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Jennifer Wong, Elliot Backer, Joseph C Keenan, Huseyin Erhan Dincer, Roy Joseph Cho Interventional Pulmonology Division, Department of Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Minnesota, Minneapolis, MN, USACorrespondence: Roy Joseph Cho Tel +1 612-626-2637Email choxx548@umn.eduPurpose: Balloon-expandable stents are commonly used for the treatment of tracheobronchial strictures. We routinely perform targeted overdilation of these stents 1– 2mm on initial deployment to prevent stent migration or allow foreshortening to target airway caliber; however, specific data on the effect of targeted overdilation is unknown.Patients and Methods: We used three iCAST stents (6× 22mm, 7× 22mm, and 10× 38mm) to perform the study. We had two sets of each size to average our results. Targeted overdilation was accomplished with Merit Elation balloons.Results: The 6 × 22 and 7 × 22 stent OD increased from 6 to 11.4mm and 7 to 11.6mm. The 10 × 38 stent demonstrated minimal OD change with overdilation (OD change of 10.4 to 12.2mm). All stents demonstrated significant foreshortening with overdilation (20.2 to 5.65mm, 19.4 to 6mm, and 30.9 to 10.2mm for 6 × 22, 7 × 22, and 10 × 38, respectively). The breakpoint was seen at near twice the stated stent OD (13.5mm, 15mm, and 15mm with 6 × 22, 7 × 22 and 10 × 38, respectively).Conclusion: We have demonstrated that iCAST stents can increase their OD with subsequent foreshortening during targeted overdilation. This data can help facilitate decisions when selecting a particular iCAST stent for a specific airway application. Additionally, we have highlighted that balloon inflation diameter does not correspond to the actual stent OD during deployment. We believe that this data offers practical information for end-users of this stent type and additional data will be needed to corroborate our findings.Keywords: airway stenosis, tracheobronchial disease, interventional pulmonary, airway stent, balloon expandable stent, airway balloon dilation

© 2018 The Authors Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ∼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+myeloid dendritic cell population in psoriatic epidermis, and IL1βhiCCL3hiCD14+monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation. Cheng et al. report single-cell RNA sequencing of normal and inflamed human epidermis, revealing a discrete set of specialized keratinocytes that exhibit a distinct composition at different anatomic sites. Myeloid dendritic cells and macrophages also vary sharply with epidermal anatomic site and inflammation, indicating dynamic programming of antigen-presenting cells.

© The Author 2013.Motivation: Tumors acquire many chromosomal amplifications, and those acquired early in the lifespan of the tumor may be not only important for tumor growth but also can be used for diagnostic purposes. Many methods infer the order of the

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with a ∼50% response rate to immune checkpoint blockade (ICB) therapy. To identify predictive biomarkers, we integrated bulk and single-cell RNA sequencing (RNA-seq) with spatial transcriptomics from a cohort of 186 samples from 116 patients, including bulk RNA-seq from 14 matched pairs pre- and post-ICB. In nonresponders, tumors show evidence of increased tumor proliferation, neuronal stem cell markers, and IL1. Responders have increased type I/II interferons and preexisting tissue resident (Trm) CD8 or Vδ1 γδ T cells that functionally converge with overlapping antigen-specific transcriptional programs and clonal expansion of public T-cell receptors. Spatial transcriptomics demonstrated colocalization of T cells with B and dendritic cells, which supply chemokines and costimulation. Lastly, ICB significantly increased clonal expansion or recruitment of Trm and Vδ1 cells in tumors specifically in responders, underscoring their therapeutic importance. These data identify potential clinically actionable biomarkers and therapeutic targets for MCC. Significance: MCC serves as a model of ICB response. We utilized the largest-to-date, multimodal MCC dataset (n = 116 patients) to uncover unique tumor-intrinsic properties and immune circuits that predict response. We identified CD8 Trm and Vδ1 T cells as clinically actionable mediators of ICB response in major histocompatibility complex-high and -low MCCs, respectively., (©2024 American Association for Cancer Research.)

Tissues are organized into anatomical and functional units at different scales. New technologies for high-dimensional molecular profiling in situ have enabled the characterization of structure-function relationships in increasing molecular detail. However, it remains a challenge to consistently identify key functional units across experiments, tissues, and disease contexts, a task that demands extensive manual annotation. Here, we present spatial cellular graph partitioning (SCGP), a flexible method for the unsupervised annotation of tissue structures. We further present a reference-query extension pipeline, SCGP-Extension, that generalizes reference tissue structure labels to previously unseen samples, performing data integration and tissue structure discovery. Our experiments demonstrate reliable, robust partitioning of spatial data in a wide variety of contexts and best-in-class accuracy in identifying expertly annotated structures. Downstream analysis on SCGP-identified tissue structures reveals disease-relevant insights regarding diabetic kidney disease, skin disorder, and neoplastic diseases, underscoring its potential to drive biological insight and discovery from spatial datasets., Competing Interests: Declaration of interests Z.W., A.K., M.M., E.A.G.B., B.C., M.K.R., A.T.M., and A.E.T. are affiliated with Enable Medicine as employees and/or shareholders. J.Z. is a member of Enable Medicine’s scientific advisory board., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Medical ultrasound has emerged as an indispensable tool within interventional pulmonology, revolutionizing diagnostic and procedural practices through its non-invasive nature and real-time visualization capabilities. By harnessing the principles of sound waves and employing a variety of transducer types, ultrasound facilitates enhanced accuracy and safety in procedures such as transthoracic needle aspiration and pleural effusion drainage, consequently leading to improved patient outcomes. Understanding the fundamentals of ultrasound physics is paramount for clinicians, as it forms the basis for interpreting imaging results and optimizing interventions. Thoracic ultrasound plays a pivotal role in diagnosing conditions like pleural effusions and pneumothorax, while also optimizing procedures such as thoracentesis and biopsy by providing precise guidance. Advanced ultrasound techniques, including endobronchial ultrasound, has transformed the evaluation and biopsy of lymph nodes, bolstered by innovative features like elastography, which contribute to increased procedural efficacy and patient safety. Peripheral ultrasound techniques, notably radial endobronchial ultrasound (rEBUS), have become essential for assessing pulmonary nodules and evaluating airway structures, offering clinicians valuable insights into disease localization and severity. Neck ultrasound serves as a crucial tool in guiding supraclavicular lymph node biopsy and percutaneous dilatational tracheostomy procedures, ensuring safe placement and minimizing associated complications. Ultrasound technology is suited for further advancement through the integration of artificial intelligence, miniaturization, and the development of portable devices. These advancements hold the promise of not only improving diagnostic accuracy but also enhancing the accessibility of ultrasound imaging in diverse healthcare settings, ultimately expanding its utility and impact on patient care. Additionally, the integration of enhanced techniques such as contrast-enhanced ultrasound and 3D imaging is anticipated to revolutionize personalized medicine by providing clinicians with a more comprehensive understanding of anatomical structures and pathological processes. The transformative potential of medical ultrasound in interventional pulmonology extends beyond mere technological advancements; it represents a paradigm shift in healthcare delivery, empowering clinicians with unprecedented capabilities to diagnose and treat pulmonary conditions with precision and efficacy. By leveraging the latest innovations in ultrasound technology, clinicians can navigate complex anatomical structures with confidence, leading to more informed decision-making and ultimately improving patient outcomes. Moreover, the portability and versatility of modern ultrasound devices enable their deployment in various clinical settings, from traditional hospital environments to remote or resource-limited areas, thereby bridging gaps in healthcare access and equity.

We performed multi-omic profiling of epidermal keratinocytes, precancerous actinic keratoses, and squamous cell carcinomas to understand the molecular transitions during skin carcinogenesis. Single-cell mutational analyses showed that most keratinocytes in normal skin had lower mutation burdens than melanocytes and fibroblasts, however keratinocytes with TP53 or NOTCH1 mutations had substantially higher mutation burdens, suggesting that these mutations prime keratinocytes for transformation by increasing their mutation rate. Mutational profiling and spatial transcriptomics on squamous cell carcinomas adjacent to actinic keratoses revealed TERT promoter and CDKN2A mutations emerging in actinic keratoses, whereas additional mutations inactivating ARID2 and activating the MAPK-pathway delineated the transition to squamous cell carcinomas. Spatial variation in gene expression patterns was common in both tumor and immune cells, with high expression of checkpoint molecules at the invasive front of tumors. In conclusion, this study documents key events during the evolution of cutaneous squamous cell carcinoma.

Genome organization is thought to underlie cell type specific gene expression, yet how it is regulated in progenitors to produce cellular diversity is unknown. In Drosophila, a developmentally-timed genome reorganization in neural progenitors terminates competence to produce early-born neurons. These events require downregulation of Distal antenna (Dan), part of the conserved pipsqueak DNA-binding superfamily. Here we find that Dan forms liquid-like condensates with high protein mobility, and whose size and subnuclear distribution are balanced with its DNA-binding. Further, we identify a LARKS domain, a structural motif associated with condensate-forming proteins. Deleting just 13 amino acids from LARKS abrogates Dan's ability to retain the early-born neural fate gene, hunchback, in the neuroblast nuclear interior and maintain competence in vivo. Conversely, domain-swapping with LARKS from known phase-separating proteins rescues Dan's effects on competence. Together, we provide in vivo evidence for condensate formation and the regulation of progenitor nuclear architecture underlying neuronal diversification., (© 2024. The Author(s).)

Introduction: During the COVID-19 pandemic, ventilator shortages necessitated the development of new, low-cost ventilator designs. The fundamental requirements of a ventilator include precise gas delivery, rapid adjustments, durability, and user-friendliness, often achieved through solenoid valves. However, few solenoid-valve assisted low-cost ventilator (LCV) designs have been published, and gas exchange evaluation during LCV testing is lacking. This study describes the development and performance evaluation of a solenoid-valve assisted low-cost ventilator (SV-LCV) in vitro and in vivo, focusing on gas exchange and respiratory mechanics., Methods: The SV-LCV, a fully open ventilator device, was developed with comprehensive hardware and design documentation, utilizing solenoid valves for gas delivery regulation. Lung simulator testing calibrated tidal volumes at specified inspiratory and expiratory times, followed by in vivo testing in a porcine model to compare SV-LCV performance with a conventional ventilator., Results: The SV-LCV closely matched the control ventilator's respiratory profile and gas exchange across all test cycles. Lung simulator testing revealed direct effects of compliance and resistance changes on peak pressures and tidal volumes, with no significant changes in respiratory rate. In vivo testing demonstrated comparable gas exchange parameters between SV-LCV and conventional ventilator across all cycles. Specifically, in cycle 1, the SV-LCV showed arterial blood gas (ABG) results of pH 7.54, PCO2 34.5 mmHg, and PO2 91.7 mmHg, compared to the control ventilator's ABG of pH 7.53, PCO2 37.1 mmHg, and PO2 134 mmHg. Cycle 2 exhibited ABG results of pH 7.53, PCO2 33.6 mmHg, and PO2 84.3 mmHg for SV-LCV, and pH 7.5, PCO2 34.2 mmHg, and PO2 93.5 mmHg for the control ventilator. Similarly, cycle 3 showed ABG results of pH 7.53, PCO2 32.1 mmHg, and PO2 127 mmHg for SV-LCV, and pH 7.5, PCO2 35.5 mmHg, and PO2 91.3 mmHg for the control ventilator., Conclusion: The SV-LCV provides similar gas exchange and respiratory mechanic profiles compared to a conventional ventilator. With a streamlined design and performance akin to commercially available ventilators, the SV-LCV presents a viable, readily available, and reliable short-term solution for overcoming ventilator supply shortages during crises., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shyu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) are two emerging research technologies that uniquely characterize gene expression microenvironments on a cellular or subcellular level. The skin, a clinically accessible tissue composed of diverse, essential cell populations, serves as an ideal target for these high-resolution investigative approaches. Using these tools, researchers are assembling a compendium of data and discoveries in healthy skin as well as a range of dermatologic pathophysiologies, including atopic dermatitis, psoriasis, and cutaneous malignancies. The ongoing advancement of single-cell approaches, coupled with anticipated decreases in cost with increased adoption, will reshape dermatologic research, profoundly influencing disease characterization, prognosis, and ultimately clinical practice., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

Background: Stent encrustation with debris and mucostasis is a significant cause of airway injury and comorbidity, leading to ~25% of stent exchanges (1-3). Previous work from our group has shown that the experimental coating can reduce mucous adhesion in bench testing and demonstrated a signal for reducing airway injury and mucostasis in a feasibility study., Objectives: The aim of this study is to continue our inquiry in a randomized, single-blinded multi-animal trial to investigate the degree of airway injury and mucostasis using silicone stents with and without this specialized coating., Methods: We modified commercially available silicone stents with a hydrophilic polymer from Toray Industries. We conducted an in vivo survival study in 6 mainstem airways (3 coated and 3 uncoated) of 3 pigs to compare the degree of airway injury and mucostasis between coated versus noncoated stented airways. Both stents were randomized to either left or right mainstem bronchus. The pathologist was blinded to the stent type., Results: We implanted a total of six 14×15 mm silicone stents (1 per mainstem bronchi) into 3 pigs. All animals survived to termination at 4 weeks. All stents were intact; however, 1 uncoated stent migrated out. On average, all the coated stents demonstrated reduced pathology and tissue injury scores (75 vs. 68.3, respectively). The average total dried mucous weight was slightly higher in the coated stents (0.07 g vs. 0.05 g; respectively)., Conclusion: Coated stents had lower airway injury compared with uncoated stents in this study. Of all the stents, 1 uncoated stent migrated out and was not included in the dried mucous weight totals. This could explain the slightly higher mucous weight in the coated stents. Nevertheless, this current study demonstrates promising results in lowering airway injury in stents incorporated with the hydrophilic coating, and future studies, including a larger number of subjects, would be needed to corroborate our findings., Competing Interests: Disclosure: R.J.C. is a consultant for Toray. The remaining authors declare no conflict of interest or other disclosures., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: A traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs)., Methods: We examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing., Results: We found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC., Conclusions: Contrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro., (© 2024. The Author(s).)

Psoriasis vulgaris and other chronic inflammatory diseases improve markedly with therapeutic blockade of interleukin-23 (IL-23) signaling, but the genetic mechanisms underlying clinical responses remain poorly understood. Using single-cell transcriptomics, we profiled immune cells isolated from lesional psoriatic skin before and during IL-23 blockade. In clinically responsive patients, a psoriatic transcriptional signature in skin-resident memory T cells was strongly attenuated. In contrast, poorly responsive patients were distinguished by persistent activation of IL-17-producing T (T17) cells, a mechanism distinct from alternative cytokine signaling or resistance isolated to epidermal keratinocytes. Even in IL-23 blockade-responsive patients, we detected a recurring set of recalcitrant, disease-specific transcriptional abnormalities. This irreversible immunological state may necessitate ongoing IL-23 inhibition. Spatial transcriptomic analyses also suggested that successful IL-23 blockade requires dampening of >90% of IL-17-induced response in lymphocyte-adjacent keratinocytes, an unexpectedly high threshold. Collectively, our data establish a patient-level paradigm for dissecting responses to immunomodulatory treatments.

Spatial transcriptomic technologies, such as the Visium platform, measure gene expression in different regions of tissues. Here, we describe new software, STmut, to visualize somatic point mutations, allelic imbalance, and copy number alterations in Visium data. STmut is tested on fresh-frozen Visium data, formalin-fixed paraffin-embedded (FFPE) Visium data, and tumors with and without matching DNA sequencing data. Copy number is inferred on all conditions, but the chemistry of the FFPE platform does not permit analyses of single nucleotide variants. Taken together, we propose solutions to add the genetic dimension to spatial transcriptomic data and describe the limitations of different datatypes., (© 2023. The Author(s).)

The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.

Competing Interests: SJJ received grants from AbbVie, Boehringer Ingelheim, Bristol Myers Squibb, Celltrion Healthcare, Daewoong, Green Cross Laboratories, Janssen Pharmaceuticals, LEO Pharma, Novartis, Pfizer, and UCB and personal fees for lectures or consultation from AbbVie, Boehringer Ingelheim (including a portion related to Asian epidemiology and treatment guidelines of GPP), Daewoong, Eli Lilly and Company, Janssen Pharmaceuticals, Kolon Pharma, LEO Pharma, Novartis, Sanofi, and Yuhan. RJC received research funding from Sun Pharma, Leo Pharma, Bristol Myers Squibb, Regeneron, and Janssen. Unless stated these competing interests are unrelated to treatments for GPP.

Single-cell RNA sequencing (scRNA-seq) allows for high-resolution analysis of transcriptionally dysregulated cell subpopulations in inflammatory diseases. However, it can be challenging to properly isolate viable immune cells from human skin for scRNA-seq due to its barrier properties. Here, we present a protocol to isolate high-viability human cutaneous immune cells. We describe steps for obtaining and enzymatically dissociating a skin biopsy specimen and isolating immune cells using flow cytometry. We then provide an overview of downstream computational techniques to analyze sequencing data. For complete details on the use and execution of this protocol, please refer to Cook et al. (2022) 1 and Liu et al. (2022). 2 ., Competing Interests: Declaration of interests J.B.C. and R.J.C. have been investigators for Sun Pharmaceutical Industries, LEO Pharmaceuticals, Sanofi, and Regeneron (grants to their institution)., (Published by Elsevier Inc.)

Competing Interests: Declaration of interests JBC and RJC are recipients of research support from Sun Pharma, the manufacturer of tildrakizumab, as well as from Regeneron, LEO Pharma, and Pfizer. TB receives research support from AbbVie, Castle, CorEvitas, Dermavant, Galderma, Mindera, Pfizer, Novartis, and Regeneron. JPN consults with Kiniksa Pharmaceuticals, Advinow Medical, and Enspectra Health. All other authors declare no competing interests.

Background: Electromagnetic navigation bronchoscopy (ENB) and robotic-assisted bronchoscopy (RAB) systems are used for pulmonary lesion sampling, and utilize a pre-procedural CT scan where an airway, or "bronchus sign", is used to map a pathway to the target lesion. However, up to 40% of pre-procedural CT's lack a "bronchus sign" partially due to surrounding emphysema or limitation in CT resolution. Recognizing that the branches of the pulmonary artery, lymphatics, and airways are often present together as the bronchovascular bundle, we postulate that a branch of the pulmonary artery ("artery sign") could be used for pathway mapping during navigation bronchoscopy when a "bronchus sign" is absent. Herein we describe the navigation success and safety of using the "artery sign" to create a pathway for pulmonary lesion sampling., Methods: We reviewed data on consecutive cases in which the "artery sign" was used for pre-procedural planning for conventional ENB (superDimension™, Medtronic) and RAB (Monarch™, Johnson & Johnson). Patients who underwent these procedures from July 2020 until July 2021 at the University of Minnesota Medical Center and from June 2018 until December 2019 at the University of Chicago Medical Center were included in this analysis (IRB #19-0011 for the University of Chicago and IRB #00013135 for the University of Minnesota). The primary outcome was navigation success, defined as successfully maneuvering the bronchoscope to the target lesion based on feedback from the navigation system. Secondary outcomes included navigation success based on radial EBUS imaging, pneumothorax, and bleeding rates., Results: A total of 30 patients were enrolled in this analysis. The median diameter of the lesions was 17 mm. The median distance of the lesion from the pleura was 5 mm. Eleven lesions were solid, 15 were pure ground glass, and 4 were mixed. All cases were planned successfully using the "artery sign" on either the superDimension™ ENB (n = 15) or the Monarch™ RAB (n = 15). Navigation to the target was successful for 29 lesions (96.7%) based on feedback from the navigation system (virtual target). Radial EBUS image was acquired in 27 cases (90%) [eccentric view in 13 (43.33%) and concentric view in 14 patients (46.66%)], while in 3 cases (10%) no r-EBUS view was obtained. Pneumothorax occurred in one case (3%). Significant airway bleeding was reported in one case (3%)., Conclusions: We describe the concept of using the "artery sign" as an alternative for planning EMN and RAB procedures when "bronchus sign" is absent. The navigation success based on virtual target or r-EBUS imaging is high and safety of sampling of such lesions compares favorably with prior reports. Prospective studies are needed to assess the impact of the "artery sign" on diagnostic yield.

The homeostatic mechanisms that fail to restrain chronic tissue inflammation in diseases, such as psoriasis vulgaris, remain incompletely understood. We profiled transcriptomes and epitopes of single psoriatic and normal skin-resident T cells, revealing a gradated transcriptional program of coordinately regulated inflammation-suppressive genes. This program, which is sharply suppressed in lesional skin, strikingly restricts Th17/Tc17 cytokine and other inflammatory mediators on the single-cell level. CRISPR-based deactivation of two core components of this inflammation-suppressive program, ZFP36L2 and ZFP36, replicates the interleukin-17A (IL-17A), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon gamma (IFNγ) elevation in psoriatic memory T cells deficient in these transcripts, functionally validating their influence. Combinatoric expression analysis indicates the suppression of specific inflammatory mediators by individual program members. Finally, we find that therapeutic IL-23 blockade reduces Th17/Tc17 cell frequency in lesional skin but fails to normalize this inflammatory-suppressive program, suggesting how treated lesions may be primed for recurrence after withdrawal of treatment., Competing Interests: Declaration of interests A.M. is a cofounder of Arsenal Biosciences, Spotlight Therapeutics, and Survey Genomics. A.M. serves on the boards of directors at Spotlight Therapeutics and Survey Genomics and is board observer (and former member of the board of directors) at Arsenal Biosciences. A.M. is a member of the scientific advisory boards of Arsenal Biosciences, Spotlight Therapeutics, Survey Genomics, and NewLimit. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, NewLimit, Survey Genomics, PACT Pharma, and Merck. A.M. has received fees from Arsenal Biosciences, Spotlight Therapeutics, NewLimit, 23andMe, PACT Pharma, Juno Therapeutics, Trizell, Vertex, Merck, Amgen, Genentech, AlphaSights, Rupert Case Management, Bernstein, and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Identifying genetic variation underlying human diseases establishes targets for therapeutic development and helps tailor treatments to individual patients. Large-scale transcriptomic profiling has extended the study of such molecular heterogeneity between patients to somatic tissues. However, the lower resolution of bulk RNA profiling, especially in a complex, composite tissue such as the skin, has limited its success. Here we demonstrate approaches to interrogate patient-level molecular variance in a chronic skin inflammatory disease, psoriasis vulgaris, leveraging single-cell RNA-sequencing of CD45 + cells isolated from active lesions. Highly psoriasis-specific transcriptional abnormalities display greater than average inter-individual variance, nominating them as potential sources of clinical heterogeneity. We find that one of these chemokines, CXCL13 , demonstrates significant correlation with severity of lesions within our patient series. Our analyses also establish that genes elevated in psoriatic skin-resident memory T cells are enriched for programs orchestrating chromatin and CDC42-dependent cytoskeleton remodeling, specific components of which are distinctly correlated with and against Th17 identity on a single-cell level. Collectively, these analyses describe systematic means to dissect cell type- and patient-level differences in cutaneous psoriasis using high-resolution transcriptional profiles of human inflammatory disease., Competing Interests: JC and RC receive support (research grants to their institution) from LEO Pharmaceuticals, Sanofi, and Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Wang, Cook, Taylor, North, Hailer, Shou, Sadik, Kim, Purdom, Cheng and Cho.)

Motivation: Single-cell sequencing technologies that simultaneously generate multimodal cellular profiles present opportunities for improved understanding of cell heterogeneity in tissues. How the multimodal information can be integrated to obtain a common cell type identification, however, poses a computational challenge. Multilayer graphs provide a natural representation of multi-omic single-cell sequencing datasets, and finding cell clusters may be understood as a multilayer graph partition problem., Results: We introduce two spectral algorithms on multilayer graphs, spectral clustering on multilayer graphs and the weighted locally linear (WLL) method, to cluster cells in multi-omic single-cell sequencing datasets. We connect these algorithms through a unifying mathematical framework that represents each layer using a Hamiltonian operator and a mixture of its eigenstates to integrate the multiple graph layers, demonstrating in the process that the WLL method is a rigorous multilayer spectral graph theoretic reformulation of the popular Seurat weighted nearest neighbor (WNN) algorithm. Implementing our algorithms and applying them to a CITE-seq dataset of cord blood mononuclear cells yields results similar to the Seurat WNN analysis. Our work thus extends spectral methods to multimodal single-cell data analysis., Availability and Implementation: The code used in this study can be found at https://github.com/jssong-lab/sc-spectrum. All public data used in the article are accurately cited and described in Materials and Methods and in Supplementary Information., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Inflammatory conditions represent the largest class of chronic skin disease, but the molecular dysregulation underlying many individual cases remains unclear. Single-cell RNA sequencing (scRNA-seq) has increased precision in dissecting the complex mixture of immune and stromal cell perturbations in inflammatory skin disease states. We single-cell-profiled CD45 + immune cell transcriptomes from skin samples of 31 patients (7 atopic dermatitis, 8 psoriasis vulgaris, 2 lichen planus (LP), 1 bullous pemphigoid (BP), 6 clinical/histopathologically indeterminate rashes, and 7 healthy controls). Our data revealed active proliferative expansion of the T reg and Trm components and universal T cell exhaustion in human rashes, with a relative attenuation of antigen-presenting cells. Skin-resident memory T cells showed the greatest transcriptional dysregulation in both atopic dermatitis and psoriasis, whereas atopic dermatitis also demonstrated recurrent abnormalities in ILC and CD8 + cytotoxic lymphocytes. Transcript signatures differentiating these rash types included genes previously implicated in T helper cell (T H 2)/T H 17 diatheses, segregated in unbiased functional networks, and accurately identified disease class in untrained validation data sets. These gene signatures were able to classify clinicopathologically ambiguous rashes with diagnoses consistent with therapeutic response. Thus, we have defined major classes of human inflammatory skin disease at the molecular level and described a quantitative method to classify indeterminate instances of pathologic inflammation. To make this approach accessible to the scientific community, we created a proof-of-principle web interface (RashX), where scientists and clinicians can visualize their patient-level rash scRNA-seq-derived data in the context of our T H 2/T H 17 transcriptional framework.

Background: There is a paucity of real-time imaging modalities available for the bronchoscopic biopsy of peripheral lung nodules. We aim to demonstrate the feasibility of the O-arm imaging system to guide real-time biopsies of peripheral lung nodules during electromagnetic navigation bronchoscopy., Methods: A retrospective review was performed at 2 academic medical centers utilizing O-arm guidance., Results: The average nodule size was 2.1×2.0 cm and were mostly solid (66%) with a positive bronchus sign (83%). O-arm imaging confirmed tool-in-lesion in all cases. The diagnostic yield was 33%. Four cases were nondiagnostic of the 6 cases performed. In these cases, necrotic tissue was the most common (75%) and showed resolution following subsequent imaging. The average 3-dimensional (3D) spin time was 23.5 seconds. The average number of 3D spins performed per case was 4.33. The average effective dose per 3D spin was 3.73 mSv., Conclusion: We have demonstrated the O-arm's feasibility with electromagnetic navigation bronchoscopy for peripheral lung nodules. The O-arm was able to confirm tool-in-lesion in all cases which added confidence to the biopsy. Four high-resolution 3D spins per case may limit the total computed tomography effective dose. We also noted that both metal and radiation scatter were minimal when appropriate radiation safety standards were met. Although additional experience and data will be required to verify the O-arm approach for routine use, our initial experience is promising., Competing Interests: Disclosure: There is no conflict of interest or other disclosures., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Aggressive squamous cell carcinomas (SCCs) present frequently in the context of chronic skin injury occurring in patients with the congenital blistering disease recessive dystrophic epidermolysis bullosa. Recently, these cancers were shown to harbor mutation signatures associated with endogenous deaminases of the active polynucleotide cytosine deaminase family, collectively termed APOBEC, and clock-like COSMIC [Catalogue of Somatic Mutations in Cancer] signatures, which are associated with normal aging and might result from cumulative DNA replication errors. We present a case of a nasal septal SCC arising in the context of recurrent injury, but also modest past tobacco use. Our genetic analysis of this tumor reveals unusually high APOBEC and clock-like but low tobacco-related COSMIC signatures, suggesting that chronic injury may have played a primary role in somatic mutation. This case report demonstrates how signature-based analyses may implicate key roles for certain mutagenic forces in individual malignancies such as head-and-neck SCC, with multiple etiological origins., Case Presentation: We report the case of a 43-year-old male former smoker who presented with congestion and swelling following a traumatic nasal fracture. During surgery, the mucosa surrounding the right nasal valve appeared abnormal, and biopsies revealed invasive keratinizing SCC. Frozen section biopsies revealed multiple areas to be positive for SCC. Gene sequencing showed loss of PTEN (exons 2-8), CDKN2A/B and TP53 (exons 8-9), MYC amplification, and BLM S338*. Exome sequencing data also revealed that 36% of mutations matched an APOBEC mutational signature (COSMIC signatures 2 and 13) and 53% of mutations matched the clock-like mutation signature (COSMIC signature 5). These proportions place this tumor in the 90th percentile bearing each signature, independently, in a reference data set combining cutaneous and The Cancer Genome Atlas (TCGA) head and neck SCC data. In contrast, few mutations harbored a tobacco-related COSMIC signature 4, representing about the 10th percentile in our reference SCC data set. The patient was treated with partial rhinectomy with local flap reconstruction, bilateral neck dissection, and adjuvant radiation therapy; the patient remains disease-free to date., Conclusion: Based on comparative mutational signature analysis, we propose that the history of tobacco use and traumatic injury may have collaborated in activating APOBEC enzymes and the clock-like mutational process, ultimately leading to cancer formation. Clinical awareness of the relationship between epithelial injury and tumorigenesis should enhance earlier detection of this particularly aggressive type of cancer.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes 1,2 . The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution 1,3,4 . Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2 + breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Inflammatory response heterogeneity has impeded high-resolution dissection of diverse immune cell populations during activation. We characterize mouse cutaneous immune cells by single-cell RNA sequencing, after inducing inflammation using imiquimod and oxazolone dermatitis models. We identify 13 CD45 + subpopulations, which broadly represent most functionally characterized immune cell types. Oxazolone pervasively upregulates Jak2 / Stat3 expression across T cells and antigen-presenting cells (APCs). Oxazolone also induces Il4 / Il13 expression in newly infiltrating basophils, and Il4ra and Ccl24, most prominently in APCs. In contrast, imiquimod broadly upregulates Il17 / Il22 and Ccl4 / Ccl5 . A comparative analysis of single-cell inflammatory transcriptional responses reveals that APC response to oxazolone is tightly restricted by cell identity, whereas imiquimod enforces shared programs on multiple APC populations in parallel. These global molecular patterns not only contrast immune responses on a systems level but also suggest that the mechanisms of new sources of inflammation can eventually be deduced by comparison to known signatures., Competing Interests: A.J.S. and S.C.B. are employees of ImmunityBio Inc, S.C.B. is an equity holder of ImmunityBio Inc. The remaining authors disclose no conflicts.

Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application.

Recent advances in next generation sequencing-based single-cell technologies have allowed high-throughput quantitative detection of cell-surface proteins along with the transcriptome in individual cells, extending our understanding of the heterogeneity of cell populations in diverse tissues that are in different diseased states or under different experimental conditions. Count data of surface proteins from the cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) technology pose new computational challenges, and there is currently a dearth of rigorous mathematical tools for analyzing the data. This work utilizes concepts and ideas from Riemannian geometry to remove batch effects between samples and develops a statistical framework for distinguishing positive signals from background noise. The strengths of these approaches are demonstrated on two independent CITE-seq data sets in mouse and human.

Immature teratoma is a subtype of malignant germ cell tumor of the ovary that occurs most commonly in the first three decades of life, frequently with bilateral ovarian disease. Despite being the second most common malignant germ cell tumor of the ovary, little is known about its genetic underpinnings. Here we performed multiregion whole-exome sequencing to interrogate the genetic zygosity, clonal relationship, DNA copy number, and mutational status of 52 pathologically distinct tumor components from ten females with ovarian immature teratomas, with bilateral tumors present in five cases and peritoneal dissemination in seven cases. We found that ovarian immature teratomas are genetically characterized by 2N near-diploid genomes with extensive loss of heterozygosity and an absence of genes harboring recurrent somatic mutations or known oncogenic variants. All components within a single ovarian tumor (immature teratoma, mature teratoma with different histologic patterns of differentiation, and yolk sac tumor) were found to harbor an identical pattern of loss of heterozygosity across the genome, indicating a shared clonal origin. In contrast, the four analyzed bilateral teratomas showed distinct patterns of zygosity changes in the right versus left sided tumors, indicating independent clonal origins. All disseminated teratoma components within the peritoneum (including gliomatosis peritonei) shared a clonal pattern of loss of heterozygosity with either the right or left primary ovarian tumor. The observed genomic loss of heterozygosity patterns indicate that diverse meiotic errors contribute to the formation of ovarian immature teratomas, with 11 out of the 15 genetically distinct clones determined to result from nondisjunction errors during meiosis I or II. Overall, these findings suggest that copy-neutral loss of heterozygosity resulting from meiotic abnormalities may be sufficient to generate ovarian immature teratomas from germ cells.

Background: We aimed to investigate the effects of incremental PEEP titration in patients with ARDS on regional diaphragmatic motion with bedside ultrasound., Methods: Dorsal diaphragmatic excursion (DDE) and ventral diaphragmatic excursion (VDE) were measured using anatomic M-mode ultrasonography of the right hemidiaphragm as PEEP was randomized to -6, -3, +3, and +6 cm H 2 O from baseline to achieve a positive transpulmonary pressure. Inter-operator variability of DDE was assessed in 10 separate subjects., Results: A total of 14 subjects ventilated for ARDS were enrolled. Subjects had a mean age of 54 ± 12 y, mean P aO 2 /F IO 2 = 137 ± 54 mm Hg, and mean sequential organ failure assessment (SOFA) score = 14 ± 1). Transpulmonary pressure, DDE, and DDE/VDE ratio increased with incremental PEEP titration (-1.15 cm H 2 O vs 3.63 cm H 2 O, P < .001; 4.9 mm vs 8.2 mm, P < .001; and 62% vs 93%, P < .001, respectively). When transpulmonary pressure became positive, a visual increase in DDE and DDE/VDE ratio 0.60 to 0.93 was observed (from 0.48 cm to 0.82 cm, R 2 = 0.87, P = .02; and R 2 = 0.93, P = .006, respectively). There was high agreement in DDE measurements between 2 ultrasonographers (intra-class correlation 0.987, P < .001)., Conclusions: DDE was affected by incremental PEEP titration toward a positive transpulmonary pressure. The ultrasound assessment using anatomic M-mode allowed for specific measurement of regional diaphragmatic excursion. This pattern of motion in the dependent regions of the diaphragm during PEEP titration in subjects with ARDS achieving a positive transpulmonary pressure may reflect a potential target for future studies in the bedside assessment for lung recruitment. (Clinical Trials.gov registration NCT02463773.)., Competing Interests: The authors have disclosed no conflicts of interest., (Copyright © 2020 by Daedalus Enterprises.)

Basosquamous carcinoma (BSC) is an aggressive skin neoplasm with the features of both basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). While genetic drivers of BCC and SCC development have been extensively characterized, BSC has not been well studied, and it remains unclear whether these tumors originally derive from BCC or SCC. In addition, it is unknown which molecular pathways mediate the reprogramming of tumor keratinocytes toward basaloid or squamatized phenotypes. We sought to characterize the genomic alterations underlying sporadic BSC to elucidate the derivation of these mixed tumors. We identifed frequent Hedgehog (Hh) pathway mutations in BSCs, implicating Hh deregulation as the primary driving event in BSC. Principal component analysis of BCC and SCC driver genes further demonstrate the genetic similarity between BCC and BSC. In addition, 45% of the BSCs harbor recurrent mutations in the SWI/SNF complex gene, ARID1A, and evolutionary analysis revealed that ARID1A mutations occur after PTCH1 but before SCC driver mutations, indicating that ARID1A mutations may bestow plasticity enabling squamatization. Finally, we demonstrate mitogen-activated protein kinase pathway activation and the loss of Hh signaling associated with the squamatization of BSCs. Overall, these results support the genetic derivation of BSCs from BCCs and highlight potential factors involved in modulating tumor reprogramming between basaloid and squamatized phenotypes., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sebaceous carcinoma (SeC) is an uncommon malignancy arising from sebaceous glands of the conjunctiva and skin. Recurrent mutations in the ZNF750 were recently identified in ocular SeC. We assessed whether ZNF750 loss is a specific feature of ocular SeC or a general feature of sebaceous tumors., Methods: Immunostaining for ZNF750 expression was performed in 54 benign and malignant sebocytic proliferations. Staining for ZNF750 was scored on a three-tier scale: positive (>75%), partially positive (5%-74%), and negative (<5%)., Results: ZNF750 expression was negative in 4/11 ocular SeC, and partially positive in 4/11 ocular SeC and 6/13 cutaneous SeC. No extraocular tumors were negative. No loss was found in sebaceous adenoma or sebaceous hyperplasia. In nine previously sequenced ocular SeCs, two lacked detectable somatic mutations in ZNF750, but showed complete loss of staining, indicating non-mutational inactivation of ZNF750., Conclusion: We show complete loss of the ZNF750 epidermal differentiation regulator in about half of ocular SeC, highlighting the most common genetic defect in this cancer type. Loss of ZNF750 expression is seen even in tumors without truncating mutations and reduced in many of the remaining ocular and cutaneous SeC. In contrast, no ZNF750 loss was detected in benign sebaceous proliferations., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Keratinocyte differentiation requires intricately coordinated spatiotemporal expression changes that specify epidermis structure and function. This article utilizes single-cell RNA-seq data from 22,338 human foreskin keratinocytes to reconstruct the transcriptional regulation of skin development and homeostasis genes, organizing them by differentiation stage and also into transcription factor (TF)-associated modules. We identify groups of TFs characterized by coordinate expression changes during progression from the undifferentiated basal to the differentiated state and show that these TFs also have concordant differential predicted binding enrichment in the super-enhancers previously reported to turn over between the two states. The identified TFs form a core subset of the regulators controlling gene modules essential for basal and differentiated keratinocyte functions, supporting their nomination as master coordinators of keratinocyte differentiation. Experimental depletion of the TFs ZBED2 and ETV4, both predicted to promote the basal state, induces differentiation. Furthermore, our single-cell RNA expression analysis reveals preferential expression of antioxidant genes in the basal state, suggesting keratinocytes actively suppress reactive oxygen species to maintain the undifferentiated state. Overall, our work demonstrates diverse computational methods to advance our understanding of dynamic gene regulation in development.

Genetic variation in the NF-κB inhibitors, ABIN1 and A20, increase risk for psoriasis. While critical for hematopoietic immune cell function, these genes are believed to additionally inhibit psoriasis by dampening inflammatory signaling in keratinocytes. We dissected ABIN1 and A20's regulatory role in human keratinocyte inflammation using an RNA sequencing-based comparative genomic approach. Here we show subsets of the IL-17 and tumor necrosis factor-α signaling pathways are robustly restricted by A20 overexpression. In contrast, ABIN1 overexpression inhibits these genes more modestly for IL-17, and weakly for tumor necrosis factor-α. Our genome-scale analysis also indicates that inflammatory program suppression appears to be the major transcriptional influence of A20/ABIN1 overexpression, without obvious influence on keratinocyte viability genes. Our findings thus enable dissection of the differing anti-inflammatory mechanisms of two distinct psoriasis modifiers, which may be directly exploited for therapeutic purposes. Importantly, we report that IL-17-induced targets of A20 show similar aberrant epidermal layer-specific transcriptional upregulation in keratinocytes from diseases as diverse as psoriasis, atopic dermatitis, and erythrokeratodermia variabilis, suggesting a contributory role for epidermal inflammation in a broad spectrum of rashes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ∼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C + CD301A + myeloid dendritic cell population in psoriatic epidermis, and IL1β hi CCL3 hi CD14 + monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Sebaceous carcinomas (SeC) are cutaneous malignancies that, in rare cases, metastasize and prove fatal. Here we report whole-exome sequencing on 32 SeC, revealing distinct mutational classes that explain both cancer ontogeny and clinical course. A UV-damage signature predominates in 10/32 samples, while nine show microsatellite instability (MSI) profiles. UV-damage SeC exhibited poorly differentiated, infiltrative histopathology compared to MSI signature SeC (p = 0.003), features previously associated with dissemination. Moreover, UV-damage SeC transcriptomes and anatomic distribution closely resemble those of cutaneous squamous cell carcinomas (SCC), implicating sun-exposed keratinocytes as a cell of origin. Like SCC, this UV-damage subclass harbors a high somatic mutation burden with >50 mutations per Mb, predicting immunotherapeutic response. In contrast, ocular SeC acquires far fewer mutations without a dominant signature, but show frequent truncations in the ZNF750 epidermal differentiation regulator. Our data exemplify how different mutational processes convergently drive histopathologically related but clinically distinct cancers.

In this issue, Albibas et al. investigate the mutational nature of p53-immunopositive patches, commonly observed in sun-damaged skin. p53-immunopositive patches have long been suspected to be lineal precursors to actinic keratoses and cutaneous squamous cell carcinomas. However, the mutations actually giving rise to p53-immunopositive patches, and their relationship to skin cancer, have never been defined. The considerable clinical and economic costs of monitoring and treating sun-damaged skin demand we better understand the evolution of these common premalignancies., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)