Scattering-Based Light-Sheet Microscopy Imaging of Human Papillomavirus-Associated Squamous Lesions of the Anal Canal: A Proof-of-Principle Study.

Demand for anal cancer screening is expected to rise following the recent publication of the Anal Cancer-HSIL Outcomes Research trial, which showed that treatment of high-grade squamous intraepithelial lesions significantly reduces the rate of progression to anal cancer. While screening for human papillomavirus-associated squamous lesions in the cervix is well established and effective, this is less true for other sites in the lower anogenital tract. Current anal cancer screening and prevention rely on high-resolution anoscopy with biopsies. This procedure has a steep learning curve for providers and may cause patient discomfort. Scattering-based light-sheet microscopy (sLSM) is a novel imaging modality with the potential to mitigate these challenges through real-time, microscopic visualization of disease-susceptible tissue. Here, we report a proof-of-principle study that establishes feasibility of dysplasia detection using an sLSM device. We imaged 110 anal biopsy specimens collected prospectively at our institution's dysplasia clinic (including 30 nondysplastic, 40 low-grade squamous intraepithelial lesion, and 40 high-grade squamous intraepithelial lesion specimens) and found that these optical images are highly interpretable and accurately recapitulate histopathologic features traditionally used for the diagnosis of human papillomavirus-associated squamous dysplasia. A reader study to assess diagnostic accuracy suggests that sLSM images are noninferior to hematoxylin and eosin images for the detection of anal dysplasia (sLSM accuracy = 0.87; hematoxylin and eosin accuracy = 0.80; P = .066). Given these results, we believe that sLSM technology holds great potential to enhance the efficacy of anal cancer screening by allowing accurate sampling of diagnostic tissue at the time of anoscopy. While the current imaging study was performed on ex vivo biopsy specimens, we are currently developing a handheld device for in vivo imaging that will provide immediate microscopic guidance to high-resolution anoscopy providers.

Using Initial Biopsies and Vertical Sections to Improve Trainees' Confidence in Performing Mohs Surgery.

BACKGROUND: Tissue preservation and tumor clearance are hallmarks of Mohs micrographic surgery, but no standardized method currently exists to guide trainees on how to balance the two. OBJECTIVE: The authors provided residents and fellows with additional histologic information to enhance their surgical decision-making without changing the standard methodology of Mohs surgery. METHODS AND MATERIALS: Trainees were provided initial biopsy slides (IS) and frozen vertical sections (VS) of the first Mohs layer. All Mohs layers were excised in standard fashion, and vertically oriented sections were taken from the layer without disturbing the surgical margins to obtain VS. Surveys were used to assess trainees' confidence in performing Mohs surgery with and without these tools. RESULTS: Trainees reported increased confidence in performing Mohs surgery when they reviewed IS before surgery and viewed VS of the first layer. CONCLUSION: Reviewing IS and VS improved trainees' confidence in performing Mohs surgery. This additional histological information was obtained while maintaining the usual steps of Mohs surgery. Objective information obtained from IS and VS may explain why trainees' confidence increased using this technique. Both IS and VS can be valuable teaching tools that may enhance trainees' ability to perform Mohs surgery.

Performance measurement part I: Foundational knowledge for measure development.

As medicine is moving toward performance and outcome-based payment and is transitioning away from productivity-based systems, value is now being appraised in healthcare through "performance measures." Over the past few decades, assessment of clinical performance in health care has been essential in ensuring safe and cost-effective patient care. The Centers for Medicare & Medicaid Services is further driving this change with measurable, outcomes-based national payer incentive payment systems. With the continually evolving requirements in health care reform focused on value-based care, there is a growing concern that clinicians, particularly dermatologists, may not understand the scientific rationale of health care quality measurement. As such, in order to help dermatologists understand the health care measurement science landscape to empower them to engage in the performance measure development and implementation process, the first article in this 2-part continuing medical education series reviews the value equation, historic and evolving policy issues, and the American Academy of Dermatology's approach to performance measurement development to provide the required foundational knowledge for performance measure developers.

Performance measurement part II: A guide to developing dermatology performance measures.

Throughout the 21st century, national and local governments, private health sectors, health insurance companies, healthcare professionals, labor unions, and consumers have been striving to develop an effective approach to evaluate, report, and improve the quality of healthcare. As medicine improves and health systems grow to meet patient needs, the performance measurement system of care effectiveness must also evolve. Continual efforts should be undertaken to effectively measure quality of care to create a more informed public, improve health outcomes, and reduce healthcare costs. As such, recent policy reform has necessitated that performance systems be implemented in healthcare, with the "performance measure" being the foundation of the system in which all of healthcare must be actively engaged in to ensure optimal care for patients. The development of performance measures can be highly complex, particularly when creating specialty-specific performance measures. To help dermatologists understand the process of creating dermatology-specific performance measures to engage in creating or implementing performance measures at the local or national levels, this article in the two-part continuing medical education series reviews the types, components, and process of developing, reviewing, and implementing performance measures.

Clinical Features of SARS-CoV-2 Infection During Pregnancy and Associated Placental Pathologies.

SUMMARY: We reviewed the clinicopathologic findings of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-exposed placentas at our institution. We identified patients diagnosed with SARS-CoV-2 during pregnancy (March-October 2020). Clinical data included gestational age at diagnosis and delivery and maternal symptoms. Hematoxylin and eosin slides were reviewed for maternal vascular malperfusion, fetal vascular malperfusion, chronic villitis, amniotic fluid infection, intervillous thrombi, fibrin deposition, and infarction. Immunohistochemistry (IHC) for coronavirus spike protein and RNA in situ hybridization (ISH) for SARS-CoV-2 was performed on a subset of blocks. A review of placentas from age-matched patients received March-October 2019 was conducted as a comparison cohort. A total of 151 patients were identified. Placentas in the 2 groups were similar in weight for gestational age and had similar rates of maternal vascular malperfusion, fetal vascular malperfusion, amniotic fluid infection, intervillous thrombi, fibrin deposition, and infarction. Chronic villitis was the only significantly different pathologic finding between cases and controls (29% of cases showed chronic villitis vs. 8% of controls, P <0.001). Overall, 146/151 (96.7%) cases were negative for IHC and 129/133 (97%) cases were negative for RNA ISH. There were 4 cases that stained positively for IHC/ISH, 2 of which showed massive perivillous fibrin deposition, inflammation, and decidual arteriopathy. Coronavirus disease 2019 (COVID-19)-positive patients were more likely to self-identify as Hispanic and more likely to have public health insurance. Our data suggests SARS-CoV-2 exposed placentas that stain positively for SARS-CoV-2 show abnormal fibrin deposition, inflammatory changes, and decidual arteriopathy. The group of patients with clinical COVID-19 are more likely to show chronic villitis. IHC and ISH evidence of viral infection is rare.

Building a pipeline to identify and engineer constitutive and repressible promoters.

To support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. We have developed a pipeline to identify genes that have stable expression across a wide range of Arabidopsis tissues at different developmental stages and have identified a number of promoters that are well expressed in both transient (Nicotiana benthamiana) and stable (Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target sites in a subset of the screened promoters, converting them into NOR logic gates. The work here establishes a pipeline to screen for additional constitutive promoters and can form the basis of constructing more complex information processing circuits in the future.

A comparative analysis of stably expressed genes across diverse angiosperms exposes flexibility in underlying promoter architecture.

Promoters regulate both the amplitude and pattern of gene expression-key factors needed for optimization of many synthetic biology applications. Previous work in Arabidopsis found that promoters that contain a TATA-box element tend to be expressed only under specific conditions or in particular tissues, while promoters that lack any known promoter elements, thus designated as Coreless, tend to be expressed more uniformly. To test whether this trend represents a conserved promoter design rule, we identified stably expressed genes across multiple angiosperm species using publicly available RNA-seq data. Comparisons between core promoter architectures and gene expression stability revealed differences in core promoter usage in monocots and eudicots. Furthermore, when tracing the evolution of a given promoter across species, we found that core promoter type was not a strong predictor of expression pattern. Our analysis suggests that core promoter types are correlative rather than causative in promoter expression patterns and highlights the challenges in finding or building constitutive promoters that will work across diverse plant species.

A comparative analysis of stably expressed genes across diverse angiosperms exposes flexibility in underlying promoter architecture.

Promoters regulate both the amplitude and pattern of gene expression-key factors needed for optimization of many synthetic biology applications. Previous work in Arabidopsis found that promoters that contain a TATA-box element tend to be expressed only under specific conditions or in particular tissues, while promoters which lack any known promoter elements, thus designated as Coreless, tend to be expressed more ubiquitously. To test whether this trend represents a conserved promoter design rule, we identified stably expressed genes across multiple angiosperm species using publicly available RNA-seq data. Comparisons between core promoter architectures and gene expression stability revealed differences in core promoter usage in monocots and eudicots. Furthermore, when tracing the evolution of a given promoter across species, we found that core promoter type was not a strong predictor of expression stability. Our analysis suggests that core promoter types are correlative rather than causative in promoter expression patterns and highlights the challenges in finding or building constitutive promoters that will work across diverse plant species.

Nuclear ß-Catenin Expression in the Context of Abnormal p53 Expression Indicates a Nonserous Histotype in Endometrial Carcinoma.

The interobserver reproducibility is poor for histotyping within the p53-abnormal molecular category of endometrial carcinomas (ECs); therefore, biomarkers that improve histologic classification are useful. ß-catenin has been proposed to have prognostic significance in specific clinicopathologic and molecular contexts. The diagnostic utility for ß-catenin expression patterns in determining the histotype of p53-abnormal ECs has not been well studied. We identified ECs molecularly classified as "p53-abnormal." The p53-abnormal classification was assigned when (1) no POLE exonuclease domain hotspot mutations identified, (2) mismatch-repair protein expression was retained, and (3) abnormal p53 expression (null or overexpression) was present. Morphology was re-reviewed and ß-catenin immunohistochemistry was scored as abnormal (nuclear) or normal (membranous, non-nuclear). Eighty ECs were identified in the "p53-abnormal" category; 27 (33.75%) were uterine serous carcinomas, and 53 were of nonserous histotype: 28 uterine carcinosarcomas (35%), 16 endometrioid carcinomas (20%), 2 clear cell carcinomas (2.5%), and 7 high-grade EC with ambiguous morphology (8.75%). All 27 uterine serous carcinomas demonstrated membranous ß-catenin staining. Of the 53 nonserous ECs, 11 (21%) showed abnormal ß-catenin expression: 6 endometrioid carcinomas, 4 uterine carcinosarcoma, and 1 high-grade EC with ambiguous morphology. The specificity of abnormal ß-catenin expression for nonserous EC is high (100%) but the sensitivity is low (21%) with positive and negative predictive values of 100% and 60%, respectively. Our data shows that abnormal ß-catenin expression in the context of p53-abnormal EC is highly specific, but not sensitive, for nonserous ECs and may be of value as part of a panel in classifying high-grade EC, particularly to exclude uterine serous carcinoma when nuclear staining is present.

64Cu-DOTATATE Uptake in a Pulmonary Hamartoma.

ABSTRACT: DOTATATE PET/CT is frequently used to evaluate indeterminant pulmonary nodules suspected to be pulmonary carcinoid. We report an unexpected case of pulmonary hamartoma demonstrating 64Cu-DOTATATE uptake in a 43-year-old woman with a slowly enlarging pulmonary nodule. Histopathological staining showed somatostatin receptor 2 expression on vascular endothelial cells and a proportion of cartilage and smooth muscle cells within the hamartoma.

Transcriptomic Profiling of Plaque Psoriasis and Cutaneous T-Cell Subsets during Treatment with Secukinumab.

The IL-17A inhibitor secukinumab is efficacious for the treatment of psoriasis. To better understand its mechanism of action, we investigated its impact on psoriatic lesions from 15 patients with moderate-to-severe plaque psoriasis undergoing secukinumab treatment. We characterized the longitudinal transcriptomic changes of whole lesional skin tissue as well as cutaneous CD4+ and CD8+ T effector cells and CD4+ T regulatory cells across 12 weeks of treatment. Secukinumab was clinically effective and reduced disease-associated overexpression of IL17A , IL17F, IL23A, IL23R, and IFNG in whole tissue as soon as 2 weeks after initiation of treatment. IL17A overexpression in T-cell subsets, primarily CD8+ T cells, was also reduced. Although secukinumab treatment resolved 89‒97% of psoriasis-associated expression differences in bulk tissue and T-cell subsets by week 12 of treatment, we observed expression differences involved in IFN signaling and metallothionein synthesis that remained unresolved at this time point as well as potential treatment-associated expression differences involved in IL-15 signaling. These changes were accompanied by shifts in broader immune cell composition on the basis of deconvolution of RNA-sequencing data. In conclusion, our study reveals several phenotypic and cellular changes within the lesion that underlie clinical improvement from secukinumab.

Intravascular adenomyomatosis: a morphologic variant of intravenous leiomyomatosis associated with endometriosis and potential for misdiagnosis.

Intravenous leiomyomatosis (IVL) is a quasi-malignant smooth muscle tumor involving lymphatic and venous spaces of the myometrium. Rare cases of IVL with admixed endometrial glands and stroma have been described, termed intravascular adenomyomatosis. We report four additional cases of intravascular adenomyomatosis and expand the clinicopathologic features of these rare tumors. Patients were 39-45 years old and presented with symptoms of dysmenorrhea, postmenopausal bleeding, or pelvic mass. All cases were associated with endometriosis. Three cases comprised intravascular bland smooth muscle tumors with plexiform features, and in some foci, the intravascular tumor contained endometrial type glands and stroma. In one case, there was extensive (>10) foci of intravascular adenomyomatosis without evidence of associated smooth muscle neoplasm but did have an endometrial polyp with adenomyomatous features. None of the cases had nuclear atypia, increased mitotic activity, or tumor cell necrosis. The endometrial stromal components were positive for CD10 and negative or weakly positive for desmin by immunohistochemistry. Two cases underwent molecular testing for JAZF1 and PHF1 rearrangements with negative results. Three patients had no evidence of disease at the time of the last follow-up, and one had persistent but stable disease 7 years after incomplete surgical removal and megestrol acetate treatment. Intravascular adenomyomatosis is a variant morphology rarely seen in IVL that lacks characteristic morphologic and molecular features of endometrial stromal sarcoma. Similar to IVL, prognosis is likely linked to completeness of surgical resection. In this study, we found that intravascular adenomyomatosis is frequently associated with endometriosis, a novel finding to add to the literature on this rare IVL variant.

Communicating Certainty in Pathology Reports.

CONTEXT.­: Pathology reports are the main modality in which results are communicated to other physicians. For various reasons, the diagnosis may be qualified on a spectrum of uncertainty. OBJECTIVE.­: To examine how communication of uncertainty is an unexamined source of possible medical error. No study to our knowledge has examined pathology reports across multiple institutions. This study seeks to identify commonly used phrases of diagnostic uncertainty and their interpreted meanings by surgical pathologists and clinicians. DESIGN.­: Anonymous surveys were completed at 3 major US academic institutions by 18 practicing staff pathologists, 12 pathology residents, 53 staff clinicians, and 50 resident/allied health professional clinicians at 5 standard tumor boards. All participants rated percentage certainty associated with 7 diagnostic terms. Pathologists answered 2 questions related to the ability to clarify a diagnosis using a comment and comfort wording pathology reports. Clinicians answered questions on how often they read a pathology report comment, if they found the comment helpful, and how comfortable they were in reading pathology reports. RESULTS.­: A wide range in percentage certainty was found for each of the 7 diagnostic phrases. Both staff and resident clinicians and residents showed wide variability in interpreting the phrases. Twenty-five of 50 staff clinicians (52%) were very comfortable reading a pathology report, whereas only 4 of 53 resident clinicians (8%) were very comfortable reading a pathology report. Twenty-four of 53 staff clinicians (63%) reported always reading the comment, yet only 20 of 53 (27%) always found the comment helpful. The phrases "diagnostic of" and "consistent with" had the strongest agreement in meaning. The weakest agreement was between "suspicious for" and "compatible with." CONCLUSIONS.­: Efforts to standardize diagnostic terms may improve communication.

Prospective molecular classification of endometrial carcinomas: institutional implementation, practice, and clinical experience.

The comprehensive genomic analysis of endometrial carcinoma (EC) by The Cancer Genome Atlas (TCGA) led to the discovery of four distinct and prognostically significant molecular subgroups. Molecular classification has the potential to improve risk-stratification when integrated with clinicopathologic features and has recently been included in national and international patient management EC guidelines. Thus, the adoption of molecular classification into routine pathologic and clinical practice is likely to grow significantly in the upcoming years. Establishing an efficient and standardized workflow for performing molecular classification on ECs, and reporting both the molecular and histologic findings in an integrative manner, is imperative. Here we describe our effort to implement rapid and routine molecular classification on all ECs diagnosed at our institution. To this effect, we performed immunohistochemistry as a surrogate marker for identifying genetic and/or epigenetic alterations in DNA mismatch repair (e.g., MLH1, PMS2, MSH6, MSH2), and TP53 genes. In addition, we have developed and employed a single-gene POLE SNaPshot assay, which is a rapid and analytically sensitive method for detecting select POLE exonuclease domain mutations (EDMs). We report our molecular testing workflow and integrative reporting system as well as the clinicopathologic and molecular features of 310 ECs that underwent routine molecular classification at our institution. The 310 ECs were molecularly classified as follows: 15 (5%) POLE mutant (POLEmut), 79 (25%) mismatch repair-deficient (MMRd), 135 (44%) no specific molecular profile (NSMP), and 81 (26%) p53 abnormal (p53abnl). This work provides an initial framework for implementing routine molecular classification of ECs.

Point-of-Need Diagnostics for Foodborne Pathogen Screening.

Foodborne illness is a major public health issue that results in millions of global infections annually. The burden of such illness sits mostly with developing countries, as access to advanced laboratory equipment and skilled lab technicians, as well as consistent power sources, is limited and expensive. Current gold standards in foodborne pathogen screening involve labor-intensive sample enrichment steps, pathogen isolation and purification, and costly readout machinery. Overall, time to detection can take multiple days, excluding the time it takes to ship samples to off-site laboratories. Efforts have been made to simplify the workflow of such tests by integrating multiple steps of foodborne pathogen screening procedures into a singular device, as well as implementing more point-of-need readout methods. In this review, we explore recent advancements in developing point-of-need devices for foodborne pathogen screening. We discuss the detection of surface markers, nucleic acids, and metabolic products using both paper-based and microfluidic devices, focusing primarily on developments that have been made between 2015 and mid-2020.

Changes in Drug List Prices and Amounts Paid by Patients and Insurers.

Importance: High out-of-pocket drug costs can cause patients to skip treatment and worsen outcomes, and high insurer drug payments could increase premiums. Drug wholesale list prices have doubled in recent years. However, because of manufacturer discounts and rebates, the extent to which increases in wholesale list prices are associated with amounts paid by patients and insurers is poorly characterized. Objective: To determine whether increases in wholesale list prices are associated with increases in amounts paid by patients and insurers for branded medications. Design, Setting, and Participants: Cross-sectional retrospective study analyzing pharmacy claims for patients younger than 65 years in the IBM MarketScan Commercial Database and pricing data from SSR Health, LLC, between January 1, 2010, and December 31, 2016. Pharmacy claims analyzed represent claims of employees and dependents participating in employer health benefit programs belonging to large employers. Rebate data were estimated from sales data from publicly traded companies. Analysis focused on the top 5 patent-protected specialty and 9 traditional brand-name medications with the highest total drug expenditures by commercial insurers nationwide in 2014. Data were analyzed from July 2017 to July 2020. Exposures: Calendar year. Main Outcomes and Measures: Changes in inflation-adjusted amounts paid by patients and insurers for branded medications. Results: In this analysis of 14.4 million pharmacy claims made by 1.8 million patients from 2010-2016, median drug wholesale list price increased by 129% (interquartile range [IQR], 78%-133%), while median insurance payments increased by 64% (IQR, 28%-120%) and out-of-pocket costs increased by 53% (IQR, 42%-82%). The mean percentage of wholesale list price accounted for by discounts increased from 17% in 2010 to 21% in 2016, and the mean percentage of wholesale list price accounted for by rebates increased from 22% in 2010 to 24% in 2016. For specialty medications, median patient out-of-pocket costs increased by 85% (IQR, 73%-88%) from 2010 to 2016 after adjustment for inflation and 42% (IQR, 25%-53%) for nonspecialty medications. During that same period, insurer payments increased by 116% for specialty medications (IQR, 100%-127%) and 28% for nonspecialty medications (IQR, 5%-34%). Conclusions and Relevance: This study's findings suggest that drug list prices more than doubled over a 7-year study period. Despite rising manufacturer discounts and rebates, these price increases were associated with large increases in patient out-of-pocket costs and insurer payments.