Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

Tert-expressing cells contribute to salivary gland homeostasis and tissue regeneration after radiation therapy.

Salivary gland homeostasis and regeneration after radiotherapy depend significantly on progenitor cells. However, the lineage of submandibular gland (SMG) progenitor cells remains less defined compared with other normal organs. Here, using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct telomerase-expressing (TertHigh) cell population located in the ductal region of the adult SMG. These TertHigh cells contribute to ductal cell generation during SMG homeostasis and to both ductal and acinar cell renewal 1 year after radiotherapy. TertHigh cells maintain self-renewal capacity during in vitro culture, exhibit resistance to radiation damage, and demonstrate enhanced proliferative activity after radiation exposure. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy, and CRISPR-activated Tert in human SMG spheres increases proliferation after radiation. RNA sequencing reveals upregulation of "cell cycling" and "oxidative stress response" pathways in TertHigh cells following radiation. Mechanistically, Tert appears to modulate cell survival through ROS levels in SMG spheres following radiation damage. Our findings highlight the significance of TertHigh cells in salivary gland biology, providing insights into their response to radiotherapy and into their use as a potential target for enhancing salivary gland regeneration after radiotherapy.

Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility.

Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

Retraction Note: Lysyl oxidase is essential for hypoxia-induced metastasis.

A Retraction to this paper has been published and can be accessed via a link at the top of the paper.

Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA.

Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We developed an unsupervised machine learning algorithm, CELESTA, which identifies the cell type of each cell, individually, using the cell's marker expression profile and, when needed, its spatial information. We demonstrate the performance of CELESTA on multiplexed immunofluorescence images of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, and validate our findings in an independent cohort. By coupling our spatial analysis with single-cell RNA-sequencing data on proximal sections of the same specimens, we identify cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of CELESTA to facilitate identification of clinically relevant interactions.

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.

Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer.

The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10-50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.

Herpes Zoster and Postherpetic Neuralgia: Changing Incidence Rates From 1994 to 2018 in the United States.

BACKGROUND: The incidence of herpes zoster (HZ) has been increasing in recent decades. Although 2 vaccines for HZ are available, there have been few studies on the incidence rates of HZ and postherpetic neuralgia (PHN) since their introduction. This study examined the incidence rates of HZ and PHN from 1994 to 2018 in the United States to determine if they have continued to increase since introduction of the HZ vaccines. METHODS: A de-identified longitudinal administrative claims database, the OptumLabs Data Warehouse, was used to assess incidence rates among individuals continuously enrolled in the database for ≥365 days with no prior history of HZ or PHN. Unstandardized and standardized incidence rates were calculated by year, 10-year age groups, sex, and race/ethnicity. RESULTS: There were 610 766 individuals with HZ (median age, 56.3; interquartile range, 43.0-68.7 years; 59.8% women; 70.6% white). From 1994 to 2018, the incidence of HZ increased from 286.0 (95% confidence interval [CI], 259.1-312.8) to 579.6 (95% CI, 554.2-605.0) cases per 100 000 person-years, an annual increase of 3.1% (95% CI, 2.5-3.6%). Since 2007, annual HZ incidence rates have decreased in individuals ≤20 and >60 years old. The overall incidence rate of PHN was 57.5 (95% CI, 56.0-59.0) cases per 100 000 person-years. The proportion of individuals with HZ who developed PHN was higher from 2007 to 2018 than from 1994 to 2006. CONCLUSIONS: HZ incidence rates have continued to increase in age groups for which HZ vaccines are not currently recommended, warranting a review of current vaccine recommendations.

Effectiveness of the Recombinant Zoster Vaccine in Adults Aged 50 and Older in the United States: A Claims-Based Cohort Study.

BACKGROUND: The recombinant zoster vaccine had over 90% efficacy in preventing herpes zoster in clinical trials. However, its effectiveness outside of a clinical trial setting has not been investigated. This study aimed to assess the effectiveness of the recombinant zoster vaccine in general practice. METHODS: A de-identified administrative claims database, the OptumLabs Data Warehouse, was used to conduct this retrospective cohort study to assess the effectiveness of the recombinant zoster vaccine against herpes zoster in nonimmunocompromised, vaccine age-eligible individuals enrolled in the database for ≥365 days. RESULTS: A total of 4 769 819 adults were included in this study, with 173 745 (3.6%) adults receiving 2 valid doses of the recombinant zoster vaccine. The incidence rate of herpes zoster was 258.8 (95% confidence interval [CI], 230.0-289.4) cases per 100 000 person-years in vaccinated persons compared with 893.1 (95% CI, 886.2-900.0) in unvaccinated persons. Recombinant zoster vaccine effectiveness was 85.5% (95% CI, 83.5-87.3%) overall, with an effectiveness of 86.8% (95% CI, 84.6-88.7%) in individuals 50 to 79 years old compared with 80.3% (95% CI, 75.1-84.3%) in individuals aged 80 and older. In patients with a history of live zoster vaccine within 5 years of study inclusion, vaccine effectiveness was 84.8% (95% CI, 75.3-90.7%). CONCLUSIONS: Recombinant zoster vaccine effectiveness against herpes zoster was high in a real-world setting. Given the low vaccine coverage and high effectiveness, a major public health effort is needed to identify and address barriers to vaccination and increase immunization rates.

Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients.

Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.

The Role of Chest Imaging in Patient Management during the COVID-19 Pandemic: A Multinational Consensus Statement from the Fleischner Society.

With more than 900 000 confirmed cases worldwide and nearly 50 000 deaths during the first 3 months of 2020, the coronavirus disease 2019 (COVID-19) pandemic has emerged as an unprecedented health care crisis. The spread of COVID-19 has been heterogeneous, resulting in some regions having sporadic transmission and relatively few hospitalized patients with COVID-19 and others having community transmission that has led to overwhelming numbers of severe cases. For these regions, health care delivery has been disrupted and compromised by critical resource constraints in diagnostic testing, hospital beds, ventilators, and health care workers who have fallen ill to the virus exacerbated by shortages of personal protective equipment. Although mild cases mimic common upper respiratory viral infections, respiratory dysfunction becomes the principal source of morbidity and mortality as the disease advances. Thoracic imaging with chest radiography and CT are key tools for pulmonary disease diagnosis and management, but their role in the management of COVID-19 has not been considered within the multivariable context of the severity of respiratory disease, pretest probability, risk factors for disease progression, and critical resource constraints. To address this deficit, a multidisciplinary panel comprised principally of radiologists and pulmonologists from 10 countries with experience managing patients with COVID-19 across a spectrum of health care environments evaluated the utility of imaging within three scenarios representing varying risk factors, community conditions, and resource constraints. Fourteen key questions, corresponding to 11 decision points within the three scenarios and three additional clinical situations, were rated by the panel based on the anticipated value of the information that thoracic imaging would be expected to provide. The results were aggregated, resulting in five main and three additional recommendations intended to guide medical practitioners in the use of chest radiography and CT in the management of COVID-19.

Incidence Rate of Herpes Zoster Ophthalmicus: A Retrospective Cohort Study from 1994 through 2018.

PURPOSE: To analyze the incidence rate (IR) of herpes zoster ophthalmicus (HZO) and differences by age, gender, race, and region from 1994 through 2018. DESIGN: Retrospective, observational cohort study. PARTICIPANTS: Patients with a new International Classification of Diseases, Ninth or Tenth Edition, codes for herpes zoster (HZ) and HZO from January 1, 1994, through December 31, 2018, in the OptumLabs Data Warehouse (OptumLabs, Cambridge, MA). METHODS: OptumLabs Data Warehouse, a longitudinal, real-world data asset with de-identified administrative claims and electronic health record data, was used to identify enrollees with continuous enrollment in the database for 365 days or more. Patients with no history of HZ or HZO and a new code for HZ and HZO were counted as incident cases. The IR of HZO was calculated by year, 10-year age groups, gender, race, and region. MAIN OUTCOME MEASURES: Differences in IR from 1994 through 2018 by 10-year age groups and gender. RESULTS: From 1994 through 2018, 633 474 cases of HZ were reported, with 49 745 (7.9%) having HZO. The incidence of HZO increased from 1994 through 2018 by an estimated 1.1 cases per 100 000 person-years annually (95% confidence interval [CI], 1.0-1.3; P < 0.001). The estimated relative increase was 3.6% annually (95% CI, 3.0%-4.1%). HZO IR increased in all ages over 10 years until 2007, then began declining in individuals younger than 21 and older than 60, stabilizing in individuals 21 to 30 years old, and increasing more slowly among individuals 31 to 60 years old. Men showed an HZO incidence rate ratio (IRR) of 0.74 compared with women. Compared with white patients, the IRRs were 0.70, 0.75, and 0.64 for Asians, black patients, and Hispanics, respectively. CONCLUSIONS: The incidence of HZO has increased 3.6% per year from 1994 to 2018 in the United States. Since 2008, HZO incidence declined in individuals younger than 21 years and older than 60 years while increasing at a lower rate in middle-aged adults. Given the continued increase, greater efforts should be made to vaccinate eligible adults 50 years of age and older. More research on earlier vaccination is warranted.

The association of smoking with ultrasound-measured kidney dimensions
.

PURPOSE: Ultrasound is considered a preferred first-line imaging technique for the assessment of kidney function. The potential relationship between tobacco smoke exposure and ultrasound-measured renal characteristics has yet to be explored. We hypothesized that exposure to tobacco smoke would be associated with reduced kidney dimensions. MATERIALS AND METHODS: This was a cross-sectional study that included all individuals over age 18 at a single site in Mojokerto, Indonesia. A questionnaire was used to assess prior history and environmental exposure, and blinded evaluators performed ultrasound assessments. Six kidney parameters (length, width, and parenchymal thickness of each kidney) were considered as dependent variables, and statistical relationships were assessed using multivariate analysis. Echogenicity was evaluated using a 5-point grading scale described previously. RESULTS: Of the 445 participants assessed, a total of 138 male and 269 female subjects were included in the final analysis. There was a statistically significant association between kidney measures and the following independent variables: pack years smoking (p < 0.001), height (p < 0.001), weight (p < 0.001), and beginning to smoke at the age of 25 or younger (p < 0.001). There was not a statistically significant association between kidney measures and hypertension (p > 0.05) or diabetes (p > 0.05). Echogenicity was similar among all smoking groups. CONCLUSION: Kidney dimensions were decreased in individuals with increased smoking history. This association is notable, particularly given that statistically significant associations were not observed between renal dimensions and hypertension or diabetes. The null findings using echogenicity are consistent with previous studies.

Evaluation of a point-of-care ultrasound curriculum taught by medical students for physicians, nurses, and midwives in rural Indonesia.

PURPOSE: In the Indonesian health-care system, nurses and midwives often serve as the primary health-care providers due to physician shortages. Seeking to address the need for medical care in resource-limited environments, some have advocated for portable equipment in the hands of health-care providers. We hypothesized that medical students are able to effectively teach point-of-care ultrasound (POCUS) to physicians, nurses, and midwives in rural Indonesia. METHODS: We conducted a prospective, observational study using health-care practitioners from a clinic and accredited school for nursing and midwifery in Mojokerto, East Java, Indonesia. Enrolled practitioners took part in a 4-week POCUS course followed by postinstructional testing. RESULTS: A total of 55 health-care practitioners completed the course. This included 19 physicians, 13 nurses, and 19 midwives. Of the 55 clinicians, 43 (72%) passed the course and 12 (28%) failed. CONCLUSIONS: Physicians, nurses, and midwives in rural Indonesia showed significant acquisition of ultrasound (US) knowledge and skills following a 4-week US course. Following training, all three groups displayed skills in practical US use during a postcourse practical examination. This is one of the first studies to assess the efficacy of medical students teaching POCUS to midwives and nurses.

The Molecular Pathogenesis of Dupuytren Disease: Review of the Literature and Suggested New Approaches to Treatment.

BACKGROUND: Ever since the classification of Dupuytren disease into the proliferative, involutional, and residual stages, extensive research has been performed to uncover the molecular underpinnings of the disease and develop better treatment modalities for patients. The aim of this article is to systematically review the basic science literature pertaining to Dupuytren disease and suggest a new approach to treatment. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines, a systematic review was conducted using the MEDLINE database to identify basic science literature on Dupuytren pathophysiology falling under 1 or more of the following categories: (1) Molecular alterations, (2) Structural alterations, and (3) Genetic predisposition. RESULTS: A total of 177 articles were reviewed of which 77 studies met inclusion criteria. Articles were categorized into respective sections outlined in the study methods. CONCLUSION: The pathophysiological changes involved in Dupuytren's disease can be divided into a number of molecular and structural alterations with genetic predisposition playing a contributory role. Understanding these changes can allow for the development of biologics which may disrupt and halt the disease process.

Test Characteristics of Specific p16 Clones in the Detection of High-grade Squamous Intraepithelial Lesions (HSIL).

p16 immunohistochemistry is recommended by the CAP-ASCCP Lower Anogenital Squamous Terminology (LAST) Standardization Project for human papillomavirus associated Lesions as an adjunct to morphologic assessment in the diagnosis of high-grade squamous intraepithelial lesion. This study evaluates the performance of different p16 clones as compared with E6H4 (CINtec) in detecting high-grade squamous intraepithelial lesion. The 54 high-quality articles addressing the performance of p16 identified by work group 4 of the LAST Project were evaluated for: specific p16 clone, scoring method, number of cases, anatomic site, and histologic diagnoses. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for each clone. Two-proportion z tests (pooled) were used to evaluate significance. In total, 32 of the 54 studies met the inclusion criteria. The most commonly used clone was E6H4 (17 studies, 3507 cases) with smaller numbers (1-4) of studies evaluating the following: 16P04, JC8, 16P07, G175-405, K5334, K5336, and 7962. p16 clones 16P04 and JC8 performed better than E6H4 with 16P04 exhibiting statistically significant higher sensitivity (94% vs. 87% for E6H4), specificity (94% vs. 81%), and positive predictive value (96% vs. 69%) while JC8 exhibited higher specificity (91% vs. 81%) and positive predictive value (88% vs. 69%). 16P07 performed similarly to E6H4 and the other 4 clones did not perform as well as E6H4. p16 clones 16P04, JC8, and 16P07 clones perform as well or better than the widely used p16 clone E6H4 (CINtec). However, further studies are indicated to determine the reproducibility of these findings and the impact of interlaboratory variation on test performance.

Three-dimensional organization of nascent rod outer segment disk membranes.

The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.

Discovery of recurrent structural variants in nasopharyngeal carcinoma.

We present the discovery of genes recurrently involved in structural variation in nasopharyngeal carcinoma (NPC) and the identification of a novel type of somatic structural variant. We identified the variants with high complexity mate-pair libraries and a novel computational algorithm specifically designed for tumor-normal comparisons, SMASH. SMASH combines signals from split reads and mate-pair discordance to detect somatic structural variants. We demonstrate a >90% validation rate and a breakpoint reconstruction accuracy of 3 bp by Sanger sequencing. Our approach identified three in-frame gene fusions (YAP1-MAML2, PTPLB-RSRC1, and SP3-PTK2) that had strong levels of expression in corresponding NPC tissues. We found two cases of a novel type of structural variant, which we call "coupled inversion," one of which produced the YAP1-MAML2 fusion. To investigate whether the identified fusion genes are recurrent, we performed fluorescent in situ hybridization (FISH) to screen 196 independent NPC cases. We observed recurrent rearrangements of MAML2 (three cases), PTK2 (six cases), and SP3 (two cases), corresponding to a combined rate of structural variation recurrence of 6% among tested NPC tissues.