Flype: Software for enabling personalized medicine.

The advent of next generation DNA sequencing (NGS) has revolutionized clinical medicine by enabling wide-spread testing for genomic anomalies and polymorphisms. With that explosion in testing, however, come several informatics challenges including managing large amounts of data, interpreting the results and providing clinical decision support. We present Flype, a web-based bioinformatics platform built by a small group of bioinformaticians working in a community hospital setting, to address these challenges by allowing us to: (a) securely accept data from a variety of sources, (b) send orders to a variety of destinations, (c) perform secondary analysis and annotation of NGS data, (d) provide a central repository for all genomic variants, (e) assist with tertiary analysis and clinical interpretation, (f) send signed out data to our EHR as both PDF and discrete data elements, (g) allow population frequency analysis and (h) update variant annotation when literature knowledge evolves. We discuss the multiple use cases Flype supports such as (a) in-house NGS tests, (b) in-house pharmacogenomics (PGX) tests, (c) dramatic scale-up of genomic testing using an external lab, (d) consumer genomics using two external partners, and (e) a variety of reporting tools. The source code for Flype is available upon request to the authors.

Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer.

Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as "stitchers," to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication-licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer.

NGS implementation for monitoring SARS-CoV-2 variants in Chicagoland: An institutional perspective, successes and challenges.

Identification of SARS-CoV-2 lineages has shown to provide invaluable information regarding treatment efficacy, viral transmissibility, disease severity, and immune evasion. These benefits provide institutions with an expectation of high informational upside with little insight in regards to practicality with implementation and execution of such high complexity testing in the midst of a pandemic. This article details our institution's experience implementing and using Next Generation Sequencing (NGS) to monitor SARS-CoV-2 lineages in the northern Chicagoland area throughout the pandemic. To date, we have sequenced nearly 7,000 previously known SARS-CoV-2 positive samples from various patient populations (e.g., outpatient, inpatient, and outreach sites) to reduce bias in sampling. As a result, our hospital was guided while making crucial decisions about staffing, masking, and other infection control measures during the pandemic. While beneficial, establishing this NGS procedure was challenging, with countless considerations at every stage of assay development and validation. Reduced staffing prompted transition from a manual to automated high throughput workflow, requiring further validation, lab space, and instrumentation. Data management and IT security were additional considerations that delayed implementation and dictated our bioinformatic capabilities. Taken together, our experience highlights the obstacles and triumphs of SARS-CoV-2 sequencing.

Corrigendum: NGS implementation for monitoring SARS-CoV-2 variants in Chicagoland: an institutional perspective, successes and challenges.

[This corrects the article DOI: 10.3389/fpubh.2023.1177695.].

Personalized medicine in a community health system: the NorthShore experience.

Genomic and personalized medicine implementation efforts have largely centered on specialty care in tertiary health systems. There are few examples of fully integrated care systems that span the healthcare continuum. In 2014, NorthShore University HealthSystem launched the Center for Personalized Medicine to catalyze the delivery of personalized medicine. Successful implementation required the development of a scalable family history collection tool, the Genetic and Wellness Assessment (GWA) and Breast Health Assessment (BHA) tools; integrated pharmacogenomics programming; educational programming; electronic medical record integration; and robust clinical decision support tools. To date, more than 225,000 patients have been screened for increased hereditary conditions, such as cancer risk, through these tools in primary care. More than 35,000 patients completed clinical genetic testing following GWA or BHA completion. An innovative program trained more than 100 primary care providers in genomic medicine, activated with clinical decision support and access to patient genetic counseling services and digital healthcare tools. The development of a novel bioinformatics platform (FLYPE) enabled the incorporation of genomics data into electronic medical records. To date, over 4,000 patients have been identified to have a pathogenic or likely pathogenic variant in a gene with medical management implications. Over 33,000 patients have clinical pharmacogenomics data incorporated into the electronic health record supported by clinical decision support tools. This manuscript describes the evolution, strategy, and successful multispecialty partnerships aligned with health system leadership that enabled the implementation of a comprehensive personalized medicine program with measurable patient outcomes through a genomics-enabled learning health system model that utilizes implementation science frameworks.

Multilaboratory evaluation of real-time PCR tests for hepatitis B virus DNA quantification.

The performance characteristics of four different assays for hepatitis B virus (HBV) quantification were assessed: the Abbott RealTime HBV IUO, the Roche Cobas AmpliPrep/Cobas TaqMan HBV test, the Roche Cobas TaqMan HBV test with HighPure system, and the Qiagen artus HBV TM ASR. Limit of detection (LOD), linear range, reproducibility, and agreement were determined using a serially diluted plasma sample from a single chronically infected subject. Each assay was tested by at least three laboratories. The LOD of the RealTime and two TaqMan assays was approximately 1.0 log(10) IU/ml; for artus HBV (which used the lowest volume of extracted DNA), it was approximately 1.5 log(10) IU/ml. The linear range spanned 1.0 to at least 7.0 log(10) IU/ml for all assays. Median values were consistently lowest for artus HBV and highest for Cobas AmpliPrep/Cobas TaqMan HBV. Assays incorporating automated nucleic acid extraction were the most reproducible; however, the overall variability was minor since the standard deviations for the means of all tested concentrations were ≤0.32 log(10) IU/ml for all assays. False-positive results were observed with all assays; the highest rates occurred with tests using manual nucleic acid extraction. The performance characteristics of these assays suggest that they are useful for management and therapeutic monitoring of chronic HBV infection.

SILO: A Computational Method for Detecting Copy Number Gain in Clinical Specimens Analyzed on a Next-Generation Sequencing Platform.

Next-generation sequencing (NGS) has proved to be a beneficial approach for genotyping solid tumor specimens and for identifying clinically actionable mutations. However, copy number variations (CNVs), which can be equally important, are often challenging to detect from NGS data. Current bioinformatics methods for CNV detection from NGS often require comparison of tumor/normal pairs and/or the sequencing of whole genome or whole exome. These approaches are currently impractical for routine clinical practice. However, clinical practice does involve repeated use of the same gene panel on a large number of specimens over a long period of time. We take advantage of this repetitiveness and present SILO: a procedure for CNV detection based on NGS on a gene panel. The SILO algorithm analyzes coverage depth of the aligned reads from a sample and predicts CNV by comparing this depth to the average depth seen in a large training set of other samples. Such comparison is robust and can reliably detect copy number gain, although it is found to be unreliable in detecting copy number losses. Successful validation of SILO on NGS data from the Ion Torrent platform with two panels is presented: a small hotspot panel and a larger cancer gene panel.

Clinical characteristics and viral load dynamics of COVID-19 in a mildly or moderately symptomatic outpatient sample.

BACKGROUND: Studies of outpatients with mild or moderate COVID-19 are uncommon. We studied: 1) association of symptoms with reverse transcriptase polymerase chain reaction (RT-PCR) test results; and 2) association of initial RT-PCR cycle threshold (Ct) in relation to duration of RT-PCR positivity in outpatients with mild or moderate COVID-19. METHODS: This was a cohort study of outpatients with confirmed COVID-19 and at least one symptom. Participants had repeat nasopharyngeal swabs and symptom checklists every 3-5 days until two consecutive RT-PCR tests were negative. RT-PCR tests were used to assess viral load. Antibody tests for COVID-19 were performed at 2 weeks, 4 weeks, and 8 weeks after symptom onset. RESULTS: Twenty-five patients (nine females) were enrolled, ranging in age from 19-58 (median age 28 years). All patients reported at least one symptom, with a median of six symptoms per patient. Symptoms persisted for 6-67 days (median duration 18 days). In all 25 patients, blood samples collected a median of 13 days after symptom onset were positive for SARS-CoV-2 antibodies in 15 (60%). After a median of 28 days following symptom onset, 23/23 patients with available samples tested positive for antibodies. The longest duration of positive RT-PCR test was 49 days from first positive PCR test (Mean = 27.4, SD = 12.5, Median = 24). Initial Ct was significantly associated with longer duration (ß = -1.3, SE = 0.3, p<0.01 per 1 cycle higher) of RT-PCR positivity. CONCLUSIONS: In mildly or moderately ill COVID-19 outpatients, RT-PCT tests remained positive for as long as 49 days and test positivity and symptom duration correlated with initial viral load.

From Testing to Decision-Making: A Data-Driven Analytics COVID-19 Response.

In March 2020, NorthShore University Health System laboratories mobilized to develop and validate polymerase chain reaction based testing for detection of SARS-CoV-2. Using laboratory data, NorthShore University Health System created the Data Coronavirus Analytics Research Team to track activities affected by SARS-CoV-2 across the organization. Operational leaders used data insights and predictions from Data Coronavirus Analytics Research Team to redeploy critical care resources across the hospital system, and real-time data were used daily to make adjustments to staffing and supply decisions. Geographical data were used to triage patients to other hospitals in our system when COVID-19 detected pavilions were at capacity. Additionally, one of the consequences of COVID-19 was the inability for patients to receive elective care leading to extended periods of pain and uncertainty about a disease or treatment. After shutting down elective surgeries beginning in March of 2020, NorthShore University Health System set a recovery goal to achieve 80% of our historical volumes by October 1, 2020. Using the Data Coronavirus Analytics Research Team, our operational and clinical teams were able to achieve 89% of our historical volumes a month ahead of schedule, allowing rapid recovery of surgical volume and financial stability. The Data Coronavirus Analytics Research Team also was used to demonstrate that the accelerated recovery period had no negative impact with regard to iatrogenic COVID-19 infection and did not result in increased deep vein thrombosis, pulmonary embolisms, or cerebrovascular accident. These achievements demonstrate how a coordinated and transparent data-driven effort that was built upon a robust laboratory testing capability was essential to the operational response and recovery from the COVID-19 crisis.

The Value and Institutional Impact of an In-System Laboratory Testing During the COVID-19 Pandemic.

In-system clinical laboratories have proven themselves to be a fundamentally important resource to their institutions during the COVID-19 pandemic of the past year. The ability to provide SARS-CoV-2 molecular testing to our hospital system allowed us to offer the best possible care to our patients, and to support neighboring hospitals and nursing homes. In-house testing led to significant revenue enhancement to the laboratory and institution, and attracted new patients to the system. Timely testing of inpatients allowed the majority who did not have COVID-19 infection to be removed from respiratory and contact isolation, conserving valuable personal protective equipment and staff resources at a time that both were in short supply. As 2020 evolved and our institution restarted delivery of routine care, the availability of in-system laboratory testing to deliver both accurate and timely results was absolutely critical. In this article, we attempt to demonstrate the value and impact of an in-system laboratory during the COVID-19 pandemic. A strong in-house laboratory service was absolutely critical to institutional operational and financial success during 2020, and will ensure resiliency in the future as well.

Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic has resulted in significant shortages of RT-PCR testing supplies including RNA extraction kits. The goal of our study was to determine if a simplified heat-RNA release method would provide comparable detection of SARS-CoV-2 without the need for nucleic acid extraction. RT-PCR results using the ChromaCode HDPCR™ SARS-CoV-2 were compared using the heat-RNA release method and an automated RNA extraction system (EMAG). The heat-RNA release method correctly identified 94 % (81/86 nasopharyngeal samples) that were positive for SARS-CoV-2. Five samples that were missed by heat-RNA release method had a mean Ct value: 35 using the automated extraction instrument, indicating a very low viral load. Our findings show that a simple heat-RNA release method is a reasonable alternative for the majority of COVID-19 positive patients and can help overcome the cost and availability issues of RNA extraction reagents.

Diagnostic Capability of Next-Generation Sequencing Fusion Analysis in Identifying a Rare CASE of TRAF1-ALK-Associated Anaplastic Large Cell Lymphoma.

Background: Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is a rare T-cell neoplasm, accounting for approximately 3% of adult non-Hodgkin lymphomas. Although NPM1 is the most common fusion partner with ALK, many others have been described, necessitating break-apart FISH studies for confirmation of the diagnosis. TNF receptor-associated factor 1 (TRAF1) is a rare ALK partner that is thought to confer a worse prognosis in patients. We describe the utility of next-generation sequencing (NGS) RNA analysis in detection of this uncommon ALK partner. Case Description: A 42-year-old male with cervical lymphadenopathy presented for excisional biopsy. Following a tissue diagnosis of ALCL, ALK+, RNA from the biopsy was extracted from Formalin-fixed paraffin-embedded (FFPE) tissue and prepared for Anchored Multiplex PCR using the Archer® FusionPlex® v2 assay, which employs unidirectional gene-specific primers using NGS to detect novel or unknown gene partners. Results: Histologic evaluation of the excised lymph node showed atypical cells, including "horseshoe/kidney"-shaped nuclei. Neoplastic cells were immunoreactive against CD30, ALK (diffuse, cytoplasmic), CD2, CD4, granzyme B, and TIA-1. A diagnosis of ALCL, ALK+ was made. The pattern of ALK immunostaining suggested a non-NPM1-associated ALK translocation pattern, prompting further investigation. NGS fusion analysis showed a translocation involving exon 7 of TRAF1 and exon 20 of ALK. Conclusion: ALK positivity suggests an overall favorable prognosis of ALCL as compared to ALK-negative cases. However, in the rare published cases of TRAF1-ALK, an aggressive clinical course has been observed, which may reflect the aggressive propensity of this particular fusion, as these cases appear to be refractory to standard chemotherapy and also to the first generation ALK inhibitors. This study highlights the advantage of using NGS in RNA-based fusion assays to detect rare translocations, which can be of some clinical importance in detecting rare but aggressive fusion partners of ALK. As these technologies become more available, there is potential to identify such changes and effectively stratify the prognosis of ALCL patients.

A Case of Severe, Nilotinib-Induced Liver Injury.

Idiosyncratic hepatotoxicity is a leading reason for the discontinuation or dose modification of Food and Drug Administration (FDA)-approved medications in the United States. We report the case of a 53-year-old woman with chronic myeloid leukemia who developed acute cholestatic hepatitis in response to the tyrosine kinase inhibitor nilotinib. Nilotinib was discontinued, and the patient's liver function tests normalized over the next 3 months. We conclude that nilotinib may cause life-threatening hepatotoxicity and recommend that patients on the medication undergo regular monitoring of their liver tests.

A prevalence study of methicillin-resistant Staphylococcus aureus colonization in emergency department health care workers.

STUDY OBJECTIVE: Few studies of the prevalence of nasal colonization of methicillin-resistant Staphylococcus aureus (MRSA) in emergency department (ED) health care workers have been conducted. To better understand the epidemiology of this pathogen, we seek to determine the MRSA nasal colonization rates in the ED health care workers in our hospital. METHODS: We conducted a prospective cohort study on a convenience sample of ED health care workers, including nurses, physicians, and technicians. Nasal swabs from subjects were analyzed with a polymerase chain reaction assay for the presence of MRSA. RESULTS: Of the 105 ED health care workers enrolled, a total of 16 (15%, 95% confidence interval 9.6% to 23%) were MRSA positive. No significant difference was observed in colonization rates between nurses, physicians, and technicians. CONCLUSION: Our ED health care workers demonstrated a high prevalence of nasal MRSA colonization compared with individuals in recent community surveillance and other studies involving ED staff.

What should we mean by empirical validation in hypnotherapy: evidence-based practice in clinical hypnosis.

This paper briefly surveys the trend of and controversy surrounding empirical validation in psychotherapy. Empirical validation of hypnotherapy has paralleled the practice of validation in psychotherapy and the professionalization of clinical psychology, in general. This evolution in determining what counts as evidence for bona fide clinical practice has gone from theory-driven clinical approaches in the 1960s and 1970s through critical attempts at categorization of empirically supported therapies in the 1990s on to the concept of evidence-based practice in 2006. Implications of this progression in professional psychology are discussed in the light of hypnosis's current quest for validation and empirical accreditation.

The Case for Laboratory Developed Procedures: Quality and Positive Impact on Patient Care.

An explosion of knowledge and technology is revolutionizing medicine and patient care. Novel testing must be brought to the clinic with safety and accuracy, but also in a timely and cost-effective manner, so that patients can benefit and laboratories can offer testing consistent with current guidelines. Under the oversight provided by the Clinical Laboratory Improvement Amendments, laboratories have been able to develop and optimize laboratory procedures for use in-house. Quality improvement programs, interlaboratory comparisons, and the ability of laboratories to adjust assays as needed to improve results, utilize new sample types, or incorporate new mutations, information, or technologies are positive aspects of Clinical Laboratory Improvement Amendments oversight of laboratory-developed procedures. Laboratories have a long history of successful service to patients operating under Clinical Laboratory Improvement Amendments. A series of detailed clinical examples illustrating the quality and positive impact of laboratory-developed procedures on patient care is provided. These examples also demonstrate how Clinical Laboratory Improvement Amendments oversight ensures accurate, reliable, and reproducible testing in clinical laboratories.

Genomic Sequencing Procedure Microcosting Analysis and Health Economic Cost-Impact Analysis: A Report of the Association for Molecular Pathology.

The increasing use of advanced nucleic acid sequencing technologies for clinical diagnostics and therapeutics has made vital understanding the costs of performing these procedures and their value to patients, providers, and payers. The Association for Molecular Pathology invested in a cost and value analysis of specific genomic sequencing procedures (GSPs) newly coded by the American Medical Association Current Procedural Terminology Editorial Panel. Cost data and work effort, including the development and use of data analysis pipelines, were gathered from representative laboratories currently performing these GSPs. Results were aggregated to generate representative cost ranges given the complexity and variability of performing the tests. Cost-impact models for three clinical scenarios were generated with assistance from key opinion leaders: impact of using a targeted gene panel in optimizing care for patients with advanced non-small-cell lung cancer, use of a targeted gene panel in the diagnosis and management of patients with sensorineural hearing loss, and exome sequencing in the diagnosis and management of children with neurodevelopmental disorders of unknown genetic etiology. Each model demonstrated value by either reducing health care costs or identifying appropriate care pathways. The templates generated will aid laboratories in assessing their individual costs, considering the value structure in their own patient populations, and contributing their data to the ongoing dialogue regarding the impact of GSPs on improving patient care.

Summary of microsatellite instability test results from laboratories participating in proficiency surveys: proficiency survey results from 2005 to 2012.

CONTEXT: The College of American Pathologists surveys are the largest laboratory peer comparison programs in the world. These programs allow laboratories to regularly evaluate their performance and improve the accuracy of the patient test results they provide. Proficiency testing is offered twice a year to laboratories performing microsatellite instability testing. These surveys are designed to emulate clinical practice, and some surveys have more challenging cases to encourage the refinement of laboratory practices. OBJECTIVE: This report summarizes the results and trends in microsatellite instability proficiency testing from participating laboratories from the inception of the program in 2005 through 2012. DESIGN: We compiled and analyzed data for 16 surveys of microsatellite instability proficiency testing during 2005 to 2012. RESULTS: The number of laboratories participating in the microsatellite instability survey has more than doubled from 42 to 104 during the 8 years analyzed. An average of 95.4% of the laboratories correctly classified each of the survey test samples from the 2005A through 2012B proficiency challenges. In the 2011B survey, a lower percentage of laboratories (78.4%) correctly classified the specimen, possibly because of overlooking subtle changes of microsatellite instability and/or failing to enrich the tumor content of the specimen to meet the limit of detection of their assay. CONCLUSIONS: In general, laboratories performed well in microsatellite instability testing. This testing will continue to be important in screening patients with colorectal and other cancers for Lynch syndrome and guiding the management of patients with sporadic colorectal cancer.