is challenging. Recommendations from AMP/ASCO/CAP/ACMG (Li et al. JMD 2017) provide guidelines for prioritizing variants based on clinical significance and actionability. Sirohi et al. (JMD 2020) reported on interrater agreement with the AMP guidelines by distributing 51 variants from solid tumor NGS to 20 participants at 10 US academic molecular diagnostics laboratories. Initial agreement based on independent assessment amongst the 20 experts was fair (Cohen κ = 0.35). To explore the potential for decision support software to streamline interpretation and improve the reliability of tier classification, we evaluated the same variants with NAVIFY® Mutation Profiler*, a CE-IVD somatic variant interpretation tool that automatically assigns tiers based on curated public evidence. METHODS: Reverse annotation from protein or coding sequence changes was completed with TransVar (Zhou et al. Nat Methods 2015) to obtain hg38 genomic coordinates for the 51 variants. Variants were prepared in VCF files and uploaded to NAVIFY Mutation Profiler (v2.0.0.22e0193) for analysis. The software assigned tier classifications using evidence in Roche curation content version 2.33.0 (released Nov 10, 2020). The default tier for each variant was compared to the tier assigned by the most experts in Sirohi et al. If there was a tie, the higher tier was used for comparison (e.g. Tier II when there was a tie between Tier II and III). RESULTS: There was good agreement (Cohen κ = 0.61) in tier assignments for 37 variants with curation content in NAVIFY Mutation Profiler compared to consensus expert curation in Sirohi et al. Most disagreements could be explained by emerging biomedical evidence, such as phase II clinical trial results for NFE2L2 in squamous cell lung carcinoma, or recommendations in NCCN guidelines. For example, ERBB2 mutations were up-classified by NAVIFY Mutation Profiler to Tier I due to NCCN therapy recommendations, including trastuzumab in combination with lapatinib or pertuzumab for ERBB2-amplified colorectal cancer and trastuzumab emtansine for non-small cell lung cancer with ERBB2 exon 20 insertions. Of the 14 variants without curation in the database, none were actionable, and the experts had classified 11 as Tier III and 3 as Tier II. CONCLUSIONS: When curation content was available, there was high agreement between the assigned tier in NAVIFY Mutation Profiler and the most common tier assigned by 20 molecular diagnostics experts in a previously published study. The continuous growth in new biomedical evidence requires staying current with the latest guidelines and clinical trial results, or the use of regularly updated databases, to accurately interpret somatic cancer variants from NGS test results. * This product is for Research Use Only; Not for use in diagnostic procedures in the US. Citation Format: Stephanie J. Yaung, Shuba Krishna, Sidney Scudder, Maximilian Schmid, John F. Palma. Comparison of variant classification between molecular diagnostics experts and decision support software [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 249.

Designing a Molecular Diagnostic Laboratory for Testing Highly Pathogenic Viruses

Designing a Molecular Diagnostic Laboratory for Testing Highly Pathogenic Viruses

Genotype\u2013phenotype correlations and novel molecular insights into the DHX30-associated neurodevelopmental disorders

BackgroundWe aimed to define the clinical and variant spectrum and to provide novel molecular insights into the DHX30-associated neurodevelopmental disorder.MethodsClinical and genetic data from affected individuals were collected through Facebook-based family support group, GeneMatcher, and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays.ResultsWe identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment, and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual harboring a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location, and type we establish two distinct clinical subtypes. DHX30 loss-of-function variants cause a milder phenotype whereas a severe phenotype is caused by HCM missense variants that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of-function with respect to SG formation. Behavioral characterization of dhx30-deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype.ConclusionsOur study highlights the usefulness of social media to define novel Mendelian disorders and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories, and research laboratories.

Contamination Assessment for Cancer Next-Generation Sequencing

The presence of allogeneic contamination impacts clinical reporting in cancer next-generation sequencing specimens. Although consensus guidelines recommend the identification of contaminating DNA as a part of quality control, implementation of contamination assessment methods in clinical molecular diagnostic laboratories has not been reported in the literature. To develop and implement a method to assess allogeneic contamination in clinical cancer next-generation sequencing specimens. We describe a method to detect contamination based on the evaluation of single-nucleotide polymorphic sites from tumor-only specimens. We validate this method and apply it to a large cohort of cancer sequencing specimens. Identification of specimen contamination was validated via in silico and in vitro mixtures, and reference range and reproducibility were established in a panel of normal specimens. The algorithm accurately detects an episode of systemic contamination due to reagent impurity. We prospectively applied this algorithm across 7571 clinical cancer specimens from a targeted next-generation sequencing panel, in which 262 specimens (3.5%) were predicted to be affected by greater than 5% contamination. Allogeneic contamination can be inferred from intrinsic cancer next-generation sequencing data without paired normal sequencing. The adoption of this approach can be useful as a quality control measure for laboratories performing clinical next-generation sequencing.

Guideline-Adherent Clinical Validation of a Comprehensive 170-Gene DNA/RNA Panel for Determination of Small Variants, Copy Number Variations, Splice Variants, and Fusions on a Next-Generation Sequencing Platform in the CLIA Setting.

We describe the clinical validation of a targeted DNA and RNA-based next-generation sequencing (NGS) assay at two clinical molecular diagnostic laboratories. This assay employs simultaneous DNA and RNA analysis of all coding exons to detect small variants (single-nucleotide variants, insertions, and deletions) in 148 genes, amplifications in 59 genes, and fusions and splice variants in 55 genes. During independent validations at two sites, 234 individual specimens were tested, including clinical formalin-fixed, paraffin-embedded (FFPE) tumor specimens, reference material, and cell lines. Samples were prepared using the Illumina TruSight Tumor 170 (TST170) kit, sequenced with Illumina sequencers, and the data were analyzed using the TST170 App. At both sites, TST170 had ≥98% success for ≥250× depth for ≥95% of covered positions. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection studies determined that inputs of ≥50 ng of DNA (with ≥3.3 ng/μl) and ≥50 ng RNA (minimum of 7 copies/ng) were optimal for high analytical sensitivity. The TST170 assay results were highly concordant with prior results using different methods across all variant categories. Optimization of nucleic acid extraction and DNA shearing, and quality control following library preparation is recommended to maximize assay success rates. In summary, we describe the validation of comprehensive and simultaneous DNA and RNA-based NGS testing using TST170 at two clinical sites.

David Katzenstein and James Hakim-In Memoriam.

The world lost two wonderful doctors who devoted their lives to the fight against HIV/AIDS in Africa. Ironically, they both died of COVID-19 within hours of each other in late January in Zimbabwe. David Katzenstein, MD, began his career in the early 1980 s working with underserved populations at a Native Tribal clinic in New Mexico and with persons suffering from AIDS at the Haight Ashbury Clinic in San Francisco, conducted CMV research at the University of Minnesota, and then took a position working as a microbiologist at the University of Zimbabwe in Harare between 1985 and 1987. In 1989, David joined the faculty at Stanford University where he remained until his retirement in 2017. Dr Katzenstein, led many important clinical and translational research studies related to HIV. With his colleagues in the AIDS Clinical Trials Group (ACTG), he led studies that resulted in the approval of life-saving antiretroviral medications. David dedicated a major portion of his academic endeavors to study HIV drug resistance, serving as director of one of the Virology Specialty Laboratories for the ACTG for many years. He was a protocol team member and leader on several ACTG studies and served on numerous ACTG committees, including the Performance Evaluation Committee and the original Virology Committee. He was co-chair of ACTG 384 that dealt with protease and/or non-nucleoside reverse transcriptase inhibitors, and was one of the first investigators to call attention to the issues of drug resistance in Africa. Recognizing the disparate impact of HIV in resource limited settings, David was a pioneer in Global HIV Research, in the fields of perinatal HIV transmission prevention, and studying different approaches to optimize antiretroviral treatment and laboratory monitoring in various settings around the world. He worked diligently to bring laboratory technology to Zimbabwe. Following his retirement from Stanford in 2017, David became the director of the Biomedical Research Training Institute in Zimbabwe where he led the molecular diagnostics laboratory to support laboratory monitoring of community-based treatment programs. In all his work, David engaged researchers and trainees from different parts of the world. Generous with his time, effort and ideas, David was a cherished mentor for many young and mid-career physicians and scientists, now scattered throughout different academic institutions and agencies in the U.S. He nurtured careers of clinicians, basic and social scientists, while encouraging multidisciplinary efforts to tackle the global HIV pandemic. He worked tirelessly and with great joy, giving of himself without reserve. James Gita Hakim, MRCP-UK, Professor of Medicine, University of Zimbabwe College of Health Sciences, to COVID-19. Dr Hakim was an accomplished investigator who made seminal contributions in HIV treatment and prevention research. He was known internationally for his accomplishments as a research leader, a builder of African research capacity, a leader of modernizing medical education in Africa, and as a mentor of young African investigators. Dr Hakim was at the forefront of clinical research on HIV/AIDS in Africa for over 20 years. He founded the University of Zimbabwe Clinical Research Centre (UZ-CRC) in 2001 as a site for international clinical trials on HIV treatment and prevention. He was a leader in pivotal international clinical trials including the landmark HIV ‘treatment as prevention’ trial HPTN 052, the MRC-funded “Development of Anti-Retroviral Therapy in Africa” (DART) trial, and the ACTG “Prospective Evaluation of Antiretroviral Therapy in Resource Limited Settings” (PEARLS) study. These studies contributed to the knowledge needed for successful implementation of antiretroviral therapy in southern Africa. Dr Hakim was as Principal Investigator of the University of Zimbabwe's program, the Medical Education Partnership Initiative (MEPI) which serves as a model for medical education in Africa. He was as a representative on the International AIDS Society (IAS) Governing Council from 2016 to 2020 and on the World Health Organization Guidelines Review Committee, the UNAIDS Advisory Committee, and on program committees for IAS Conference on HIV Science, the Conference on Retroviruses and Opportunistic Infections (CROI). Dr Hakim was Co-chair of the World AIDS Conference, Amsterdam, Netherlands in 2018. James Hakim's life demonstrated how investment in biomedical research and modern medical education can pay large dividends and be transformative for Africa. The world is better for the lives you lived, David and James. May light perpetual shine upon you.

Validation of a Next-Generation Sequencing–Based T-Cell Receptor Gamma Gene Rearrangement Diagnostic Assay: Transitioning from Capillary Electrophoresis to Next-Generation Sequencing

Assessment of T-cell receptor γ gene (TRG) rearrangements is an importants consideration in the diagnostic workup of lymphoproliferative diseases. Although fragment analysis by PCR and capillary electrophoresis (CE) is the current standard of such assessment in clinical molecular diagnostic laboratories, it does not provide sequence information and is only semi-quantitative. Next-generation sequencing (NGS)-based assays are an attractive alternative to the conventional fragment size-based methods, given that they generate results with specific clonotype sequence information and allow for more accurate quantitation. The present study evaluated various test parameters and performance characteristics of a commercially available NGS-based TRG gene-rearrangement assay by testing 101 clinical samples previously characterized by fragment analysis. The NGS TRG assay showed an overall accuracy of 83% and an analytical specificity of 100%. The concordance rates were 88% to 95% in the Vγ1-8, Vγ10, and Vγ11 gene families, but lower in the Vγ9 gene family. This difference was mostly attributed to the incomplete polyclonal symmetry resulting from the two-tube CE assay versus the one-tube design of the NGS assay. The NGS assay also demonstrated strengths in distinguishing clonotypes of the same fragment size. This clinical validation demonstrated robust performance of the NGS-based TRG assay and identified potential pitfalls associated with CE assay design that are important for understanding the observed discrepancies with the CE-based assay.