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Background The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT–qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. Methods We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT–qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. Results After optimization, SwabExpress has a low limit of detection at 2–4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT–qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. Conclusion SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

PURPOSE: Desmoplastic infantile astrocytoma (DIA) and desmoplastic infantile ganglioglioma (DIG) are classified together as grade I neuronal and mixed neuronal-glial tumor of the central nervous system by the World Health Organization (WHO). These tumors are rare and have not been well characterized in terms of clinical outcomes. We aimed to identify clinical predictors of mortality and tumor recurrence/progression by performing an individual patient data meta-analysis (IPDMA) of the literature. METHODS: A systematic literature review from 1970 to 2020 was performed, and individualized clinical data for patients diagnosed with DIA/DIG were extracted. Aggregated data were excluded from collection. Outcome measures of interest were mortality and tumor recurrence/progression, as well as time-to-event (TTE) for each of these. Participants without information on these outcome measures were excluded. Cox regression survival analyses were performed to determine predictors of mortality and tumor recurrence / progression. RESULTS: We identified 98 articles and extracted individual patient data from 188 patients. The cohort consisted of 58.9% males with a median age of 7 months. The majority (68.1%) were DIGs, while 24.5% were DIAs and 7.5% were non-specific desmoplastic infantile tumors; DIAs presented more commonly in deep locations (p = 0.001), with leptomeningeal metastasis (p = 0.001), and was associated with decreased probability of gross total resection (GTR; p = 0.001). Gender, age, and tumor pathology were not statistically significant predictors of either mortality or tumor recurrence/progression. On multivariate survival analysis, GTR was a predictor of survival (HR = 0.058; p = 0.007) while leptomeningeal metastasis at presentation was a predictor of mortality (HR = 3.27; p = 0.025). Deep tumor location (HR = 2.93; p = 0.001) and chemotherapy administration (HR = 2.02; p = 0.017) were associated with tumor recurrence/progression. CONCLUSION: Our IPDMA of DIA/DIG cases reported in the literature revealed that GTR was a predictor of survival while leptomeningeal metastasis at presentation was associated with mortality. Deep tumor location and chemotherapy were associated with tumor recurrence / progression.

Infantile fibrosarcoma (IFS)/cellular congenital mesoblastic nephroma (cCMN) commonly harbors the classic ETV6-NTRK3 translocation. However, there are recent reports of mesenchymal tumors with IFS-like morphology harboring fusions of other receptor tyrosine kinases or downstream effectors, including NTRK1/2/3, MET, RET, and RAF1 fusions as well as one prior series with BRAF fusions. Discovery of these additional molecular drivers contributes to a more integrated diagnostic approach and presents important targets for therapy. Here we report the clinicopathologic and molecular features of 14 BRAF-altered tumors, of which 5 had BRAF point mutations and 10 harbored one or more BRAF fusions. Of the BRAF fusion-positive tumors, one harbored two BRAF fusions (FOXN3-BRAF, TRIP11-BRAF) and another harbored three unique alternative splice variants of EPB41L2-BRAF. Tumors occurred in ten males and four females, aged from birth to 32 years (median 6 months). Twelve were soft tissue based; two were visceral including one located in the kidney (cCMN). All neoplasms demonstrated ovoid to short spindle cells most frequently arranged haphazardly or in intersecting fascicles, often with collagenized stroma and a chronic inflammatory infiltrate. No specific immunophenotype was observed; expression of CD34, S100, and SMA was variable. To date, this is the largest cohort of BRAF-altered spindle cell neoplasms with IFS-like morphology, including not only seven novel BRAF fusion partners but also the first description of oncogenic BRAF point mutations in these tumors.

Objectives To assess current pathology resident training in genomic and molecular pathology. Methods The Training Residents in Genomics (TRIG) Working Group has developed survey questions for the pathology Resident In-Service Examination (RISE) since 2012. Responses to these questions, as well as knowledge questions, were analyzed. Results A total of 2,529 residents took the 2019 RISE. Since 2013, there has been an increase in postgraduate year 4 (PGY4) respondents indicating training in genomic medicine (58% to approximately 80%) but still less than almost 100% each year for molecular pathology. In 2019, PGY4 residents indicated less perceived knowledge and ability related to both genomic and traditional molecular pathology topics compared with control areas. Knowledge question results supported this subjective self-appraisal. Conclusions The RISE is a powerful tool for assessing the current state and also trends related to resident training in genomic pathology. The results show progress but also the need for improvement in not only genomic pathology but traditional molecular pathology training as well.

Introduction Beyond focal radiation, there is no consensus standard therapy for pediatric high-grade glioma (pHGG) and outcomes remain dismal. We describe the largest molecularly-characterized cohort of children with pHGG treated with a 3-drug maintenance regimen of temozolomide, irinotecan, and bevacizumab (TIB) following radiation. Methods We retrospectively reviewed 36 pediatric patients treated with TIB at Seattle Children's Hospital from 2009 to 2018 and analyzed survival using the Kaplan-Meier method. Molecular profiling was performed by targeted DNA sequencing and toxicities, steroid use, and palliative care utilization were evaluated. Results Median age at diagnosis was 10.9 years (18 months-18 years). Genetic alterations were detected in 26 genes and aligned with recognized molecular subgroups including H3 K27M-mutant (12), H3F3A G34-mutant (2), IDH-mutant (4), and hypermutator profiles (4). Fifteen patients (42%) completed 12 planned cycles of maintenance. Side effects associated with chemotherapy delays or modifications included thrombocytopenia (28%) and nausea/vomiting (19%), with temozolomide dosing most frequently modified. Median event-free survival (EFS) and overall survival (OS) was 16.2 and 20.1 months, with shorter survival seen in DIPG (9.3 and 13.3 months, respectively). Survival at 1, 2, and 5 years was 80%, 10% and 0% for DIPG and 85%, 38%, and 16% for other pHGG. Conclusion Our single-center experience demonstrates tolerability of this 3-drug regimen, with prolonged survival in DIPG compared to historical single-agent temozolomide. pHGG survival was comparable to analogous 3-drug regimens and superior to historical agents; however, cure was rare. Children with pHGG remain excellent candidates for the study of novel therapeutics combined with standard therapy.

Cirrhotic explanted livers occasionally have unexpected periodic acid-Schiff-diastase (PASD)-positive globules within the hepatocyte cytoplasm. It is often unclear whether this finding is a nonspecific consequence of cirrhosis or is indicative of an underlying alpha-1-antitrypsin deficiency (A1ATD) contributing to the cirrhosis. In this study, explanted livers were retrospectively evaluated for histopathology (including PASD status with confirmatory alpha-1-antitrypsin [A1AT] immunohistochemistry [IHC]), and chart review provided etiology of liver failure and general clinical parameters. Real-time polymerase chain reaction was used to detect A1AT genotype (SERPINA1 S and Z alleles) by melting curve analysis on liver explant tissue from selected cases. Of 196 explanted livers, 21 (11%) had PASD+ globules, which were significantly enriched in patients with a clinical diagnosis of nonalcoholic steatohepatitis (NASH; 47%) compared with other causes (P

BACKGROUND AND OBJECTIVES Pediatric sepsis quality improvement in emergency departments has been well described and associated with improved survival. Acute care (non-ICU inpatient) units differ in important ways, and optimal approaches to improving sepsis processes and outcomes in this setting are not yet known. Our objective was to increase the proportion of acute care sepsis cases in our health system with initial antibiotic order-to-administration time ≤60 minutes by 20% from a baseline of 43% to 52% by December 2020. METHODS Employing the Model for Improvement with broad stakeholder engagement, we developed and implemented interventions aimed at effective intervention for sepsis cases on acute care units. We analyzed process and outcome metrics over time using statistical process control charts. We used descriptive statistics to explore differences in antibiotic order-to-administration time and inform ongoing improvement. RESULTS We cared for 187 patients with sepsis over the course of our initiative. The proportion within our goal antibiotic order-to-administration time rose from 43% to 64% with evidence of special cause variation after our interventions. Of all patients, 66% experienced ICU transfer and 4% died. CONCLUSIONS We successfully decreased antibiotic order-to-administration time. We also introduced a novel model for sepsis response systems that integrates interventions designed for the complexities of acute care settings. We demonstrated impactful local improvements in the acute care setting where quality improvement reports and success have previously been limited.

BackgroundTesting programs have been utilized as part of SARS-CoV-2 mitigation strategies on university campuses, and it is not known which strategies successfully identify cases and contain outbreaks.ObjectiveEvaluation of a testing program to control SARS-CoV-2 transmission at a large university.DesignProspective longitudinal study using remote contactless enrollment, daily mobile symptom and exposure tracking, and self-swab sample collection. Individuals were tested if the participant was (1) exposed to a known case, developed new symptoms, or reported high-risk behavior, (2) a member of a group experiencing an outbreak, or (3) at baseline upon enrollment.SettingAn urban, public university during Autumn quarter of 2020ParticipantsStudents, staff, and faculty.MeasurementsSARS-CoV-2 PCR testing was conducted, and viral genome sequencing was performed.ResultsWe enrolled 16,476 individuals, performed 29,783 SARS-CoV-2 tests, and detected 236 infections. Greek community affiliation was the strongest risk factor for testing positive. 75.0% of positive cases reported at least one of the following: symptoms (60.8%), exposure (34.7%), or high-risk behaviors (21.5%). 88.1% of viral genomes (52/59) sequenced from Greek-affiliated students were genetically identical to at least one other genome detected, indicative of rapid SARS-CoV-2 spread within this group, compared to 37.9% (11/29) of genomes from non-Greek students and employees.LimitationsObservational study.ConclusionIn a setting of limited resources during a pandemic, we prioritized testing of individuals with symptoms and high-risk exposure during outbreaks. Rapid spread of SARS- CoV-2 occurred within outbreaks without evidence of further spread to the surrounding community. A testing program focused on high-risk populations may be effective as part of a comprehensive university-wide mitigation strategy to control the SARS-CoV-2 pandemic.

Background Placental derived cell‐free DNA (cfDNA), widely utilized for prenatal screening, may serve as a biomarker for preeclampsia. To determine whether cfDNA parameters are altered in preeclampsia, we conducted a case‐control study using prospectively collected maternal plasma (n=20 preeclampsia, n=22 normal) using our in‐house validated prenatal screening assay. Methods and Results Isolated cfDNA was quantified, sequenced using Illumina NextSeq 500, and the placental‐derived fraction was determined. Clinical and test characteristics were compared between preeclampsia and controls, followed by comparisons within the preeclampsia cohort dichotomized by cfDNA concentration. Lastly, cfDNA parameters in preeclampsia were correlated with markers of disease severity. Maternal age, body mass index, gestational age at delivery, cesarean rate, and neonatal birthweight were expectedly different between groups ( P ≤0.05). The placental‐derived cfDNA fraction did not differ between groups (21.4% versus 16.9%, P =0.06); however, total cfDNA was more than 10 times higher in preeclampsia (1235 versus 106.5 pg/µL, P P =0.01). The dichotomized preeclampsia group with the highest cfDNA concentration delivered earlier (33.2 versus 36.6 weeks, P =0.02) and had lower placental‐derived fractions (9.1% versus 21.4%, P =0.04). Among preeclampsia cases, higher total cfDNA correlated with earlier gestational age at delivery ( P =0.01) and higher maximum systolic blood pressure ( P =0.04). Conclusions At diagnosis, total cfDNA is notably higher in preeclampsia, whereas the placental derived fraction remains similar to healthy pregnancies. In preeclampsia, higher total cfDNA correlates with earlier gestational age at delivery and higher systolic blood pressure. These findings may indicate increased release of cfDNA from maternal tissue injury.

Pediatric mesenchymal tumors harboring variant NTRK fusions (ETV6-negative) are being increasingly described; however, the histologic and clinical features of these variant NTRK tumors and their relationship to classic infantile fibrosarcoma are not well characterized. A better understanding of the clinicopathologic features of these tumors is necessary, and would aid in both early diagnosis and treatment. Therefore, the aim of this study was to characterize a series of pediatric NTRK-rearranged mesenchymal tumors, including classic ETV6-NTRK3 fused tumors and tumors with variant (non-ETV6) NTRK fusions. The clinical features, morphology, immunophenotype, and genetics of 12 classic ETV6-NTRK3 fused infantile fibrosarcoma and 18 variant NTRK-rearranged mesenchymal tumors were evaluated. For both classic and variant groups, the age at diagnosis ranged from birth to 15 years (median, 4 mo) with no sex predilection; the most common sites involved were the extremities and trunk. The rate of local recurrence and metastasis were not significantly different (recurrence rate: 11% classic, 40% variant; metastatic rate: 18% classic, 25% variant). Classic and variant NTRK tumors had an overlapping spectrum of histologic features, containing haphazardly arranged primitive cells in a myxoid background and/or spindle cells in long fascicles. Both groups showed diffuse pan-TRK expression by immunohistochemistry. Otherwise, the immunoprofile was nonspecific, but similar between both groups. No statistical difference was seen in any clinicopathologic feature between the classic ETV6-NTRK3 and variant fusion cohorts. Pediatric NTRK-rearranged mesenchymal tumors with both classic and variant fusions likely represent a spectrum of disease with shared, recognizable cliniopathologic features.

Purpose Next-generation sequencing is gaining acceptance as a clinical tool to aid diagnosis and guide treatment of pediatric cancer. Prior pilot studies have evaluated the feasibility and utility of clinical genomic profiling in a subset of selected patients with brain tumors. Here, we report an unselected prospective cohort study to evaluate the clinical use of universal targeted sequencing in pediatric patients with brain tumors. Methods We applied a universal sequencing protocol for all tumors of the CNS undergoing diagnostic workup at Seattle Children’s Hospital during the study period of November 2015 to November 2016. All tumors were sequenced using the UW-OncoPlex platform, which is a multiplexed targeted deep gene sequencing panel that detects genetic alterations in 262 cancer-related genes performed in a College of American Pathologists–accredited Clinical Laboratory Improvements Amendments–certified laboratory. Results Eighty-eight patients underwent diagnostic evaluation during the study period, of which 85 tumors (95%) yielded sufficient DNA for sequencing, including 59 newly diagnosed and 26 relapsed. Clinically relevant genetic alterations were identified in 68 of 85 patients (80%). Of these, 57 (67%) had disease-defining or disease-modifying mutations, 44 (52%) had potentially targetable mutations, and 31 (36%) had mutations requiring germline follow-up. As of the last follow-up, seven patients had been prescribed targeted agents on the basis of sequencing results, and nine had confirmed deleterious germline mutations. Conclusion Clinically validated molecular profiling of pediatric brain tumors aids diagnosis and treatment of patients with a variety of high- and low-grade primary and relapsed pediatric brain tumors.

Unsupervised upper respiratory specimen collection is a key factor in the ability to massively scale SARS-CoV-2 testing. But there is concern that unsupervised specimen collection may produce inferior samples. Across two studies that included unsupervised at-home mid-turbinate specimen collection, ∼1% of participants used the wrong end of the swab. We found that molecular detection of respiratory pathogens and a human biomarker were comparable between specimens collected from the handle of the swab and those collected correctly. Older participants were more likely to use the swab backwards. Our results suggest that errors made during home-collection of nasal specimens do not preclude molecular detection of pathogens and specialized swabs may be an unnecessary luxury during a pandemic.

In October 2020, an outbreak of at least 50 COVID-19 cases was reported surrounding individuals employed at or visiting the White House. Here, we applied genomic epidemiology to investigate the origins of this outbreak. We enrolled two individuals with exposures linked to the White House COVID-19 outbreak into an IRB-approved research study and sequenced their SARS-CoV-2 infections. We find these viral sequences are identical to each other, but are distinct from over 190,000 publicly available SARS-CoV-2 genomes. These genomes fall as part of a lineage circulating in the USA since April or May 2020 and detected in Virginia and Michigan. Looking forwards, sequencing of additional community SARS-CoV-2 infections collected in the USA prior to October 2020 may shed further light on its geographic ancestry. In sequencing of SARS-CoV-2 infections collected after October 2020, it may be possible to identify infections that likely descend from the White House COVID-19 outbreak.

Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs). These oncohistone mutations dominantly inhibit histone H3K27 trimethylation and silencing, but it is unknown how oncohistone type affects gliomagenesis. We show that the genomic distributions of H3.1 and H3.3 oncohistones in human patient-derived DMG cells are consistent with the DNA replication-coupled deposition of histone H3.1 and the predominant replication-independent deposition of histone H3.3. Although H3K27 trimethylation is reduced for both oncohistone types, H3.3K27M-bearing cells retain some domains, and only H3.1K27M-bearing cells lack H3K27 trimethylation. Neither oncohistone interferes with PRC2 binding. Using Drosophila as a model, we demonstrate that inhibition of H3K27 trimethylation occurs only when H3K27M oncohistones are deposited into chromatin and only when expressed in cycling cells. We propose that oncohistones inhibit the H3K27 methyltransferase as chromatin patterns are being duplicated in proliferating cells, predisposing them to tumorigenesis.

Detection of Covid-19 through a Citywide Surveillance Platform The Seattle Flu study, initiated in 2018, mailed kits for home collection of midnasal swabs to people reporting respiratory symptoms. ...

Background Inequitable use of next-generation sequencing (NGS) testing for cancer risk and treatment can contribute to heath disparity. Consequently, it is important to assess the population receiving this testing. In this article, we characterize the population receiving both germline and somatic NGS testing for cancer predisposition and precision oncology at the Genetics and Solid Tumors Laboratory of the University of Washington Medical Center. Methods The general demographics, including ancestry, of patients receiving somatic testing to identify genes related to cancer treatment or prognosis, diagnosis, or germline testing for heritable cancer risk from January 2015 to July 2017 were characterized. Ancestry was determined using single nucleotide variant data and documented pedigree. The demographics of the patient population receiving testing were compared with a reference population comprising patients receiving care from the University of Washington Medical Center with a diagnosis of malignant neoplasm of breast, ovary, colon, rectum, or prostate between January 2015 and May 2018. Results A total of 2210 unique patients were included in this study. Women composed 66% of our total tested population. Patients of European ancestry composed 78% of the tested cohort. The percentages of American Indian/Alaskan Native and Native Hawaiian/Other Pacific Islander in the cohort receiving NGS testing were significantly different than their respective distributions in the reference cohort. Conclusions Characterizing the demographics of patients receiving NGS testing for cancer predisposition and precision oncology using single nucleotide variant data and documented pedigree may help identify potential health disparities.

Structured AbstractBackgroundThe urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse transcription PCR (RT-qPCR) (1). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce (2). To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction.MethodsWe evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral activation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance.ResultsAfter optimization, SwabExpress has a low limit of detection at 2-4 molecules/uL, 100% sensitivity, and 99.4% specificity when compared side-by-side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time.ConclusionSwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

Pharmacy benefit can be purchased as part of an integrated medical and pharmacy health package-a carve-in model-or purchased separately and administered by an external pharmacy benefit manager-a carve-out model. Limited peer-reviewed information is available assessing differences in use and medical costs among carve-in versus carve-out populations.To compare total medical costs per member per year (PMPY) and utilization between commercially self-insured members receiving carve-in to those receiving carve-out pharmacy benefits overall and by 7 chronic condition subgroups.This study used deidentified data of members continuously enrolled in Cambia Health Solutions self-insured Blue plans without benefit changes from 2017 through 2018. Cambia covers 1.6 million members in Oregon, Washington, Idaho, and Utah. The medical cost PMPY comparison was performed using multivariable general linear regression with gamma distribution adjusting for age, gender, state, insured group size, case or disease management enrollment, 7 chronic diseases, risk score (illness severity proxy), and plan paid to total paid ratio (benefit richness proxy). Medical event objectives were assessed using multivariable logistic regression comparing odds of hospitalization and emergency department (ED) visit adjusting for the same covariates. Sensitivity analyses repeated the medical cost PMPY comparison excluding high-cost members, greater than $250,000 annually. Chronic condition subgroup analyses were performed using the same methods separately for members having asthma, coronary artery disease, chronic obstructive pulmonary disease, heart failure, diabetes mellitus, depression, and rheumatoid arthritis.There were 205,835 carve-in and 125,555 carve-out members meeting study criteria. Average age (SD) was 34.2 years (18.6) and risk score (SD) 1.1 (2.3) for carve-in versus 35.2 years (19.3) and 1.1 (2.4), respectively, for carve-out. Members with carve-in benefits had lower medical costs after adjustment (4%,These findings suggest that integrated, carve-in pharmacy and medical benefits are associated with lower medical costs, fewer hospitalizations, and fewer ED visits. This study focused on associations, and defining causation was not in scope. Possible reasons for these findings include plan access to both medical and pharmacy data and data-informed care management and coordination. Future research should include investigation of integrated data use and its effect across the spectrum of integrated health plan offerings, provider partnerships, and analytic strategies, as well as inclusion of analyzing pharmacy costs to encompass total cost of care.This study received no external funding. The study was jointly conducted by employees of Cambia Health Solutions and Prime Therapeutics, a pharmacy benefit manager servicing Cambia Health Solutions. Smith, Lam, Lockwood, and Pegus are employees of Cambia Health Solutions. Qiu and Gleason are employees of Prime Therapeutics.

Genetic data are rapidly becoming part of tumor classification and are integral to prognosis and predicting response to therapy. Current molecular tumor profiling relies heavily on tissue resection or biopsy. Tissue profiling has several disadvantages in tumors of the central nervous system, including the challenge associated with invasive biopsy, the heterogeneous nature of many malignancies where a small biopsy can underrepresent the mutational profile, and the frequent lack of obtaining a repeat biopsy, which limits routine monitoring to assess therapy response and/or tumor evolution. Circulating tumor, cell-free DNA (cfDNA), has been proposed as a liquid biopsy to address some limitations of tissue-based genetics. In cancer patients, a portion of cfDNA is tumor-derived and may contain somatic genetic alterations. In central nervous system (CNS) neoplasia, plasma tumor-derived cfDNA is very low or absent, likely due to the blood brain barrier. Interrogating cfDNA in cerebrospinal fluid (CSF) has several advantages. Compared to blood, CSF is paucicellular and therefore predominantly lacks non-tumor cfDNA; however, patients with CNS-limited tumors have significantly enriched tumor-derived cfDNA in CSF. In patients with metastatic CNS disease, mutations in CSF cfDNA are most concordant with the intracranial process. CSF cfDNA can also occasionally uncover additional genetic alterations absent in concurrent biopsy specimens, reflecting tumor heterogeneity. Although CSF is enriched for tumor-derived cfDNA, absolute quantities are low. Highly sensitive, targeted methods including next-generation sequencing and digital PCR are required to detect mutations in CSF cfDNA. Additional technical and bioinformatic approaches also facilitate enhanced ability to detect tumor mutations in CSF cfDNA.

Human cytomegalovirus (CMV) infections comprise a leading cause of newborn impairments worldwide and are pervasive concerns among the immunocompromised. Quantification of CMV viral loads is increasingly used to guide definitions of CMV disease but standardization of CMV quantitation remains problematic, mostly due to differences in qPCR amplicon sizes between clinical laboratories. Here, we used plasma cfDNA sequencing data from 2,208 samples sent for non-invasive prenatal aneuploidy screening to detect CMV and precisely measure the length of CMV fragments in human plasma. CMV reads were identified in 120 (5.4%) samples. Median cfDNA fragment size derived from CMV was significantly shorter than cfDNA derived from human chromosomes (103 vs 172 bp, p rd percentile of human cfDNA. Sequencing of cfDNA from seven plasma samples from transplant patients positive for CMV confirmed the extraordinarily short nature of CMV cfDNA fragment size with a median length of 149 bp. We further show that these high-resolution measurements of CMV DNA fragment size accurately predict measured discrepancies in serum viral load measurements by different qPCR assays. These results highlight the exceptionally fragmented nature of CMV cfDNA and illustrate the promise of plasma cfDNA sequencing for quantitating viral loads through detection of fragments that would be unrecoverable by qPCR.

Hemoglobin (Hb) is the iron-containing protein within red blood cells, responsible for oxygen transport and delivery. Functional Hb is a tetramer consisting of four globular protein subunits organized as two heterodimers. Proportions of these subunits change over time during embryogenesis. Hb abnormalities may be categorized as structural hemoglobinopathies or thalassemias. Structural hemoglobinopathies occur when there is a mutation in a monomer, leading to a structurally abnormal Hb subunit. Thalassemias occur when there is a quantitative decrease in the overall amount of an Hb subunit. Both abnormalities may have significant clinical implications. Detection of Hb abnormalities relies heavily on the clinical laboratory. A variety of biochemical and molecular techniques are applied for characterization of hemoglobinopathies. Diagnostic testing for Hb disorders is frequently performed in the evaluation of anemia and microcytosis, pulmonary hypertension, and preoperative anesthesia evaluation. Testing is more recently increasing in volume due to implementation of prenatal and population-based screening programs.

Desmoplastic infantile ganglioglioma (DIG) and desmoplastic infantile astrocytoma (DIA) are extremely rare tumors that typically arise in infancy; however, these entities have not been well characterized in terms of genetic alterations or clinical outcomes. Here, through a multi-institutional collaboration, the largest cohort of DIG/DIA to date is examined using advanced laboratory and data processing techniques. Targeted DNA exome sequencing and DNA methylation profiling were performed on tumor specimens obtained from different patients (n = 8) diagnosed histologically as DIG/DIGA. Two of these cases clustered with other tumor entities, and were excluded from analysis. The remaining 16 cases were confirmed to be DIG/DIA by histology and by DNA methylation profiling. Somatic BRAF gene mutations were discovered in 7 instances (43.8%); 4 were BRAFV600E mutations, and 3 were BRAFV600D mutations. Three instances of malignant transformation were found, and sequencing of the recurrence demonstrated a new TP53 mutation in one case, new ATRX deletion in one case, and in the third case, the original tumor harbored an EML4–ALK fusion, also present at recurrence. DIG/DIA are distinct pathologic entities that frequently harbor BRAFV600 mutations. Complete surgical resection is the ideal treatment, and overall prognosis is excellent. While, the small sample size and incomplete surgical records limit a definitive conclusion about the risk of tumor recurrence, the risk appears quite low. In rare cases with wild-type BRAF, malignant progression can be observed, frequently with the acquisition of other genetic alterations. Implications: DIG/DIA are a distinct molecular entity, with a subset frequently harboring either BRAFV600E or BRAFV600D mutations. Mol Cancer Res; 16(10); 1491–8. ©2018 AACR.

We describe a case of acute liver failure and myopericarditis due to herpes simplex virus–1 (HSV-1) in an immunocompetent adult. We estimate that, at the height of viremia, the patient contained a quantity of HSV-1 virions approaching that of human cells. The patient recovered with acyclovir that was dose-adjusted for neurotoxicity and developed a vigorous anti-HSV-1 T-cell response.

Herein, we examined if acute or chronic resistance exercise affected markers of skeletal muscle long interspersed nuclear element-1 (LINE-1) retrotransposon activity. In study 1, 10 resistance-trained college-aged men performed three consecutive daily back squat sessions, and vastus lateralis biopsies were taken before (Pre), 2 h following session 1 (Post1), and 3 days following session 3 (Post2). In study 2, 13 untrained college-aged men performed a full-body resistance training program (3 days/wk), and vastus lateralis biopsies were taken before ( week 0) and ~72 h following training cessation ( week 12). In study 1, LINE-1 mRNA decreased 42–48% at Post1 and 2 ( P < 0.05), and reverse transcriptase (RT) activity trended downward at Post2 (−37%, P = 0.067). In study 2, LINE-1 mRNA trended downward at week 12 (−17%, P = 0.056) while LINE-1 promoter methylation increased (+142%, P = 0.041). Open reading frame (ORF)2p protein expression (−24%, P = 0.059) and RT activity (−26%, P = 0.063) also trended downward by week 12. Additionally, changes in RT activity versus satellite cell number were inversely associated ( r = −0.725, P = 0.008). Follow-up in vitro experiments demonstrated that 48-h treatments with lower doses (1 μM and 10 μM) of efavirenz and nevirapine (non-nucleoside RT inhibitors) increased myoblast proliferation ( P < 0.05). However, we observed a paradoxical decrease in myoblast proliferation with higher doses (50 μM) of efavirenz and delavirdine. This is the first report suggesting that resistance exercise downregulates markers of skeletal muscle LINE-1 activity. Given our discordant in vitro findings, future research is needed to thoroughly assess whether LINE-1-mediated RT activity enhances or blunts myoblast, or primary satellite cell, proliferative capacity.

Maternal biologic factors can affect the fetal fraction in cell-free DNA-based prenatal screening assays, thereby limiting the effectiveness. Higher rates of indeterminate results because of a low fetal fraction have been described in cases of maternal autoimmune disease in pregnancy. Existing studies are confounded by the concomitant maternal use of anticoagulants, which may independently influence the test characteristics.This study aimed to evaluate the differences in fetal fraction, indeterminate results, and total cell-free DNA concentration among women with an autoimmune disease in comparison with controls, using our in-house developed, noninvasive prenatal screening platform in the absence of maternal anticoagulation use.This was a retrospective, single institution cohort study of a previously validated, cell-free DNA-based, noninvasive prenatal screening assay using a low-pass whole-genome sequencing platform between 2017 and 2019. A diagnosis of an autoimmune disease included systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and others. Immunomodulator therapies included biologics, corticosteroids, hydroxychloroquine, azathioprine, and intravenous immunoglobulin. Women who were using anticoagulants were excluded. We evaluated the association between autoimmune disease and fetal cell-free DNA fraction, indeterminate results, and total cell-free DNA concentration using univariate and multivariate analyses, stratified according to immunomodulator therapy and adjusted for body mass index, fetal sex, and gestational age at sample collection.A total of 1445 patients met inclusion criteria. Of those, 43 women had a confirmed autoimmune disease, with 25 of those not on immunomodulator therapy and 18 on immunomodulator therapy. The mean fetal fraction for women with an autoimmune disease was significantly lower than for controls (9.7% vs 11.9%; P=.004). The rate of indeterminate results was significantly higher among women with an autoimmune disease than among controls (16.3% vs 3.5%; P.001). The total cell-free DNA concentration was not statistically different between the groups (94.8 pg/µL for women with an autoimmune disease vs 83.9 pg/µL for controls; P=.06). In a logistic regression, women with an autoimmune disease had significantly higher odds of receiving an indeterminate result than controls, (adjusted odds ratio, 5.3; 95% confidence interval, 2.0-14.2). Linear regression analysis showed a significant negative association between having an autoimmune disease and the fetal cell-free DNA fraction (aβ, -2.1; 95% confidence interval, -3.4 to -0.6). When stratifying by treatment status, the mean fetal fraction was 9.8%, 9.6%, and 11.9% for women with an autoimmune disease not on immunomodulator therapy, women with an autoimmune disease on immunomodulator therapy, and the controls, respectively (P=.02). The rate of indeterminate results increased in a stepwise fashion from 3.5% to 11.1% to 20.0% for controls, women with an autoimmune disease on immunomodulator therapy, and women with an autoimmune disease not on immunomodulator therapy, respectively (P.001). Logistic regression analysis demonstrated higher odds of an indeterminate result for women with an autoimmune disease not on immunomodulator therapy than for controls, (adjusted odds ratio, 7.3; 95% confidence interval, 2.3-22.5). There was a negative association between women with an autoimmune disease not on immunomodulator therapy and the fetal fraction when compared with controls (aβ, -2.2; 95% confidence interval, -4.2 to -0.3).Women with an autoimmune disease have lower fetal cell-free DNA fractions and higher rates of indeterminate results than women without an autoimmune disease. There was no difference in total cell-free DNA concentration. Treatment of maternal autoimmune diseases with immunomodulator therapy may decrease the indeterminate result rate.

Importance The association between COVID-19 symptoms and SARS-CoV-2 viral levels in children living in the community is not well understood. Objective To characterize symptoms of pediatric COVID-19 in the community and analyze the association between symptoms and SARS-CoV-2 RNA levels, as approximated by cycle threshold (Ct) values, in children and adults. Design, Setting, and Participants This cross-sectional study used a respiratory virus surveillance platform in persons of all ages to detect community COVID-19 cases from March 23 to November 9, 2020. A population-based convenience sample of children younger than 18 years and adults in King County, Washington, who enrolled online for home self-collection of upper respiratory samples for SARS-CoV-2 testing were included. Exposures Detection of SARS-CoV-2 RNA by reverse transcription–polymerase chain reaction (RT-PCR) from participant-collected samples. Main Outcomes and Measures RT-PCR–confirmed SARS-CoV-2 infection, with Ct values stratified by age and symptoms. Results Among 555 SARS-CoV-2–positive participants (mean [SD] age, 33.7 [20.1] years; 320 were female [57.7%]), 47 of 123 children (38.2%) were asymptomatic compared with 31 of 432 adults (7.2%). When symptomatic, fewer symptoms were reported in children compared with adults (mean [SD], 1.6 [2.0] vs 4.5 [3.1]). Symptomatic individuals had lower Ct values (which corresponded to higher viral RNA levels) than asymptomatic individuals (adjusted estimate for children, −3.0; 95% CI, −5.5 to −0.6;P = .02; adjusted estimate for adults, −2.9; 95% CI, −5.2 to −0.6;P = .01). The difference in mean Ct values was neither statistically significant between symptomatic children and symptomatic adults (adjusted estimate, −0.7; 95% CI, −2.2 to 0.9;P = .41) nor between asymptomatic children and asymptomatic adults (adjusted estimate, −0.6; 95% CI, −4.0 to 2.8;P = .74). Conclusions and Relevance In this community-based cross-sectional study, SARS-CoV-2 RNA levels, as determined by Ct values, were significantly higher in symptomatic individuals than in asymptomatic individuals and no significant age-related differences were found. Further research is needed to understand the role of SARS-CoV-2 RNA levels and viral transmission.

A 32-year-old G3P2002 (3 pregnancies, 2 births at full term, 0 preterm, 0 abortions, 2 still living) pregnant woman of Filipino ancestry was referred to a tertiary institution for severe intrauterine growth restriction (IUGR)4 at 20 weeks' gestation. Ultrasound showed fetal size uniformly less than expected, with measurements

Objectives The rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented. UW-OncoPlex addresses this through the adoption of a modular panel capable of redesign as significant alterations are identified. We describe the validation of OncoPlex version 6 (OPXv6) for the detection of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), structural variants (SVs), microsatellite instability (MSI), and tumor mutational burden (TMB) in a panel of 340 genes. Design One hundred twelve samples with diverse diagnoses were comprised of formalin-fixed-paraffin-embedded tissue, fresh-frozen tissue, plasma, peripheral blood, bone marrow, saliva, and cell-line DNA. Libraries were prepared from genomic and cell-free DNA, hybridized to a custom panel of xGen Lockdown probes, and sequenced on Illumina platforms. Sequences were processed through a custom bioinformatics pipeline, and variant calls were compared to prior orthogonal clinical results. Results Accuracy was 99% for SNVs ≥5% allele frequency, 98% for indels, 97% for SVs, 99% for CNVs, 100% for MSI, and 100% for TMB (compared to previous OncoPlex versions). Library preparation turnaround time decreased by 40%, and sequencing quality improved with a 2.5-fold increase in average sequencing coverage and 4-fold increase in percent on-target. Conclusions OPXv6 demonstrates improvements over prior UW-OncoPlex versions including reduced capture cost, improved sequencing quality, and decreased time to results. The modular capture probe design also provides a nimble laboratory response in addressing the expansions necessary to meet the needs of the continuously evolving field of molecular oncology., Highlights • OncoPlex version 6 is a targeted, comprehensive next generation sequencing panel. • The panel of 340 genes includes full coverage of BRCA1/2 and mismatch repair genes. • While sequencing quality increased, library preparation time and cost decreased. • The panel is modular and capable of rapid redesign and expansion of targeted genes.

Congenital human cytomegalovirus (CMV) infections are the leading cause of newborn hearing and central nervous system impairments worldwide. Currently, routine prenatal screening for congenital CMV is not performed in the United States and confirmation in suspected perinatal cases requires invasive sampling by amniocentesis. We hypothesized that detection of CMV from maternal cell-free DNA (cfDNA) plasma could provide a non-invasive indicator of congenital CMV infection. We analyzed sequence data from 2,208 individuals undergoing routine non-invasive prenatal aneuploidy screening at the University of Washington. CMV reads were identified in 117 (5.3%) samples. Positive samples were stratified based on CMV reads per million sample reads (RPM), resulting in ten samples being classified as strong positive (RPM > 0.3) and 107 as intermediate positive (0.01rd percentile of human cfDNA insert size. In addition, CMV cfDNA fragment lengths were distributed over a wider range than human cfDNA reads. These studies reveal the highly fragmented nature of CMV cfDNA and offer precise measurements of its length: these features likely explain discrepancies in serum CMV viral loads measurements determined by different qPCR assays, despite widespread efforts to standardize results. More work is required to determine how detection of CMV from maternal cfDNA can be best used as tool for congenital CMV screening or diagnosis.

INTRODUCTION: Targeted sequencing of somatic DNA obtained from pediatric brain tumor tissue is of clinical utility in forming an integrated molecular and histologic diagnosis as well as evaluating therapeutic targets. The evaluation of cell-free DNA (cfDNA) from blood or cerebrospinal fluid (CSF) has not yet been clinically validated in pediatric brain tumors. METHODS: Using a clinically validated targeted 262 next-generation gene sequencing platform, we evaluated cfDNA in 15 subjects with CSF and plasma samples paired with pediatric brain tumor tissue including medulloblastoma (n=7), atypical teratoid-rhabdoid tumor (n=2), diffuse midline glioma (n=4), pilocytic astrocytoma and pleomorphic xanthoastrocytoma (n=1 each). Informed consent was obtained from all subjects and this study was approved by the institutional review board. CSF was obtained at the time of surgery or from ventriculostomy in 3 cases each, and from diagnostic lumbar puncture in the remainder. RESULTS: cfDNA from CSF was successfully isolated from 8/15 (53%) subjects, and was sufficient for sequencing in 6/8 samples. Somatic mutations and copy-number changes found in cfDNA from CSF corresponded to changes found by tumor sequencing in all cases. Tumor-derived cfDNA was detected in as little as 200 microliters of CSF, and in the absence of cytological evidence of tumor. Tumor-derived cfDNA was not detected in plasma samples. CONCLUSION: The evaluation of cfDNA from CSF shows promise for clinical applications in pediatric brain tumor diagnosis and disease monitoring. A longitudinal prospective study of cfDNA detection in CSF for disease monitoring is planned.

The role of social support in assisting youth in developed countries cope with their HIV diagnosis has been examined through a vast body of research, yet, there remains a gap in research around the effects of social support among youth living in sub-Saharan African countries including Kenya. This study aimed to examine the role of social support among Kenyan youth living with HIV, specifically with regard to the variations in influences of this social support. We conducted semi-structured focus group discussions with youth (ages 18 to 27) living in the informal urban settlement of Kibera in Nairobi, Kenya (n = 53). Data analysis followed a phenomenological inquiry framework, and seven major categories of perceived social support influences were identified: (1) linkage to services, (2) antiretroviral (ARV) adherence, (3) self-acceptance of HIV status, (4) healthy and positive living, (5) understanding of what it means to be living with HIV, (6) HIV status disclosure, and (7) family and occupational strengthening. The findings from this study suggest that Kenyan youth living with HIV can benefit from social support in a multitude of ways and can occur across several socio-ecological levels. Future research should further examine these influences, specifically regarding intervention development across socio-ecological levels.

Background: Lab tests such as activated partial thromboplastin time (aPTT) or anti–factor Xa (anti-Xa) levels are typically used to monitor intravenous unfractionated heparin (IV heparin), with recent evidence suggesting that anti-Xa levels may provide a more accurate measure of anticoagulation. Objective: The Lexington Veterans Affairs Health Care System transitioned from using aPTT to anti-Xa levels in January 2017. This study was conducted to evaluate the efficacy and safety of this change. Methods: This was a retrospective cohort study comparing all patients receiving IV heparin per protocol for at least 24 hours from August 1, 2016, to January 31, 2017 (aPTT group), and February 1, 2017, to July 31, 2017 (anti-Xa group). The primary objective was a comparison of IV heparin doses required to achieve goal range between the 2 cohorts. Secondary objectives included a comparison of time to therapeutic goal, percentage of time within goal range, number of rate changes until therapeutic goal, and adverse outcomes, such as number of bleeds. Results: A total of 155 patients were included in this study. Significantly higher IV heparin doses were required to achieve therapeutic goal in the anti-Xa group, despite significantly fewer IV heparin rate changes required. Anti-Xa monitoring was not associated with an increased risk of adverse events. Conclusion and Relevance: Significantly higher IV heparin doses were required to achieve therapeutic anti-Xa levels after transitioning from an aPTT-based protocol in the largely unstudied veteran population. However, the transition from aPTT to anti-Xa monitoring appears safe and efficacious in these patients.

This project investigated whey protein and/or carbohydrate supplementation effects on musculoskeletal injury (MSI) outcomes. Four groups of Initial Entry Training soldiers consumed either: (1) one protein (38.6 g, 293 kcal), (2) one carbohydrate (63.4 g, 291 kcal), (3) two protein (77.2 g, 586 kcal), or (4) two carbohydrate servings/day (126.8 g, 582 kcal) after physical training and before bed, or before bed only. Odds Ratio, Chi-square and Wilcoxon ranked-sum test compared supplementation/no supplementation, number of servings, and protein/carbohydrate for MSI and limited/missed duty rates and limited/missed training days. Non-matched pairs group averages were compared to 2015/2016 historical data. Non-supplemented soldiers were approximately 5&times, more likely to sustain a MSI (&chi, 2 = 58.48, p &lt, 0.001) and 4&times, more likely to miss training (&chi, 2 = 9.73, p = 0.003) compared to two servings. Non-supplemented soldiers missed five additional training days compared to two servings (W = 6059.5, p = 0.02). Soldiers consuming one serving were approximately 3&times, more likely to sustain a MSI than two servings (&chi, 2 = 9.55, p = 0.002). There was no difference in limited/missed duty rates or limited/missed training days between consuming one or two servings. There was no difference between consuming one serving versus no supplementation or protein versus carbohydrate supplementation for any outcome variable. Soldiers consuming 2 servings/day of protein or carbohydrate had lower MSI rates, limited/missed duty rates, and limited/ missed training days compared to non-supplemented soldiers.

Purpose of this retrospective study was to investigate if serum markers, men's age, interval since vasectomy, BMI, testicular size and smoking could predict the success of epididymal or testicular sperm aspiration (PESA/TESA) in vasectomized men. Forty-four consecutively performed PESA/TESA procedures were reviewed retrospectively. Motile sperm was retrieved from 77.3% of PESA/TESA procedures. Mean serum Inhibin-B (Inh-B) level tended to be higher in men who had motile sperm retrieved compared to those who had not (180.3 versus 126.2 pg/ml, p = 0.05). Univariate analysis identified serum Inh-B to be the only predictor of PESA/TESA success (r = 0.32, CI: 0.006-0.584, p = 0.046). Serum FSH, LH, T levels, age, BMI, smoking status and interval since vasectomy did not correlate with the PESA/TESA outcome. Inh-B could modestly discriminate between successful and unsuccessful PESA/TESA (AUC= 0.70) with high positive (89.5%) but low negative prediction (36.8%); 58.6% sensitivity and 77.7% specificity at the optimum cut-off level of 166 pg/ml. Positive outcome was only 50% when the Inh-B level was below 100 pg/ml. It is concluded that a high serum Inh-B might reliably predict successful PESA/TESA in vasectomized men. More invasive sperm retrieval procedures could be reserved for men with very low Inh-B or failed PESA/TESA. Future studies with adequate power may confirm our findings.

e13681 Background: Cell-free tumor DNA or circulating tumor DNA tests are increasingly used in clinical care to detect somatic mutations from solid tumors. However, data on laboratory performance characteristics using standardized samples is limited. Methods: Well-characterized reference materials were used for the College of American Pathologists (CAP) cell-free tumor DNA proficiency testing surveys, which consisted of stabilized DNA fragmented to simulate cell-free DNA in a synthetic plasma matrix. For the 2018A, 2018B, 2019A and 2019B surveys, laboratories tested for hotspot mutations (single and dinucleotide sequence changes) in EGFR, BRAF, KRAS, NRAS, and IDH1 at variant allele fractions ranging from 0.1% - 1.0%. As per CAP proficiency testing standards, results were scored according to the known mutation(s) engineered at designated variant allele fractions in each PT sample. Nine laboratories were excluded from analysis because they provided incomplete results. Statistical significance was calculated using a multivariate logistic regression model. Results: In 2018 and 2019, 101 laboratories submitted survey results for at least one proficiency testing mailing. There were 5088 total proficiency testing responses for EGFR, BRAF, KRAS, NRAS, and IDH1 mutations across 12 different samples. For the 3585 responses submitted for BRAF, KRAS, NRAS, and IDH1, sensitivity ranged from 94.6 – 100%, while specificity exceeded 99%. There were no significant differences in performance between analytical methodologies for BRAF, KRAS, NRAS, and IDH1 mutations. Performance characteristics for EGFR mutations among the 1503 responses showed a combined sensitivity of 87.1% and specificity of 98.7%. For laboratories detecting mutations in EGFR, next-generation sequencing methods exhibited a sensitivity (true positivity) of 95.7% while the sensitivity of non-NGS methods was lower at 81.7% ( P= 0.02). Conclusions: These findings demonstrate high sensitivity and specificity for clinical laboratories performing cell-free tumor DNA tests. For EGFR mutations, NGS outperformed non-NGS methods. These data suggest excellent overall agreement among laboratories performing clinical cell-free tumor DNA tests. Further investigation across variant allele fractions and additional variant types is warranted.

We investigated the effects of whey protein (WP) supplementation on body composition and physical performance in soldiers participating in Army Initial Entry Training (IET). Sixty-nine, male United States Army soldiers volunteered for supplementation with either twice daily whey protein (WP, 77 g/day protein, ~580 kcal/day, n = 34, age = 19 &plusmn, 1 year, height = 173 &plusmn, 6 cm, weight = 73.4 &plusmn, 12.7 kg) or energy-matched carbohydrate (CHO) drinks (CHO, 127 g/day carbohydrate, ~580 kcal/day, n = 35, age = 19 &plusmn, 5 cm, weight = 72.3 &plusmn, 10.9 kg) for eight weeks during IET. Physical performance was evaluated using the Army Physical Fitness Test during weeks two and eight. Body composition was assessed using 7-site skinfold assessment during weeks one and nine. Post-testing push-up performance averaged 7 repetitions higher in the WP compared to the CHO group (F = 10.1, p &lt, 0.001) when controlling for baseline. There was a significant decrease in fat mass at post-training (F = 4.63, p = 0.04), but no significant change in run performance (F = 3.50, p = 0.065) or fat-free mass (F = 0.70, p = 0.41). Effect sizes for fat-free mass gains were large for both the WP (Cohen&rsquo, s d = 0.44) and CHO (Cohen&rsquo, s d = 0.42) groups. WP had a large effect on fat mass (FM) loss (Cohen&rsquo, s d = &minus, 0.67), while CHO had a medium effect (Cohen&rsquo, 0.40). Twice daily supplementation with WP improved push-up performance and potentiated reductions in fat mass during IET training in comparison to CHO supplementation.

It is currently unclear as to whether sex hormones are significantly affected by soy or whey protein consumption. Additionally, estrogenic signaling may be potentiated via soy protein supplementation due to the presence of phytoestrogenic isoflavones. Limited also evidence suggests that whey protein supplementation may increase androgenic signaling. Therefore, the purpose of this study was to examine the effects of soy protein concentrate (SPC), whey protein concentrate (WPC), or placebo (PLA) supplementation on serum sex hormones, androgen signaling markers in muscle tissue, and estrogen signaling markers in subcutaneous (SQ) adipose tissue of previously untrained, college-aged men (n = 47, 20 ± 1 yrs) that resistance trained for 12 weeks. Fasting serum total testosterone increased pre- to post-training, but more so in subjects consuming WPC (p 0.05), whereas serum 17β-estradiol remained unaltered. SQ estrogen receptor alpha (ERα) protein expression and hormone-sensitive lipase mRNA increased with training regardless of supplementation. Muscle androgen receptor (AR) mRNA increased while ornithine decarboxylase mRNA (a gene target indicative of androgen signaling) decreased with training regardless of supplementation (p 0.05). No significant interactions of supplement and time were observed for adipose tissue ERα/β protein levels, muscle tissue AR protein levels, or mRNAs in either tissue indicative of altered estrogenic or androgenic activity. Interestingly, WPC had the largest effect on increasing type II muscle fiber cross sectional area values (Cohen's d = 1.30), whereas SPC had the largest effect on increasing this metric in type I fibers (Cohen's d = 0.84). These data suggest that, while isoflavones were detected in SPC, chronic WPC or SPC supplementation did not appreciably affect biomarkers related to muscle androgenic signaling or SQ estrogenic signaling. The noted fiber type-specific responses to WPC and SPC supplementation warrant future research.

It is currently unclear as to whether sex hormones are significantly affected by soy or whey protein consumption. Additionally, estrogenic signaling may be potentiated via soy protein supplementation due to the presence of phytoestrogenic isoflavones. Limited evidence suggests that whey protein supplementation may increase androgenic signalling. Therefore, the purpose of this study was to examine the effects of soy protein concentrate (SPC), whey protein concentrate (WPC), or placebo (PLA) supplementation on serum sex hormones, androgen signaling markers in muscle tissue, and estrogen signaling markers in subcutaneous (SQ) adipose tissue of previously untrained, college-aged men (n = 47, 20 ± 1 yrs) that resistance trained for 12 weeks. Fasting serum total testosterone increased pre- to post-training, but more so in subjects consuming WPC (p

BackgroundWe sought to examine how 12 weeks of resistance exercise training (RET) affected skeletal muscle myofibrillar and sarcoplasmic protein levels along with markers of mitochondrial physiology in high versus low anabolic responders.MethodsUntrained college-aged males were classified as anabolic responders in the top 25th percentile (high-response cluster (HI);n= 13, dual x-ray absorptiometry total body muscle mass change (Δ) = +3.1 ± 0.3 kg, Δ vastus lateralis (VL) thickness = +0.59 ± 0.05 cm, Δ muscle fiber cross sectional area = +1,426 ± 253 μm2) and bottom 25th percentile (low-response cluster (LO);n= 12, +1.1 ± 0.2 kg, +0.24 ± 0.07 cm, +5 ± 209 μm2;p< 0.001 for all Δ scores compared to HI). VL muscle prior to (PRE) and following RET (POST) was assayed for myofibrillar and sarcoplasmic protein concentrations, myosin and actin protein content, and markers of mitochondrial volume. Proteins related to myofibril formation, as well as whole lysate PGC1-α protein levels were assessed.ResultsMain effects of cluster (HI > LO,p= 0.018, Cohen’sd= 0.737) and time (PRE > POST,p= 0.037, Cohen’sd= −0.589) were observed for citrate synthase activity, although no significant interaction existed (LO PRE = 1.35 ± 0.07 mM/min/mg protein, LO POST = 1.12 ± 0.06, HI PRE = 1.53 ± 0.11, HI POST = 1.39 ± 0.10). POST myofibrillar myozenin-1 protein levels were up-regulated in the LO cluster (LO PRE = 0.96 ± 0.13 relative expression units, LO POST = 1.25 ± 0.16, HI PRE = 1.00 ± 0.11, HI POST = 0.85 ± 0.12; within-group LO increasep= 0.025, Cohen’sd= 0.691). No interactions or main effects existed for other assayed markers.DiscussionOur data suggest myofibrillar or sarcoplasmic protein concentrations do not differ between HI versus LO anabolic responders prior to or following a 12-week RET program. Greater mitochondrial volume in HI responders may have facilitated greater anabolism, and myofibril myozenin-1 protein levels may represent a biomarker that differentiates anabolic responses to RET. However, mechanistic research validating these hypotheses is needed.

Next-generation sequencing (NGS) panels, while typically used clinically to identify somatic tumor mutations to guide therapeutics, can identify possible germline mutations. Incidental identification of a cancer predisposition syndrome has psychosocial and management implications for the patient and family beyond routine oncology care. We implemented a pilot integrating a genetic counselor (GC) within a multidisciplinary brain tumor clinic to facilitate follow-up germline testing on at-risk patients based on tumor testing results and to develop a clinical process for paired tumor plus blood testing for suspected hereditary cancer predisposition syndromes. From November 2015 to present, 116 brain tumors underwent clinical targeted molecular tumor profiling. Testing identified 35 (30%) patients with possible germline variants based on results and family history. Of those, 16 patients have had follow-up germline testing coordinated to date; 8 patients tested positive for an inherited cancer predisposition syndrome (RAD51D, TP53 (2), BAP1, MSH2, SUFU (2), ATM). Five patients tested negative on follow-up germline testing. Two patients were previously identified to have Neurofibromatosis 1 and one patient was previously identified to have Neurofibromatosis 2. Paired tumor testing has been coordinated for two patients so far and identified a pathogenic germline TP53 variant in one patient and a likely pathogenic germline SUFU variant in the other. Molecular diagnostics are now standard for pediatric brain tumors, and an embedded GC can improve the coordination of care for children with brain tumors. Timely counseling and diagnosis of a hereditary cancer syndrome has important health care implications for both the patient and the family.

Activating neurotrophic receptor kinase (NTRK) fusions define certain pediatric mesenchymal tumors, including infantile fibrosarcoma and cellular mesoblastic nephroma. Traditionally, molecular confirmation of these fusions has included either fluorescent in situ hybridization for ETV6 rearrangements or reverse-transcriptase polymerase chain reaction for the classic ETV6-NTRK3 fusion. However, these methods overlook variant NTRK rearrangements, which are increasingly appreciated as recurrent events in a subset of pediatric mesenchymal tumors. New therapeutic agents successfully target these fusions and may prevent morbid surgeries in very young children, making recognition of tumors harboring NTRK rearrangements of increasing importance. We evaluated the performance of immunohistochemical (IHC) staining using pan-Trk and TrkA antibodies in 79 pediatric mesenchymal tumors. Negative controls included pediatric mesenchymal tumors not harboring (n=28) or not expected to harbor (n=22) NTRK fusions. NTRK rearrangements were detected predominantly by DNA-based next-generation sequencing assays, specifically UW OncoPlex and UCSF500 Cancer Gene Panel. Pan-Trk IHC (EPR17341) was 97% sensitive and 98% specific for the presence of an NTRK rearrangement, and TrkA IHC (EP1058Y) was 100% sensitive and 63% specific for the presence of an NTRK rearrangement. Tumors with NTRK1 or NTRK2 rearrangements showed cytoplasmic staining, whereas tumors with NTRK3 rearrangements showed nuclear +/- cytoplasmic staining. We conclude that pan-Trk IHC is a highly sensitive and specific marker for NTRK rearrangements in pediatric mesenchymal tumors.

Purpose.—Clinical use of analytical tests to assess genomic variants in circulating tumor DNA (ctDNA) is increasing. This joint review from the American Society of Clinical Oncology and the College of American Pathologists summarizes current information about clinical ctDNA assays and provides a framework for future research.Methods.—An Expert Panel conducted a literature review on the use of ctDNA assays for solid tumors, including preanalytical variables, analytical validity, interpretation and reporting, and clinical validity and utility.Results.—The literature search identified 1338 references. Of those, 390, plus 31 references supplied by the Expert Panel, were selected for full-text review. There were 77 articles selected for inclusion.Conclusions.—The evidence indicates that testing for ctDNA is optimally performed on plasma collected in cell stabilization or EDTA tubes, with EDTA tubes processed within 6 hours of collection. Some ctDNA assays have demonstrated clinical validity and utility with certain types of advanced cancer; however, there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer. Evidence shows discordance between the results of ctDNA assays and genotyping tumor specimens, and supports tumor tissue genotyping to confirm undetected results from ctDNA tests. There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection. There is no evidence of clinical validity or clinical utility to suggest that ctDNA assays are useful for cancer screening, outside of a clinical trial. Given the rapid pace of research, reevaluation of the literature will shortly be required, along with the development of tools and guidance for clinical practice.