Modifications of Kompetitive Allele-Specific PCR (KASP) Genotyping for Detection of Rare Alleles.

KASP is commonly used to genotype bi-allelic SNPs and In/Dels, and the standard protocol works well when both alleles are nearly equally prevalent in the DNA template. To detect rare alleles in bulked samples or to distinguish more than three genotypes, such as tri-allelic loci or mutations across orthologous genes in polyploids, adjustments to the protocol and/or data analysis are required. In this chapter, we present modified protocols for these non-traditional applications, including reaction conditions that enhance the fluorophore signal from rare alleles, resulting in increased KASP assay sensitivity. We also describe alternative KASP data analysis approaches that increase statistical certainty of genotyping calls. Furthermore, this increased assay sensitivity enables high-throughput genotyping using KASP, as samples can be pooled and tested in a single reaction. For example, rare alleles can be detected in mixed seed pools when present in ratios as low as 1 in 200. The assay modifications presented here expand the options available for complex genotyping, and retain KASP's advantages of being cheap, fast, and accurate.

The Interleukin-1 Receptor-Associated Kinase 4 Inhibitor PF-06650833 Blocks Inflammation in Preclinical Models of Rheumatic Disease and in Humans Enrolled in a Randomized Clinical Trial.

OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

COPDGene® 2019: Redefining the Diagnosis of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) remains a major cause of morbidity and mortality. Present-day diagnostic criteria are largely based solely on spirometric criteria. Accumulating evidence has identified a substantial number of individuals without spirometric evidence of COPD who suffer from respiratory symptoms and/or increased morbidity and mortality. There is a clear need for an expanded definition of COPD that is linked to physiologic, structural (computed tomography [CT]) and clinical evidence of disease. Using data from the COPD Genetic Epidemiology study (COPDGene®), we hypothesized that an integrated approach that includes environmental exposure, clinical symptoms, chest CT imaging and spirometry better defines disease and captures the likelihood of progression of respiratory obstruction and mortality. METHODS: Four key disease characteristics - environmental exposure (cigarette smoking), clinical symptoms (dyspnea and/or chronic bronchitis), chest CT imaging abnormalities (emphysema, gas trapping and/or airway wall thickening), and abnormal spirometry - were evaluated in a group of 8784 current and former smokers who were participants in COPDGene® Phase 1. Using these 4 disease characteristics, 8 categories of participants were identified and evaluated for odds of spirometric disease progression (FEV1 > 350 ml loss over 5 years), and the hazard ratio for all-cause mortality was examined. RESULTS: Using smokers without symptoms, CT imaging abnormalities or airflow obstruction as the reference population, individuals were classified as Possible COPD, Probable COPD and Definite COPD. Current Global initiative for obstructive Lung Disease (GOLD) criteria would diagnose 4062 (46%) of the 8784 study participants with COPD. The proposed COPDGene® 2019 diagnostic criteria would add an additional 3144 participants. Under the new criteria, 82% of the 8784 study participants would be diagnosed with Possible, Probable or Definite COPD. These COPD groups showed increased risk of disease progression and mortality. Mortality increased in patients as the number of their COPD characteristics increased, with a maximum hazard ratio for all cause-mortality of 5.18 (95% confidence interval [CI]: 4.15-6.48) in those with all 4 disease characteristics. CONCLUSIONS: A substantial portion of smokers with respiratory symptoms and imaging abnormalities do not manifest spirometric obstruction as defined by population normals. These individuals are at significant risk of death and spirometric disease progression. We propose to redefine the diagnosis of COPD through an integrated approach using environmental exposure, clinical symptoms, CT imaging and spirometric criteria. These expanded criteria offer the potential to stimulate both current and future interventions that could slow or halt disease progression in patients before disability or irreversible lung structural changes develop.

Digital Breast Tomosynthesis Implementation: Considerations for Emerging Breast Cancer Screening Bundled Payment Models.

PURPOSE: Bundled payments have been touted as mechanisms to optimize quality and costs. A recent feasibility study evaluating bundled payments for screening mammography episodes predated widespread adoption of digital breast tomosynthesis (DBT). We explore a similar model reflecting emerging acceptance of DBT in breast cancer screening. METHODS: Using 4-year data for 59,094 screening episodes from two large facilities within a large academic health system, we utilized published methodology to calibrate Medicare national allowable reference prices for women undergoing screening mammography before and after practice-wide implementation of DBT. RESULTS: Excluding DBT, Medicare-normalized bundled prices for traditional breast imaging 364 days downstream to screening mammography are extremely similar pre- and post-DBT implementation ($182.86 in 2013; $182.68 in 2015). The addition of DBT increased a DBT-inclusive bundled price by $53.16 (an amount lower than the $56.13 Medicare allowable fee for screening DBT) but was associated with significantly reduced recall rates (13.0% versus 9.4%; P < .0001). Without or with DBT, screening episode bundled prices remained sensitive to bundle-included services and varied little by patient age, race, or insurance status. CONCLUSIONS: Prior non-DBT approaches to bundled payment models for breast cancer screening remain viable as DBT becomes the standard of care, with bundle prices varying little by patient age, race, or insurance status. Higher DBT-inclusive bundled prices, however, highlight the need to explore societal costs more broadly (eg, reduced time away from work from fewer recalls) as bundled payment models evolve.

Are Lymph Node Characteristics on Axillary Ultrasound Associated with ≥3 Positive Lymph Nodes in Patients Managed by the American College of Surgeons Oncology Group Z0011 Trial Criteria?

Patients often receive axillary ultrasound-biopsy (AUS-B) before clinical evaluation. One positive biopsy in the absence of palpable disease rarely indicates additional nodal involvement, but it eliminates patients from being managed by the American College of Surgeons Oncology Group Z0011 trial criteria. To determine which patients may benefit from AUS-B, we analyzed whether characteristics on AUS were associated with large-volume axillary disease and, thus, the need for axillary lymph node (LN) dissection. A retrospective review identified patients who met Z0011 criteria and underwent AUS. Clinicopathologic and ultrasound characteristics were compared between patients with ≤2 versus ≥3 positive LNs. Two hundred and seven patients with cT1-2N0 tumors underwent preoperative AUS and breast-conserving surgery. On multivariate analysis, three AUS combinations were associated with ≥3 positive LNs: cortical thickness (CT) > 4 mm + loss of fatty hilum + round shape (P = 0.0218), CT > 4 mm + loss of fatty hilum (P = 0.0211), and CT > 4 mm + round shape (P = 0.0155). Preoperative axillary LN biopsy in patients with a single abnormal LN characteristic on AUS may be unnecessary because a positive finding will eliminate management according to Z0011 criteria. Cortical thickness >4 mm combined with any other abnormal characteristic was associated with ≥3 positive LNs, supporting the performance of AUS-B in this population.

Associations of County-level Radiologist and Mammography Facility Supply with Screening Mammography Rates in the United States.

RATIONALE AND OBJECTIVES: The present study aims to assess associations of Medicare beneficiary screening mammography rates with local mammography facility and radiologist availability. MATERIALS AND METHODS: Mammography screening rates for Medicare fee-for-service beneficiaries were obtained for US counties using the County Health Rankings data set. County-level certified mammography facility counts were obtained from the United States Food and Drug Administration. County-level mammogram-interpreting radiologist and breast imaging subspecialist counts were determined using Centers for Medicare & Medicaid Services fee-for-service claims files. Spearman correlations and multivariable linear regressions were performed using counties' facility and radiologist counts, as well as counts normalized to counties' Medicare fee-for-service beneficiary volume and land area. RESULTS: Across 3035 included counties, average screening mammography rates were 60.5% ± 8.2% (range 26%-88%). Correlations between county-level screening rates and total mammography facilities, facilities per 100,000 square mile county area, total mammography-interpreting radiologists, and mammography-interpreting radiologists per 100,000 county-level Medicare beneficiaries were all weak (r = 0.22-0.26). Correlations between county-level screening rates and mammography rates per 100,000 Medicare beneficiaries, total breast imaging subspecialist radiologists, and breast imaging subspecialist radiologists per 100,000 Medicare beneficiaries were all minimal (r = 0.06-0.16). Multivariable analyses overall demonstrated radiologist supply to have a stronger independent effect than facility supply, although effect sizes remained weak for both. CONCLUSION: Mammography facility and radiologist supply-side factors are only weakly associated with county-level Medicare beneficiary screening mammography rates, and as such, screening mammography may differ from many other health-care services. Although efforts to enhance facility and radiologist supply may be helpful, initiatives to improve screening mammography rates should focus more on demand-side factors, such as patient education and primary care physician education and access.

Screening Mammography Utilization and Medicare Beneficiaries' Perceptions of Their Primary Care Physicians.

RATIONALE AND OBJECTIVES: To assess associations between screening mammography utilization and Medicare beneficiaries' relationships with, and impressions of, their primary care physicians. MATERIALS AND METHODS: Using the Medicare Current Beneficiary Survey Access to Care Public Use File, we retrospectively studied responses from a national random cross section of Medicare beneficiaries surveyed in 2013 regarding perceptions of their primary care physicians and their screening mammography utilization. Statistical analysis accounted for subject weighting factors to estimate national screening utilization. RESULTS: Among 7492 female Medicare beneficiaries, 62.0% (95% confidence interval 59.8%-64.2%) underwent screening mammography. Utilization was higher for beneficiaries having (vs. not) a regular medical practice or clinic (63.2% vs. 34.6%) and a usual physician (63.8% vs. 50.3%). Utilization was higher for beneficiaries very satisfied (vs. very dissatisfied) with the overall quality of care they received (66.0% vs. 35.8%), their ease of getting to a doctor (67.7% vs. 43.2%), and their physician's concerns for their health (65.7% vs. 53.4%), as well as for beneficiaries strongly agreeing (vs. strongly disagreeing) that their physician is competent (66.0% vs. 54.1%), understands what is wrong (66.3% vs. 47.1%), answers all questions (67.0% vs. 46.7%), and fosters confidence (66.0% vs. 50.6%). Independent predictors of screening mammography utilization (P < .05) were satisfaction with quality of care, having a regular practice or clinic, and satisfaction with ease of getting to their physician. CONCLUSIONS: Screening mammography utilization is higher among Medicare beneficiaries with established primary physician relationships, particularly when those relationships are favorable. To optimize screening mammography utilization, breast imagers are encouraged to support initiatives to enhance high-quality primary care relationships.

Variation in Screening Mammography Rates Among Medicare Advantage Plans.

PURPOSE: Prior studies have shown higher screening mammography rates for beneficiaries in capitated managed care Medicare Advantage (MA) plans compared with traditional fee-for-service Medicare. The aim of this study was to explore variation in screening mammography rates at the level of MA managed care plans. METHODS: Using the 2016 MA Healthcare Effectiveness Data and Information Set Public Use File, screening mammography rates were identified for all 385 reporting MA plans. Associations were explored with a range of plan characteristics from this file, as well as from the CMS Part C and Part D Medicare Star Ratings Data File, Medicare Advantage Plan Directory, and Medicare Monthly Enrollment by Plan File. RESULTS: Overall MA plan screening rates were high (mean, 72.6 ± 9.4%) but varied substantially among plans (range, 14.3%-91.8%). Screening rates were higher in nonprofit versus for-profit plans (77.3% versus 71.8%, P < .001), as well as in health maintenance organization or local preferred provider organization plans versus private fee-for-service or regional preferred provider organization plans (71.9%-73.2% versus 65.5%-66.8%, P = .001). Among parent organizations with five or more plans, screening rates were highest for Kaiser Foundation (median, 88.4%) and lowest for Molina Healthcare (median, 65.3%). Screening rates showed small but significant associations with plans' contract lengths, enrolled populations, and counties served. Screening rates showed strong associations (r = 0.796-0.798) with colorectal cancer screening and annual flu vaccine rates and showed moderate associations (r = 0.283-0.365) with ambulatory and preventive care visits, osteoporosis screenings, body mass index assessments, and nonrecommended prostate-specific antigen screenings after age 70. CONCLUSIONS: Screening mammography rates vary considerably among MA plans. With increased federal interest in promoting the MA program, enhanced transparency will be necessary to ensure appropriate Medicare beneficiary participation decision making.

Update on Image-Guided Percutaneous Ablation of Breast Cancer.

OBJECTIVE: The purpose of this article is to delineate the potential techniques for percutaneous ablation of breast cancer, discuss the advantages and disadvantages of each technique, and provide results from recent studies on these technologies. The techniques discussed are cryotherapy, laser irradiation, microwave irradiation, radiofrequency ablation, high-intensity focused ultrasound ablation, and irreversible electroporation. CONCLUSION: Although percutaneous ablation techniques have some promising potential for less-invasive treatment of breast cancer, larger multicenter trials are needed to confirm their efficacy, especially in comparison with the reference standard of lumpectomy. The use of these techniques also leads to other remaining unanswered questions, including how to manage the axilla and which patients are the best candidates for these treatments.

Soluble Fn14 Is Detected and Elevated in Mouse and Human Kidney Disease.

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tissue injury to mediate local tissue responses including inflammation and tissue remodeling. We found that in various models of kidney disease, Fn14 expression (mRNA and protein) is upregulated in the kidney. These models include: lupus nephritis mouse models (Nephrotoxic serum Transfer Nephritis and MRL.Faslpr/lpr), acute kidney injury models (Ischemia reperfusion injury and Folic acid injury), and a ZSF-1 diabetic nephropathy rat model. Fn14 expression levels correlate with disease severity as measured by disease histology. We have also shown for the first time the detection of soluble Fn14 (sFn14) in the urine and serum of mice. Importantly, we found the sFn14 levels are markedly increased in the diseased mice and are correlated with disease biomarkers including proteinuria and MCP-1. We have also detected sFn14 in human plasma and urine. Moreover, sFn14 levels, in urine are significantly increased in DN patients and correlated with proteinuria and MCP-1 levels. Thus our data not only confirm the up-regulation of Fn14/TWEAK pathway in kidney diseases, but also suggest a novel mechanism for its regulation by the generation of sFn14. The correlation of sFn14 levels and disease severity suggest that sFn14 may serve as a potential biomarker for both acute and chronic kidney diseases.

Does the 6-min walk test correlate with the exercise stress test in children?

Is there a correlation between the 6-min walk and aerobic fitness in children? We studied healthy and cystic fibrosis (CF) subjects age 8-20 years using the 6-min walk test, treadmill graded exercise stress test, and spirometry. Six-minute walk distance (6MWD) and the product of 6MWD and body weight (6MWORK) were related to aerobic capacity. Data were analyzed using Student's t-test and Pearson correlation. 13 healthy subjects [9 females, mean age 15.8 +/- 3.6 years, % predicted forced expiratory volume in one second (FEV(1)) 105 +/- 12%, 6MWD 557 +/- 73 m, peak oxygen uptake (V' O2 max) 41.4 +/- 7.2 ml/kg/min, and heart rate (HR) at V' O2 max 180 +/- 10] and 11 CF subjects (3 females, mean age 14.3 +/- 3.8 years, FEV(1) 67 +/- 25.9%, 6MWD 468 +/- 68 m, V' O2 max 27.0 +/- 8.1 ml/kg, and HR at V' O2 max 163 +/- 22] were studied. 6MWD correlates with V' O2 max in normal subjects (r = 0.59, P < 0.05) but not in CF subjects (r = 0.09, NS). 6MWORK correlates with V' O2 max in CF subjects (r = 0.65, P < 0.05) but not in normal subjects (r = 0.278, NS). We conclude that the 6MWD corresponds with aerobic fitness in normal pediatric subjects and 6MWORK corresponds with aerobic fitness in CF subjects. We speculate that 6MWORK is superior to 6MWD for assessment of aerobic fitness in children with CF.

A fluorescent assay suitable for inhibitor screening and vanin tissue quantification.

Vanin-1 is a pantetheinase that catalyzes the hydrolysis of pantetheine to produce pantothenic acid (vitamin B5) and cysteamine. Reported here is a highly sensitive fluorescent assay using a novel fluorescently labeled pantothenate derivative. The assay has been used for characterization of a soluble version of human vanin-1 recombinant protein, identification and characterization of hits from high-throughput screening (HTS), and quantification of vanin pantothenase activity in cell lines and tissues. Under optimized assay conditions, we quantified vanin pantothenase activity in tissue lysate and found low activity in lung and liver but high activity in kidney. We demonstrated that the purified recombinant vanin-1 consisting of the extracellular portion without the glycosylphosphatidylinositol (GPI) linker was highly active with an apparent K(m) of 28 microM for pantothenate-7-amino-4-methylcoumarin (pantothenate-AMC), which was converted to pantothenic acid and AMC based on liquid chromatography-mass spectrometry (LC-MS) analysis. The assay also performed well in a 384-well microplate format under initial rate conditions (10% conversion) with a signal-to-background ratio (S/B) of 7 and a Z factor of 0.75. Preliminary screening of a library of 1280 pharmaceutically active compounds identified inhibitors with novel chemical scaffolds. This assay will be a powerful tool for target validation and drug lead identification and characterization.

Mast cell-dependent contraction of human airway smooth muscle cell-containing collagen gels: influence of cytokines, matrix metalloproteases, and serine proteases.

In asthma, mast cells infiltrate the airway smooth muscle cell layer and secrete proinflammatory and profibrotic agents that contribute to airway remodeling. To study the effects of mast cell activation on smooth muscle cell-dependent matrix contraction, we developed coculture systems of human airway smooth muscle cells (HASM) with primary human mast cells derived from circulating progenitors or with the HMC-1 human mast cell line. Activation of primary human mast cells by IgE receptor cross-linking or activation of HMC-1 cells with C5a stimulated contraction of HASM-embedded collagen gels. Contractile activity could be transferred with conditioned medium from activated mast cells, implicating involvement of soluble factors. Cytokines and proteases are among the agents released by activated mast cells that may promote a contractile response. Both IL-13 and IL-6 enhanced contraction in this model and the activity of IL-13 was ablated under conditions leading to expression of the inhibitory receptor IL-13Ralpha2 on HASM. In addition to cytokines, matrix metalloproteinases (MMPs), and serine proteases induced matrix contraction. Inhibitor studies suggested that, although IL-13 could contribute to contraction driven by mast cell activation, MMPs were critical mediators of the response. Both MMP-1 and MMP-2 were strongly expressed in this system. Serine proteases also contributed to contraction induced by mast cell-activating agents and IL-13, most likely by mediating the proteolytic activation of MMPs. Hypercontractility is a hallmark of smooth muscle cells in the asthmatic lung. Our findings define novel mechanisms whereby mast cells may modulate HASM-driven contractile responses.