Automated HL7v2 LRI informatics framework for streamlining genomics-EHR data integration.

While VCF formatted files are the lingua franca of next-generation sequencing, most EHRs do not provide native VCF support. As a result, labs often must send non-structured PDF reports to the EHR. On the other hand, while FHIR adoption is growing, most EHRs support HL7 interoperability standards, particularly those based on the HL7 Version 2 (HL7v2) standard. The HL7 Version 2 genomics component of the HL7 Laboratory Results Interface (HL7v2 LRI) standard specifies a formalism for the structured communication of genomic data from lab to EHR. We previously described an open-source tool (vcf2fhir) that converts VCF files into HL7 FHIR format. In this report, we describe how the utility has been extended to output HL7v2 LRI data that contains both variants and variant annotations (e.g., predicted phenotypes and therapeutic implications). Using this HL7v2 converter, we implemented an automated pipeline for moving structured genomic data from the clinical laboratory to EHR. We developed an open source hl7v2GenomicsExtractor that converts genomic interpretation report files into a series of HL7v2 observations conformant to HL7v2 LRI. We further enhanced the converter to produce output conformant to Epic's genomic import specification and to support alternative input formats. An automated pipeline for pushing standards-based structured genomic data directly into the EHR was successfully implemented, where genetic variant data and the clinical annotations are now both available to be viewed in the EHR through Epic's genomics module. Issues encountered in the development and deployment of the HL7v2 converter primarily revolved around data variability issues, primarily lack of a standardized representation of data elements within various genomic interpretation report files. The technical implementation of a HL7v2 message transformation to feed genomic variant and clinical annotation data into an EHR has been successful. In addition to genetic variant data, the implementation described here releases the valuable asset of clinically relevant genomic annotations provided by labs from static PDFs to calculable, structured data in EHR systems.

CYP2D6-guided opioid therapy for adults with cancer pain: A randomized implementation clinical trial.

INTRODUCTION: The CYP2D6 enzyme metabolizes opioids commonly prescribed for cancer-related pain, and CYP2D6 polymorphisms may contribute to variability in opioid response. We evaluated the feasibility of implementing CYP2D6-guided opioid prescribing for patients with cancer and reported pilot outcome data. METHODS: Adult patients from two cancer centers were prospectively enrolled into a hybrid implementation-effectiveness clinical trial and randomized to CYP2D6-genotype-guided opioid selection, with clinical recommendations, or usual care. Implementation metrics, including provider response, medication changes consistent with recommendations, and patient-reported pain and symptom scores at baseline and up to 8 weeks, were assessed. RESULTS: Most (87/114, 76%) patients approached for the study agreed to participate. Of 85 patients randomized, 71% were prescribed oxycodone at baseline. The median (range) time to receive CYP2D6 test results was 10 (3-37) days; 24% of patients had physicians acknowledge genotype results in a clinic note. Among patients with CYP2D6-genotype-guided recommendations to change therapy (n = 11), 18% had a change congruent with recommendations. Among patients who completed baseline and follow-up questionnaires (n = 48), there was no difference in change in mean composite pain score (-1.01 ± 2.1 vs. -0.41 ± 2.5; p = 0.19) or symptom severity at last follow-up (3.96 ± 2.18 vs. 3.47 ± 1.78; p = 0.63) between the usual care arm (n = 26) and genotype-guided arm (n = 22), respectively. CONCLUSION: Our study revealed high acceptance of pharmacogenetic testing as part of a clinical trial among patients with cancer pain. However, provider response to genotype-guided recommendations was low, impacting assessment of pain-related outcomes. Addressing barriers to utility of pharmacogenetics results and clinical recommendations will be critical for implementation success.

Reimbursement of pharmacogenetic tests at a tertiary academic medical center in the United States.

Introduction: Pharmacogenetics (PGx) has the potential to improve health outcomes but cost of testing is a barrier for equitable access. Reimbursement by insurance providers may lessen the financial burden for patients, but the extent to which PGx claims are covered in clinical practice has not been well-characterized in the literature. Methods: A retrospective analysis of outpatient claims submitted to payers for PGx tests from 1/1/2019 through 12/31/2021 was performed. A reimbursement rate was calculated and compared across specific test types (e.g., single genes, panel), payers, indication, and the year the claim was submitted. Results: A total of 1,039 outpatient claims for PGx testing were analyzed. The overall reimbursement rate was 46% and ranged from 36%-48% across payers. PGx panels were reimbursed at a significantly higher rate than single gene tests (74% vs. 43%, p < 0.001). Discussion: Reimbursement of claims for PGx testing is variable based on the test type, indication, year the claim was submitted, number of diagnosis codes submitted, and number of unique diagnosis codes submitted. Due to the highly variable nature of reimbursement, cost and affordability should be discussed with each patient.

Defining critical illness using immunological endotypes in patients with and without sepsis: a cohort study.

BACKGROUND: Sepsis is a heterogenous syndrome with limited therapeutic options. Identifying immunological endotypes through gene expression patterns in septic patients may lead to targeted interventions. We investigated whether patients admitted to a surgical intensive care unit (ICU) with sepsis and with high risk of mortality express similar endotypes to non-septic, but still critically ill patients using two multiplex transcriptomic metrics obtained both on admission to a surgical ICU and at set intervals. METHODS: We analyzed transcriptomic data from 522 patients in two single-site, prospective, observational cohorts admitted to surgical ICUs over a 5-year period ending in July 2020. Using an FDA-cleared analytical platform (nCounter FLEX®, NanoString, Inc.), we assessed a previously validated 29-messenger RNA transcriptomic classifier for likelihood of 30-day mortality (IMX-SEV-3) and a 33-messenger RNA transcriptomic endotype classifier. Clinical outcomes included all-cause mortality, development of chronic critical illness, and secondary infections. Univariate and multivariate analyses were performed to assess for true effect and confounding. RESULTS: Sepsis was associated with a significantly higher predicted and actual hospital mortality. At enrollment, the predominant endotype for both septic and non-septic patients was adaptive, though with significantly different distributions. Inflammopathic and coagulopathic septic patients, as well as inflammopathic non-septic patients, showed significantly higher frequencies of secondary infections compared to those with adaptive endotypes (p < 0.01). Endotypes changed during ICU hospitalization in 57.5% of patients. Patients who remained adaptive had overall better prognosis, while those who remained inflammopathic or coagulopathic had worse overall outcomes. For severity metrics, patients admitted with sepsis and a high predicted likelihood of mortality showed an inflammopathic (49.6%) endotype and had higher rates of cumulative adverse outcomes (67.4%). Patients at low mortality risk, whether septic or non-septic, almost uniformly presented with an adaptive endotype (100% and 93.4%, respectively). CONCLUSION: Critically ill surgical patients express different and evolving immunological endotypes depending upon both their sepsis status and severity of their clinical course. Future studies will elucidate whether endotyping critically ill, septic patients can identify individuals for targeted therapeutic interventions to improve patient management and outcomes.

Defining critical illness using immunological endotypes in patients with and without of sepsis: A cohort study.

Background: Sepsis is a heterogenous syndrome with limited therapeutic options. Identifying characteristic gene expression patterns, or endotypes, in septic patients may lead to targeted interventions. We investigated whether patients admitted to a surgical ICU with sepsis and with high risk of mortality express similar endotypes to non-septic, but still critically ill patients using two multiplex transcriptomic metrics obtained both on admission to a surgical intensive care unit (ICU) and at set intervals. Methods: We analyzed transcriptomic data from 522 patients in two single-site, prospective, observational cohorts admitted to surgical ICUs over a 5-year period ending in July 2020 . Using an FDA-cleared analytical platform (nCounter FLEX ® , NanoString, Inc.), we assessed a previously validated 29-messenger RNA transcriptomic classifier for likelihood of 30-day mortality (IMX-SEV-3) and a 33-messenger RNA transcriptomic endotype classifier. Clinical outcomes included all-cause (in-hospital, 30-, 90-day) mortality, development of chronic critical illness (CCI), and secondary infections. Univariate and multivariate analyses were performed to assess for true effect and confounding. Results: Sepsis was associated with a significantly higher predicted and actual hospital mortality. At enrollment, the predominant endotype for both septic and non-septic patients was adaptive , though with significantly different distributions. Inflammopathic and coagulopathic septic patients, as well as inflammopathic non-septic patients, showed significantly higher frequencies of secondary infections compared to those with adaptive endotypes (p<0.01). Endotypes changed during ICU hospitalization in 57.5% of patients. Patients who remained adaptive had overall better prognosis, while those who remained inflammopathic or coagulopathic had worse overall outcomes. For severity metrics, patients admitted with sepsis and a high predicted likelihood of mortality showed an inflammopathic (49.6%) endotype and had higher rates of cumulative adverse outcomes (67.4%). Patients at low mortality risk, whether septic or non-septic, almost uniformly presented with an adaptive endotype (100% and 93.4%, respectively). Conclusion : Critically ill surgical patients express different and evolving immunological endotypes depending upon both their sepsis status and severity of their clinical course. Future studies will elucidate whether endotyping critically ill, septic patients can identify individuals for targeted therapeutic interventions to improve patient management and outcomes.

Current clinical practices and challenges in molecular testing: a GOAL Consortium Hematopathology Working Group report.

While molecular testing of hematologic malignancies is now standard of care, there is variability in practice and testing capabilities between different academic laboratories, with common questions arising on how to best meet clinical expectations. A survey was sent to hematopathology subgroup members of the Genomics Organization for Academic Laboratories consortium to assess current and future practice and potentially establish a reference for peer institutions. Responses were received from 18 academic tertiary-care laboratories regarding next-generation sequencing (NGS) panel design, sequencing protocols and metrics, assay characteristics, laboratory operations, case reimbursement, and development plans. Differences in NGS panel size, use, and gene content were reported. Gene content for myeloid processes was reported to be generally excellent, while genes for lymphoid processes were less well covered. The turnaround time (TAT) for acute cases, including acute myeloid leukemia, was reported to range from 2 to 7 calendar days to 15 to 21 calendar days, with different approaches to achieving rapid TAT described. To help guide NGS panel design and standardize gene content, consensus gene lists based on current and future NGS panels in development were generated. Most survey respondents expected molecular testing at academic laboratories to continue to be viable in the future, with rapid TAT for acute cases likely to remain an important factor. Molecular testing reimbursement was reported to be a major concern. The results of this survey and subsequent discussions improve the shared understanding of differences in testing practices for hematologic malignancies between institutions and will help provide a more consistent level of patient care.

Characterization of Reference Materials for TPMT and NUDT15: A GeT-RM Collaborative Project.

Pharmacogenetic testing is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the TPMT and NUDT15 variants included in clinical tests. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Material (GeT-RM) coordination program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 19 DNA samples derived from Coriell cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using a variety of commercially available and laboratory developed tests and/or Sanger sequencing. Of the 12 samples characterized for TPMT, newly identified variants include TPMT∗2, ∗6, ∗12, ∗16, ∗21, ∗24, ∗32, ∗33, and ∗40; for the 7 NUDT15 reference material samples, newly identified variants are NUDT15∗2, ∗3, ∗4, ∗5, ∗6, and ∗9. In addition, a novel haplotype, TPMT∗46, was identified in this study. Preexisting data on an additional 11 Coriell samples, as well as some supplemental testing, were used to create comprehensive reference material panels for TPMT and NUDT15. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

Implementing a pragmatic clinical trial to tailor opioids for acute pain on behalf of the IGNITE ADOPT PGx investigators.

Opioid prescribing for postoperative pain management is challenging because of inter-patient variability in opioid response and concern about opioid addiction. Tramadol, hydrocodone, and codeine depend on the cytochrome P450 2D6 (CYP2D6) enzyme for formation of highly potent metabolites. Individuals with reduced or absent CYP2D6 activity (i.e., intermediate metabolizers [IMs] or poor metabolizers [PMs], respectively) have lower concentrations of potent opioid metabolites and potentially inadequate pain control. The primary objective of this prospective, multicenter, randomized pragmatic trial is to determine the effect of postoperative CYP2D6-guided opioid prescribing on pain control and opioid usage. Up to 2020 participants, age ≥8 years, scheduled to undergo a surgical procedure will be enrolled and randomized to immediate pharmacogenetic testing with clinical decision support (CDS) for CYP2D6 phenotype-guided postoperative pain management (intervention arm) or delayed testing without CDS (control arm). CDS is provided through medical record alerts and/or a pharmacist consult note. For IMs and PM in the intervention arm, CDS includes recommendations to avoid hydrocodone, tramadol, and codeine. Patient-reported pain-related outcomes are collected 10 days and 1, 3, and 6 months after surgery. The primary outcome, a composite of pain intensity and opioid usage at 10 days postsurgery, will be compared in the subgroup of IMs and PMs in the intervention (n = 152) versus the control (n = 152) arm. Secondary end points include prescription pain medication misuse scores and opioid persistence at 6 months. This trial will provide data on the clinical utility of CYP2D6 phenotype-guided opioid selection for improving postoperative pain control and reducing opioid-related risks.

Evaluation of a Multivalent Transcriptomic Metric for Diagnosing Surgical Sepsis and Estimating Mortality Among Critically Ill Patients.

Importance: Rapid and accurate discrimination of sepsis and its potential severity currently require multiple assays with slow processing times that are often inconclusive in discerning sepsis from sterile inflammation. Objective: To analyze a whole-blood, multivalent, host-messenger RNA expression metric for estimating the likelihood of bacterial infection and 30-day mortality and compare performance of the metric with that of other diagnostic and prognostic biomarkers and clinical parameters. Design, Setting, and Participants: This prospective diagnostic and prognostic study was performed in the surgical intensive care unit (ICU) of a single, academic health science center. The analysis included 200 critically ill adult patients admitted with suspected sepsis (cohort A) or those at high risk for developing sepsis (cohort B) between July 1, 2020, and July 30, 2021. Exposures: Whole-blood sample measurements of a custom 29-messenger RNA transcriptomic metric classifier for likelihood of bacterial infection (IMX-BVN-3) or 30-day mortality (severity) (IMX-SEV-3) in a clinical-diagnostic laboratory setting using an analysis platform (510[k]-cleared nCounter FLEX; NanoString, Inc), compared with measurement of procalcitonin and interleukin 6 (IL-6) plasma levels, and maximum 24-hour sequential organ failure assessment (SOFA) scores. Main Outcomes and Measures: Estimated sepsis and 30-day mortality performance. Results: Among the 200 patients included (124 men [62.0%] and 76 women [38.0%]; median age, 62.5 [IQR, 47.0-72.0] years), the IMX-BVN-3 bacterial infection classifier had an area under the receiver operating characteristics curve (AUROC) of 0.84 (95% CI, 0.77-0.90) for discriminating bacterial infection at ICU admission, similar to procalcitonin (0.85 [95% CI, 0.79-0.90]; P = .79) and significantly better than IL-6 (0.67 [95% CI, 0.58-0.75]; P < .001). For estimating 30-day mortality, the IMX-SEV-3 metric had an AUROC of 0.81 (95% CI, 0.66-0.95), which was significantly better than IL-6 levels (0.57 [95% CI, 0.37-0.77]; P = .006), marginally better than procalcitonin levels (0.65 [95% CI, 0.50-0.79]; P = .06), and similar to the SOFA score (0.76 [95% CI, 0.62-0.91]; P = .48). Combining IMX-BVN-3 and IMX-SEV-3 with procalcitonin or IL-6 levels or SOFA scores did not significantly improve performance. Among patients with sepsis, IMX-BVN-3 scores decreased over time, reflecting the resolution of sepsis. In 11 individuals at high risk (cohort B) who subsequently developed sepsis during their hospital course, IMX-BVN-3 bacterial infection scores did not decline over time and peaked on the day of documented infection. Conclusions and Relevance: In this diagnostic and prognostic study, a novel, multivalent, transcriptomic metric accurately estimated the presence of bacterial infection and risk for 30-day mortality in patients admitted to a surgical ICU. The performance of this single transcriptomic metric was equivalent to or better than multiple alternative diagnostic and prognostic metrics when measured at admission and provided additional information when measured over time.

Design and rationale of GUARDD-US: A pragmatic, randomized trial of genetic testing for APOL1 and pharmacogenomic predictors of antihypertensive efficacy in patients with hypertension.

RATIONALE AND OBJECTIVE: APOL1 risk alleles are associated with increased cardiovascular and chronic kidney disease (CKD) risk. It is unknown whether knowledge of APOL1 risk status motivates patients and providers to attain recommended blood pressure (BP) targets to reduce cardiovascular disease. STUDY DESIGN: Multicenter, pragmatic, randomized controlled clinical trial. SETTING AND PARTICIPANTS: 6650 individuals with African ancestry and hypertension from 13 health systems. INTERVENTION: APOL1 genotyping with clinical decision support (CDS) results are returned to participants and providers immediately (intervention) or at 6 months (control). A subset of participants are re-randomized to pharmacogenomic testing for relevant antihypertensive medications (pharmacogenomic sub-study). CDS alerts encourage appropriate CKD screening and antihypertensive agent use. OUTCOMES: Blood pressure and surveys are assessed at baseline, 3 and 6 months. The primary outcome is change in systolic BP from enrollment to 3 months in individuals with two APOL1 risk alleles. Secondary outcomes include new diagnoses of CKD, systolic blood pressure at 6 months, diastolic BP, and survey results. The pharmacogenomic sub-study will evaluate the relationship of pharmacogenomic genotype and change in systolic BP between baseline and 3 months. RESULTS: To date, the trial has enrolled 3423 participants. CONCLUSIONS: The effect of patient and provider knowledge of APOL1 genotype on systolic blood pressure has not been well-studied. GUARDD-US addresses whether blood pressure improves when patients and providers have this information. GUARDD-US provides a CDS framework for primary care and specialty clinics to incorporate APOL1 genetic risk and pharmacogenomic prescribing in the electronic health record. TRIAL REGISTRATION: ClinicalTrials.govNCT04191824.

Impact of the ABCD-GENE Score on Clopidogrel Clinical Effectiveness after PCI: A Multi-Site, Real-World Investigation.

The Age, Body mass index, Chronic kidney disease, Diabetes mellitus, and CYP2C19 GENEtic variants (ABCD-GENE) score was developed to identify patients at risk for diminished antiplatelet effects with clopidogrel after percutaneous coronary intervention (PCI). The objective of this study was to validate the ability of the ABCD-GENE score to predict the risk for atherothrombotic events in a diverse, real-world population of clopidogrel-treated patients who underwent PCI and received clinical CYP2C19 genotyping to guide antiplatelet therapy. A total of 2,341 adult patients who underwent PCI, were genotyped for CYP2C19, and received treatment with clopidogrel across four institutions were included (mean age 64 ± 12 years, 35% women, and 20% Black). The primary outcome was major atherothrombotic events, defined as the composite of all-cause death, myocardial infarction, ischemic stroke, stent thrombosis, or revascularization for unstable angina within 12 months following PCI. Major adverse cardiovascular events (MACE), defined as the composite of cardiovascular death, myocardial infarction, ischemic stroke, or stent thrombosis, was assessed as the secondary outcome. Outcomes were compared between patients with an ABCD-GENE score ≥ 10 vs. < 10. The risk of major atherothrombotic events was higher in patients with an ABCD-GENE score ≥ 10 (n = 505) vs. < 10 (n = 1,836; 24.6 vs. 14.7 events per 100 patient-years, adjusted hazard ratio (HR) 1.66, 95% confidence interval (CI), 1.23-2.25, P < 0.001). The risk for MACE was also higher among patients with a score ≥ 10 vs. < 10 (16.7 vs. 10.1 events per 100 patient-years, adjusted HR 1.59, 95% CI 1.11-2.30, P = 0.013). Our diverse, real-world data demonstrate diminished clopidogrel effectiveness in post-PCI patients with an ABCD-GENE score ≥ 10.

Severe Acute Respiratory Syndrome Coronavirus 2 Delta Vaccine Breakthrough Transmissibility in Alachua County, Florida.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant has caused a dramatic resurgence in infections in the United Sates, raising questions regarding potential transmissibility among vaccinated individuals. METHODS: Between October 2020 and July 2021, we sequenced 4439 SARS-CoV-2 full genomes, 23% of all known infections in Alachua County, Florida, including 109 vaccine breakthrough cases. Univariate and multivariate regression analyses were conducted to evaluate associations between viral RNA burden and patient characteristics. Contact tracing and phylogenetic analysis were used to investigate direct transmissions involving vaccinated individuals. RESULTS: The majority of breakthrough sequences with lineage assignment were classified as Delta variants (74.6%) and occurred, on average, about 3 months (104 ±â€…57.5 days) after full vaccination, at the same time (June-July 2021) of Delta variant exponential spread within the county. Six Delta variant transmission pairs between fully vaccinated individuals were identified through contact tracing, 3 of which were confirmed by phylogenetic analysis. Delta breakthroughs exhibited broad viral RNA copy number values during acute infection (interquartile range, 1.2-8.64 Log copies/mL), on average 38% lower than matched unvaccinated patients (3.29-10.81 Log copies/mL, P < .00001). Nevertheless, 49% to 50% of all breakthroughs, and 56% to 60% of Delta-infected breakthroughs exhibited viral RNA levels above the transmissibility threshold (4 Log copies/mL) irrespective of time after vaccination. CONCLUSIONS: Delta infection transmissibility and general viral RNA quantification patterns in vaccinated individuals suggest limited levels of sterilizing immunity that need to be considered by public health policies. In particular, ongoing evaluation of vaccine boosters should specifically address whether extra vaccine doses curb breakthrough contribution to epidemic spread.

Low-frequency variants in mildly symptomatic vaccine breakthrough infections presents a doubled-edged sword.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) has raised questions regarding vaccine protection against SARS-CoV-2 infection, transmission, and ongoing virus evolution. Twenty-three mildly symptomatic "vaccination breakthrough" infections were identified as early as January 2021 in Alachua County, Florida, among individuals fully vaccinated with either the BNT162b2 (Pfizer) or the Ad26 (Janssen/J&J) vaccines. SARS-CoV-2 genomes were successfully generated for 11 of the vaccine breakthroughs, and 878 individuals in the surrounding area and were included for reference-based phylogenetic investigation. These 11 individuals were characterized by infection with VOCs, but also low-frequency variants present within the surrounding population. Low-frequency mutations were observed, which have been more recently identified as mutations of interest owing to their location within targeted immune epitopes (P812L) and association with increased replicative capacity (L18F). We present these results to posit the nature of the efficacy of vaccines in reducing symptoms as both a blessing and a curse-as vaccination becomes more widespread and self-motivated testing reduced owing to the absence of severe symptoms, we face the challenge of early recognition of novel mutations of potential concern. This case study highlights the critical need for continued testing and monitoring of infection and transmission among individuals regardless of vaccination status.

Deleterious mutations in esophageal carcinoma cuniculatum detected by next generation sequencing.

Esophageal carcinoma cuniculatum (ECC) is a rare form of extremely well-differentiated squamous cell carcinoma of esophagus that is often misdiagnosed preoperatively. The molecular changes underlying ECC remain unknown. This study aimed to explore the molecular signature of ECC using next-generation sequencing (NGS). Five cases of ECC were collected from our pathology database from 2014 to 2019. One patient received chemoradiation and the remaining four patients were treatment-naïve. Areas of normal squamous mucosa, non-invasive component, and invasive component of ECC were circled and macrodissected. Genomic DNA extracted from the macrodissected tissue was sequenced using GatorSeq NGS Panel. Deleterious mutations, predicted by Sorting Intolerant from Tolerant (SIFT), were identified using tumor/normal pairs and annotated by amino acid change. The normal-appearing squamous mucosa in the ECC harbored recurrent deleterious somatic mutations in ROS1 and POLE genes. ECC tumor-specific deleterious mutations were identified on TP53, NOTCH1, and PIK3CA genes. Our results support a mutually exclusive pattern in NOTCH1 and PIK3CA mutation. Non-invasive and invasive components in ECC had identical mutation profiles. Chemoradiation therapy led to disappearance of NOTCH1 mutation in one ECC case. Our results suggest molecular testing may help pre-operative diagnosis, and provide therapeutic targets in patients with advanced or unresectable ECC.

Synchronous Basal Cell Carcinoma and Squamous Cell Carcinoma of Nasal Vestibule With Novel Unique Variants Identified by Whole-exome Sequencing.

BACKGROUND/AIM: It is estimated that nonmelanoma skin cancer (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), affects more than 3 million Americans each year. Translation of next-generation sequencing (NGS) data into identification of new potential targets for therapeutic applications may be helpful. Whole-exome sequencing (WES) is a widely used NGS method that involves sequencing the protein-coding regions of the genome. CASE REPORT: We report a case of a 65-year-old female smoker who was found to have two 6 mm lesions in her left nasal vestibule. Biopsies demonstrated synchronous BCC and SCC. The patient underwent surgical excision of both cancers with safe margins followed by plastic reconstruction. WES was performed on both cancers and 16 alterations including BRCA2 (p.P389S), FAM5C (S420L), KMT2A (P855L), and SMO (L412F), as unique for BCC, and 4 alterations including TP53 (p.H179Q) and CDKN2A (p.P114L), as unique for SCC, were identified. CONCLUSION: We report the first documented case with unique genetic alterations in two distinct and synchronous skin BCC and SCC arising from the same nasal vestibule of a patient. This adds to the growing field of data regarding genetic variants in characterizing malignancies and potentially for targeted therapies.

GATM-Mediated Creatine Biosynthesis Enables Maintenance of FLT3-ITD-Mutant Acute Myeloid Leukemia.

FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), with the most common mutation being internal tandem duplications (ITD). The presence of FLT3-ITD in AML carries a particularly poor prognosis and renders therapeutic resistance. New druggable targets are thus needed in this disease. In this study, we demonstrate the effects of de novo creatine biosynthesis upregulation by FLT3-ITD on AML sustainability. Our data show that FLT3-ITD constitutively activates the STAT5 signaling pathway, which upregulates the expression of glycine amidinotransferase (GATM), the first rate-limiting enzyme of de novo creatine biosynthesis. Pharmacologic FLT3-ITD inhibition reduces intracellular creatinine levels through transcriptional downregulation of genes in the de novo creatine biosynthesis pathway. The same reduction can be achieved by cyclocreatine or genetic GATM knockdown with shRNA and is reflected in significant decrease of cell proliferation and moderate increase of cell apoptosis in FLT3-ITD-mutant cell lines. Those effects are at least partially mediated through the AMPK/mTOR signaling pathway. This study uncovers a previously uncharacterized role of creatine metabolic pathway in the maintenance of FLT3-ITD-mutant AML and suggests that targeting this pathway may serve as a promising therapeutic strategy for FLT3-ITD-positive AML. IMPLICATIONS: FLT3-ITD mutation in AML upregulates de novo creatine biosynthesis that we show can be suppressed to diminish the proliferation and survival of blast cells.

Overlapping but Disparate Inflammatory and Immunosuppressive Responses to SARS-CoV-2 and Bacterial Sepsis: An Immunological Time Course Analysis.

Both severe SARS-CoV-2 infections and bacterial sepsis exhibit an immunological dyscrasia and propensity for secondary infections. The nature of the immunological dyscrasias for these differing etiologies and their time course remain unclear. In this study, thirty hospitalized patients with SARS-CoV-2 infection were compared with ten critically ill patients with bacterial sepsis over 21 days, as well as ten healthy control subjects. Blood was sampled between days 1 and 21 after admission for targeted plasma biomarker analysis, cellular phenotyping, and leukocyte functional analysis via enzyme-linked immunospot assay. We found that circulating inflammatory markers were significantly higher early after bacterial sepsis compared with SARS-CoV-2. Both cohorts exhibited profound immune suppression through 21 days (suppressed HLA-DR expression, reduced mononuclear cell IFN-gamma production), and expanded numbers of myeloid-derived suppressor cells (MDSCs). In addition, MDSC expansion and ex vivo production of IFN-gamma and TNF-alpha were resolving over time in bacterial sepsis, whereas in SARS-CoV-2, immunosuppression and inflammation were accelerating. Despite less severe initial physiologic derangement, SARS-CoV-2 patients had similar incidence of secondary infections (23% vs 30%) as bacterial sepsis patients. Finally, COVID patients who developed secondary bacterial infections exhibited profound immunosuppression evident by elevated sPD-L1 and depressed HLA-DR. Although both bacterial sepsis and SARS-CoV-2 are associated with inflammation and immune suppression, their immune dyscrasia temporal patterns and clinical outcomes are different. SARS-CoV-2 patients had less severe early inflammation and organ dysfunction but had persistent inflammation and immunosuppression and suffered worse clinical outcomes, especially when SARS-CoV-2 infection was followed by secondary bacterial infection.

A Transcriptomic Severity Metric That Predicts Clinical Outcomes in Critically Ill Surgical Sepsis Patients.

OBJECTIVES: Clinically deployable methods for the rapid and accurate prediction of sepsis severity that could elicit a meaningful change in clinical practice are currently lacking. We evaluated a whole-blood, multiplex host-messenger RNA expression metric, Inflammatix-Severity-2, for identifying septic, hospitalized patients' likelihood of 30-day mortality, development of chronic critical illness, discharge disposition, and/or secondary infections. DESIGN: Retrospective, validation cohort analysis. SETTING: Single, academic health center ICU. PATIENTS: Three hundred thirty-five critically ill adult surgical patients with sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Whole blood was collected in PAXgene Blood RNA collection tubes at 24 hours after sepsis diagnosis and analyzed using a custom 29-messenger RNA classifier (Inflammatix-Severity-2) in a Clinical Laboratory Improvement Amendments certified diagnostic laboratory using the NanoString FLEX platform. Among patients meeting Sepsis-3 criteria, the Inflammatix-Severity-2 severity score was significantly better (p < 0.05) at predicting secondary infections (area under the receiver operating curve 0.71) and adverse clinical outcomes (area under the receiver operating curve 0.75) than C-reactive protein, absolute lymphocyte counts, total WBC count, age, and Charlson comorbidity index (and better, albeit nonsignificantly, than interleukin-6 and Acute Physiology and Chronic Health Evaluation II). Using multivariate logistic regression analysis, only combining the Charlson comorbidity index (area under the receiver operating curve 0.80) or Acute Physiology and Chronic Health Evaluation II (area under the receiver operating curve 0.81) with Inflammatix-Severity-2 significantly improved prediction of adverse clinical outcomes, and combining with the Charlson comorbidity index for predicting 30-day mortality (area under the receiver operating curve 0.79). CONCLUSIONS: The Inflammatix-Severity-2 severity score was superior at predicting secondary infections and overall adverse clinical outcomes compared with other common metrics. Combining a rapidly measured transcriptomic metric with clinical or physiologic indices offers the potential to optimize risk-based resource utilization and patient management adjustments that may improve outcomes in surgical sepsis. Hospitalized patients who are septic and present with an elevated IMX-SEV2 severity score and preexisting comorbidities may be ideal candidates for clinical interventions aimed at reducing the risk of secondary infections and adverse clinical outcomes.

The Hologic Aptima SARS-CoV-2 assay enables high ratio pooling saving reagents and improving turnaround time.

BACKGROUND: The Hologic Aptima™ TMA SARS-CoV-2 assay was employed to test pooled nasopharyngeal (NP) samples to evaluate the performance of pooled sample testing and characterize variables influencing results. METHODS: Results on 1033 previously tested NP samples were retrieved to characterize the relative light units (RLU) of SARS-CoV-2-positive samples in the tested population. The pooling strategy of combining 10 SARS-CoV-2 samples into one pool (10/1) was used in this study. The results were compared with neat sample testing using the same Aptima™ TMA SARS-CoV-2 assay and also the CDC RT-PCR and the Cepheid SARS-CoV-2 assays. RESULTS: The Aptima assay compares favorably with both CDC RT-PCR and the Cepheid SARS-CoV-2 assays. Once samples are pooled 10 to 1 as in our experiments, the resulting signal strength of the assay suffers. A divide opens between pools assembled from strong-positive versus only weak-positive samples. Pools of the former can be reliably detected with positive percent agreement (PPA) of 95.2%, while pools of the latter are frequently misclassified as negative with PPA of 40%. When the weak-positive samples with kRLU value lower than 1012 constitute 3.4% of the total sample profile, the assay PPA approaches 93.4% suggesting that 10/1 pooled sample testing by the Aptima assay is an effective screening tool for SARS-CoV-2. CONCLUSION: Performing pooled testing, one should monitor the weak positives with kRLU lower than 1012 or quantification cycle (Cq) value higher than 35 on an ongoing basis and adjust pooling approaches to avoid reporting false negatives.

Pathology Informatics and Robotics Strategies for Improving Efficiency of COVID-19 Pooled Testing.

The global rise of the coronavirus disease 2019 pandemic resulted in an exponentially increasing demand for severe acute respiratory syndrome coronavirus 2 testing, which resulted in shortage of reagents worldwide. This shortage has been further worsened by screening of asymptomatic populations such as returning employees, students, and so on, as part of plans to reopen the economy. To optimize the utilization of testing reagents and human resources, pool testing of populations with low prevalence has emerged as a promising strategy. Although pooling is an effective solution to reduce the number of reagents used for testing, the process of pooling samples together and tracking them throughout the entire workflow is challenging. To be effective, samples must be tracked into each pool, pool-tested and reported individually. In this article, we address these challenges using robotics and informatics.