A Multiplex SNaPshot Assay is a Rapid and Cost-Effective Method for Detecting POLE Exonuclease Domain Mutations in Endometrial Carcinoma.

Determining the replicative DNA polymerase epsilon ( POLE) mutation status in endometrial carcinomas (ECs) has important clinical implications given that the majority of "ultramutated" tumors harboring pathogenic exonuclease domain mutations in POLE ( POLE mut) have a favorable prognosis, even among high-grade histotypes. Currently, there are no specific morphologic or immunophenotypic features that allow accurate detection of POLE mut tumors without molecular testing. Consequently, identifying POLE mut tumors has been challenging without employing costly and/or time-consuming DNA sequencing approaches. Here we developed a novel SNaPshot assay to facilitate routine and efficient POLE mutation testing in EC. The SNaPshot assay interrogates 15 nucleotide sites within exons 9, 11, 13, and 14 encoding the POLE exonuclease domain. The variant sites were selected based on recurrence, evidence of functional impact, association with high tumor mutation burden and/or detection in EC clinical outcome studies. Based on the pathogenic somatic variants reported in the literature, the assay is predicted to have a clinical sensitivity of 90% to 95% for ECs. Validation studies showed 100% specificity and sensitivity for the variants covered, with expected genotypic results for both the positive (n=11) and negative (n=20) patient controls on multiple repeat tests and dilution series. Analytic sensitivity was conservatively approximated at a 10% variant allele fraction (VAF), with documented detection as low as 5% VAF. As expected, the SNaPshot assay demonstrated greater sensitivity than Sanger sequencing for VAFs below 20%, an important characteristic for somatic mutation detection. Here we have developed and validated the first SNaPshot assay to detect hotspot POLE mutations. While next-generation sequencing and Sanger sequencing-based approaches have also been used to detect POLE mutations, a SNaPshot approach provides useful balance of analytical sensitivity, cost-effectiveness, and efficiency in a high-volume case load setting.

The temporal scaling of Caenorhabditis elegans ageing.

The process of ageing makes death increasingly likely, involving a random aspect that produces a wide distribution of lifespan even in homogeneous populations. The study of this stochastic behaviour may link molecular mechanisms to the ageing process that determines lifespan. Here, by collecting high-precision mortality statistics from large populations, we observe that interventions as diverse as changes in diet, temperature, exposure to oxidative stress, and disruption of genes including the heat shock factor hsf-1, the hypoxia-inducible factor hif-1, and the insulin/IGF-1 pathway components daf-2, age-1, and daf-16 all alter lifespan distributions by an apparent stretching or shrinking of time. To produce such temporal scaling, each intervention must alter to the same extent throughout adult life all physiological determinants of the risk of death. Organismic ageing in Caenorhabditis elegans therefore appears to involve aspects of physiology that respond in concert to a diverse set of interventions. In this way, temporal scaling identifies a novel state variable, r(t), that governs the risk of death and whose average decay dynamics involves a single effective rate constant of ageing, kr. Interventions that produce temporal scaling influence lifespan exclusively by altering kr. Such interventions, when applied transiently even in early adulthood, temporarily alter kr with an attendant transient increase or decrease in the rate of change in r and a permanent effect on remaining lifespan. The existence of an organismal ageing dynamics that is invariant across genetic and environmental contexts provides the basis for a new, quantitative framework for evaluating the manner and extent to which specific molecular processes contribute to the aspect of ageing that determines lifespan.

Morphological, immunophenotypical and molecular features of hypermutation in colorectal carcinomas with mutations in DNA polymerase ε (POLE).

AIMS: Colorectal carcinomas (CRC) with mismatch repair (MMR) deficiency have increased tumour mutation burden and respond to immune check-point inhibitor therapy. The Cancer Genome Atlas identified hypermutated CRCs with somatic mutations in DNA polymerase ε (POLE) with mutation burdens exceeding that of MMR-deficient CRCs. METHODS AND RESULTS: To identify the morphological, immunophenotypical and molecular features of POLE-mutated CRCs, 63 consecutive MMR-intact CRCs were evaluated by Sanger sequencing for POLE exonuclease domain mutations in exons 9, 11, 13 and 14 and confirmed by next-generation sequencing. Tumour immune microenvironment and IMMUNOSCORE®1 were assessed in POLE-mutated CRCs using immunohistochemistry to detect CD3+ /CD8+ tumour-infiltrating lymphocytes and compared to 59 non-POLE mutated MMR-intact CRC, 10 non-POLE mutated MMR-deficient CRCs and 223 normal colonic mucosa. CONCLUSIONS: A total of 4.8% CRC (four MMR-intact primary and one MMR-intact metastasis) harboured POLE mutations in amino acid 286 in exon 9 (p.P286R) or exon 13 (p.V411L). POLE-mutated CRCs arose in the transverse colon and rectum, were male-predominant, younger and showed increased tumour-infiltrating lymphocytes and immune cells at the tumour-stromal interface. The patient with metastatic POLE-mutated CRC was placed on PD-1 inhibitor treatment with marked and sustained response. These data indicate that POLE-mutated CRCs have hypermutated phenotypes despite MMR-intact status, with mutation burdens higher than that in microsatellite-unstable CRCs. Given the recent approval for treatment of microsatellite-unstable cancer with immune check-point inhibitors, assessment of POLE status may help to guide therapeutic decisions for hypermutated tumours with intact MMR that would otherwise be missed by routine testing.

Intestinal stem cell ablation reveals differential requirements for survival in response to chemical challenge.

The Drosophila intestine is maintained by multipotent intestinal stem cells (ISCs). Although increased intestinal stem cell (ISC) proliferation has been correlated with a decrease in longevity, there is some discrepancy regarding whether a decrease or block in proliferation also has negative consequences. Here we identify headcase (hdc) as a novel marker of ISCs and enteroblasts (EBs) and demonstrate that Hdc function is required to prevent ISC/EB loss through apoptosis. Hdc depletion was used as a strategy to ablate ISCs and EBs in order to test the ability of flies to survive without ISC function. While flies lacking ISCs showed no major decrease in survival under unchallenged conditions, flies depleted of ISCs and EBs exhibited decreased survival rates in response to damage to mature enterocytes (EC) that line the intestinal lumen. Our findings indicate that constant renewal of the intestinal epithelium is not absolutely necessary under normal laboratory conditions, but it is important in the context of widespread chemical-induced damage when significant repair is necessary.

Intraoperative Pancreatic Cancer Detection using Tumor-Specific Multimodality Molecular Imaging.

BACKGROUND: Operative management of pancreatic ductal adenocarcinoma (PDAC) is complicated by several key decisions during the procedure. Identification of metastatic disease at the outset and, when none is found, complete (R0) resection of primary tumor are key to optimizing clinical outcomes. The use of tumor-targeted molecular imaging, based on photoacoustic and fluorescence optical imaging, can provide crucial information to the surgeon. The first-in-human use of multimodality molecular imaging for intraoperative detection of pancreatic cancer is reported using cetuximab-IRDye800, a near-infrared fluorescent agent that binds to epidermal growth factor receptor. METHODS: A dose-escalation study was performed to assess safety and feasibility of targeting and identifying PDAC in a tumor-specific manner using cetuximab-IRDye800 in patients undergoing surgical resection for pancreatic cancer. Patients received a loading dose of 100 mg of unlabeled cetuximab before infusion of cetuximab-IRDye800 (50 mg or 100 mg). Multi-instrument fluorescence imaging was performed throughout the surgery in addition to fluorescence and photoacoustic imaging ex vivo. RESULTS: Seven patients with resectable pancreatic masses suspected to be PDAC were enrolled in this study. Fluorescence imaging successfully identified tumor with a significantly higher mean fluorescence intensity in the tumor (0.09 ± 0.06) versus surrounding normal pancreatic tissue (0.02 ± 0.01), and pancreatitis (0.04 ± 0.01; p < 0.001), with a sensitivity of 96.1% and specificity of 67.0%. The mean photoacoustic signal in the tumor site was 3.7-fold higher than surrounding tissue. CONCLUSIONS: The safety and feasibilty of intraoperative, tumor-specific detection of PDAC using cetuximab-IRDye800 with multimodal molecular imaging of the primary tumor and metastases was demonstrated.

Diagnostic Performance of Margin Features in Thyroid Nodules in Prediction of Malignancy.

OBJECTIVE: The purpose of this study was to evaluate thyroid nodule margins for specific morphologic features and determine the diagnostic performance of these features in differentiating papillary carcinoma from benign thyroid nodules. MATERIALS AND METHODS: Nodules measuring 1-3 cm in largest diameter that had been evaluated with high-resolution ultrasound (12-18 MHz) and ultrasound-guided biopsy with definitive pathologic diagnosis were analyzed. Three blinded board-certified readers evaluated high-resolution images of each nodule for jagged edges, lobulated borders, and curved borders along their margins. Reader interpretations were correlated with the pathologic diagnosis to determine the diagnostic performance of each feature. A board-certified pathologist analyzed 10 randomly selected nodules with jagged edges by slide review to evaluate for structural correlation with the imaging finding. RESULTS: The diagnostic performance of jagged edges in papillary carcinoma of the thyroid was 67.4% sensitive and 78.3% specific (odds ratio, 7.44; p < 0.001) for malignancy. Jagged edges correlated with infiltrative variant expansion at slide review. Lobulated borders had sensitivity of 76.1% and specificity of 60.9% for papillary carcinoma (odds ratio, 4.95; p = 0.001) for malignancy. Curved borders were not a significant predictor of papillary carcinoma. CONCLUSION: Jagged edges and lobulated borders of thyroid nodule margins are statistically significant predictors of papillary carcinoma of the thyroid. Jagged edges correlate with infiltrative-type expansion and may be useful predictors of more aggressive papillary carcinomas.

A Drought-Inducible Transcription Factor Delays Reproductive Timing in Rice.

The molecular mechanisms underlying photoperiod or temperature control of flowering time have been recently elucidated, but how plants regulate flowering time in response to other external factors, such as water availability, remains poorly understood. Using a large-scale Hybrid Transcription Factor approach, we identified a bZIP transcriptional factor, O. sativa ABA responsive element binding factor 1 (OsABF1), which acts as a suppressor of floral transition in a photoperiod-independent manner. Simultaneous knockdown of both OsABF1 and its closest homologous gene, OsbZIP40, in rice (Oryza sativa) by RNA interference results in a significantly earlier flowering phenotype. Molecular and genetic analyses demonstrate that a drought regime enhances expression of the OsABF1 gene, which indirectly suppresses expression of the Early heading date 1 (Ehd1) gene that encodes a key activator of rice flowering. Furthermore, we identified a drought-inducible gene named OsWRKY104 that is under the direct regulation of OsABF1 Overexpression of OsWRKY104 can suppress Ehd1 expression and confers a later flowering phenotype in rice. Together, these findings reveal a novel pathway by which rice modulates heading date in response to the change of ambient water availability.

Signaling mechanisms of plant cryptochromes in Arabidopsis thaliana.

Cryptochromes (CRY) are flavoproteins that direct a diverse array of developmental processes in response to blue light in plants. Conformational changes in CRY are induced by the absorption of photons and result in the propagation of light signals to downstream components. In Arabidopsis, CRY1 and CRY2 serve both distinct and partially overlapping functions in regulating photomorphogenic responses and photoperiodic flowering. For example, both CRY1 and CRY2 regulate the abundance of transcription factors by directly reversing the activity of E3 ubiquitin ligase on CONSTITUTIVE PHOTOMORPHOGENIC 1 and SUPPRESSOR OF PHYA-105 1 complexes in a blue light-dependent manner. CRY2 also specifically governs a photoperiodic flowering mechanism by directly interacting with a transcription factor called CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX. Recently, structure/function analysis of CRY1 revealed that the CONSTITUTIVE PHOTOMORPHOGENIC 1 independent pathway is also involved in CRY1-mediated inhibition of hypocotyl elongation. CRY1 and CRY2 thus not only share a common pathway but also relay light signals through distinct pathways, which may lead to altered developmental programs in plants.

Fatal West Nile Virus Encephalitis in a Heart Transplant Recipient.

The diagnosis of encephalitis is particularly challenging in immunocompromised patients. We report here a case of fatal West Nile virus encephalitis confounded by the presence of budding yeast in the cerebrospinal fluid (CSF) from a patient who had undergone heart transplantation for dilated cardiomyopathy 11 months prior to presentation of neurologic symptoms.

Monoclonal antibody pairs against SARS-CoV-2 for rapid antigen test development.

BACKGROUND: The focus on laboratory-based diagnosis of coronavirus disease 2019 (COVID-19) warrants alternative public health tools such as rapid antigen tests. While there are a number of commercially available antigen tests to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), all cross-react with the genetically similar SARS-CoV-1 or require an instrument for results interpretation. METHODOLOGY/PRINCIPAL FINDINGS: We developed and validated rapid antigen tests that use pairs of murine-derived monoclonal antibodies (mAbs), along with gold nanoparticles, to detect SARS-CoV-2 with or without cross-reaction to SARS-CoV-1 and other coronaviruses. In this development, we demonstrate a robust antibody screening methodology for the selection of mAb pairs that can recognize SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. Linear epitope mapping of the mAbs helped elucidate SARS-CoV-2 S and N interactions in lateral flow chromatography. A candidate rapid antigen test for SARS-CoV-2 N was validated using nasal swab specimens that were confirmed positive or negative by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Test results were image-captured using a mobile phone and normalized signal pixel intensities were calculated; signal intensities were inversely correlated to RT-PCR cycle threshold (Ct) value. CONCLUSION/SIGNIFICANCE: Overall, our results suggest that the rapid antigen test is optimized to detect SARS-CoV-2 N during the acute phase of COVID-19. The rapid antigen tests developed in this study are alternative tools for wide scale public health surveillance of COVID-19.

Capturing the often-elusive diagnosis of idiopathic myointimal hyperplasia of mesenteric veins.

Only 50 cases of idiopathic myointimal hyperplasia of the mesenteric veins (IMHMV) have been reported since 1991 when it was first described. This rare etiology for chronic colonic ischemia is often debilitating to the patient's quality of life, and no effective medical treatment is available. IMHMV is frequently confused with inflammatory bowel disease because the most common presenting symptoms include abdominal pain, diarrhea, and hematochezia. Surgical resection is curative; however, the diagnosis is rarely reached preoperatively. In the present report, we have described the seventh patient with a diagnosis of IMHMV before surgery and included a literature review to help clinicians recognize this condition.

Eosinophilic Colitis in Recurrent Sigmoid Volvulus.

Eosinophilic colitis (EC) falls along the spectrum of a series of inflammatory gastrointestinal disorders in which eosinophils infiltrate the gut without known tissue eosinophilia. Eosinophilic gastrointestinal disorders include eosinophilic esophagitis, eosinophilic gastritis, and the least common EC. The presentation of EC is extremely variable in mucosal, submucosal, and transmural inflammation. We present a case of recurrent volvulus with histologic findings of eosinophilia.

Recurrent Candida-Associated Esophageal Strictures in an Immunocompetent Patient.

Esophageal stricture due to Candida esophagitis is a rare complication reported among immunocompromised patients in only limited case reports. We describe a unique case of a 36-year-old man with chronic mucocutaneous candidiasis without underlying immunocompromise who experienced over 10 years of recurrent dysphagia due to esophageal strictures from candidiasis. His symptoms were initially believed to be due to eosinophilic esophagitis; however, numerous biopsies from the esophagus were negative for eosinophils. Several upper endoscopies, however, did reveal fungal elements consistent with Candida spp. He experienced recurring episodes of dysphagia and persistent esophageal stricture, requiring multiple antifungal courses and endoscopic balloon dilatations.

Central echogenic areas in thyroid nodules: Diagnostic performance in prediction of papillary cancer.

PURPOSE: To determine the diagnostic performance of the "central echogenic area" sonographic finding in differentiating papillary carcinomas from benign nodules and to how this finding may be used to improve fine needle aspiration(FNA) technique/utilization. MATERIALS AND METHODS: We retrospectively analyzed ultrasound guided FNAs of thyroid nodules between 1 and 3 cm for central echogenic areas. 92 patients (evenly distributed benign vs papillary carcinoma) were evaluated by a blinded reader for areas of non-shadowing homogenously echogenic centers within the nodules and correlated with FNA proven pathologic diagnosis. A selection of nodules with the central echogenic area finding were selected for further slide review to establish a pathologic basis for the finding. RESULTS: Diagnostic performance of the "central echogenic area" feature in papillary thyroid cancers was 52.2% sensitive and 91.3% specific for papillary thyroid carcinoma with a PPV of 85.7% and NPV of 65.6%. There was a significant correlation with a p < 0.01 between the central echogenic area finding and papillary carcinoma. On pathologic slide review, nodules with central echogenic areas consistently demonstrated a central scar with conglomerate fibrosis and very few viable cells. CONCLUSION: Despite its relatively low sensitivity, the central echogenic area finding is highly specific for papillary carcinoma of the thyroid and can be a useful sonographic finding in decisions regarding FNA. Additionally, due to the paucity of cells and high density of conglomerate fibrosis, central echogenic areas should be avoided during FNA to decrease the chance of an inadequate sample collection.

Histologic Features of Gastrointestinal Tract Biopsies in IgA Vasculitis (Henoch-Schönlein Purpura).

Immunoglobulin A (IgA) vasculitis or Henoch-Schönlein purpura (HSP) typically occurs in the pediatric population, although rare cases also occur in adults. Gastrointestinal (GI) involvement is common. The "classic" histologic finding in IgA vasculitis (HSP) is leukocytoclastic vasculitis (LCV); other histologic features in biopsies of IgA vasculitis (HSP) have only been rarely described. The pathology archival files at our institution were searched for GI biopsies from patients with IgA vasculitis (HSP). Slides were retrieved and histologic and clinical features were reviewed. We identified 16 patients with IgA vasculitis (HSP) with a GI biopsy series, including both adult and pediatric patients. The most common histologic abnormality was lamina propria hemorrhage (all cases) with many cases also showing lamina propria fibrin deposition with red cell sludging and nuclear debris (7 cases). Twelve of the 16 duodenal biopsies had acute duodenitis; 3 of which were severe and erosive. Several also had an eosinophilic infiltrate. Seven of the 9 jejunal and/or ileal biopsies had acute jejunitis or ileitis. An acute colitis or proctitis was observed in 9/12 colorectal biopsies. Four biopsies contained LCV; in each of these cases, the involved vessels were small capillaries within the lamina propria. Only 1 biopsy contained deeper submucosal vessels, but they were uninvolved. Sites involved by LCV included the colorectum (2 cases), colorectum and terminal ileum, terminal ileum only, duodenum, and jejunum (1 case each). All patients presented with abdominal pain; 13/16 developed a rash, 1 following the index biopsy. Other presenting symptoms included diarrhea and/or hematochezia (8 cases), nausea/vomiting (5 cases), and intussusception (1 case). Four patients had concurrent skin biopsies showing LCV; only 1 of these patients had LCV on GI biopsy. Indications for biopsy included nonspecific presenting symptoms, absence of rash at presentation, and/or failure to respond adequately to steroid therapy. Biopsies are commonly performed in patients with or without suspected IgA vasculitis (HSP) to rule out infection, inflammatory bowel disease, and less commonly, vasculitis. In general, vasculitis is not commonly observed in GI biopsies of patients with IgA vasculitis (HSP), and the spectrum of findings includes neutrophilic infiltrate within the small bowel and colon, with the duodenum most commonly affected. While the clinical and histologic findings may mimic early inflammatory bowel disease, the presence of predominant small bowel involvement, especially erosive duodenitis, should raise suspicion for IgA vasculitis (HSP). Biopsies should be obtained before steroid therapy is initiated, if possible.

Head and Neck Cancer in Haiti: A Case Series from Hopital de L'Universite d'Etat d'Haiti.

This manuscript characterizes the demographics, presenting symptoms and risk factors of patients diagnosed with head and neck cancer at Hopital de L'Universite d'Etat d'Haiti (HUEH), Haiti's single largest healthcare facility. We conducted a prospective study of patients who presented to HUEH between January and March of 2016 with a lesion of the head or neck suspicious for cancer. All patients who met eligibility criteria received a biopsy, which was interpreted by a Haitian pathologist and when the specimen was available was confirmed by a team of pathologists from Stanford University. A total of 34 participants were identified. The biopsy-confirmed diagnoses were squamous cell carcinoma (n=7), benign (n=7), large cell lymphoma (n=2), ameloblastoma (n=2), pleomorphic adenoma (n=1), and adenocarcinoma (n=1). Fourteen patients were unavailable for biopsy. Patients with head and neck cancer had a mean age of 63.4 years, were majority male (62.5%), waited on average 10.9 months to seek medical attention, and most commonly presented with T-stage 3 or higher disease (87.5%). By characterizing patterns of head and neck cancer at HUEH we hope to facilitate efforts to improve early detection, diagnosis, and management of this important public health condition.

Determination of Tumor Margins with Surgical Specimen Mapping Using Near-Infrared Fluorescence.

For many solid tumors, surgical resection remains the gold standard and tumor-involved margins are associated with poor clinical outcomes. Near-infrared (NIR) fluorescence imaging using molecular agents has shown promise for in situ imaging during resection. However, for cancers with difficult imaging conditions, surgical value may lie in tumor mapping of surgical specimens. We thus evaluated a novel approach for real-time, intraoperative tumor margin assessment. Twenty-one adult patients with biopsy-confirmed squamous cell carcinoma arising from the head and neck (HNSCC) scheduled for standard-of-care surgery were enrolled. Cohort 1 (n = 3) received panitumumab-IRDye800CW at an intravenous microdose of 0.06 mg/kg, cohort 2A (n = 5) received 0.5 mg/kg, cohort 2B (n = 7) received 1 mg/kg, and cohort 3 (n = 6) received 50 mg. Patients were followed 30 days postinfusion and adverse events were recorded. Imaging was performed using several closed- and wide-field devices. Fluorescence was histologically correlated to determine sensitivity and specificity. In situ imaging demonstrated tumor-to-background ratio (TBR) of 2 to 3, compared with ex vivo specimen imaging TBR of 5 to 6. We obtained clear differentiation between tumor and normal tissue, with a 3-fold signal difference between positive and negative specimens (P < 0.05). We achieved high correlation of fluorescence intensity with tumor location with sensitivities and specificities >89%; fluorescence predicted distance of tumor tissue to the cut surface of the specimen. This novel method of detecting tumor-involved margins in surgical specimens using a cancer-specific agent provides highly sensitive and specific, real-time, intraoperative surgical navigation in resections with complex anatomy, which are otherwise less amenable to image guidance.Significance: This study demonstrates that fluorescence can be used as a sensitive and specific method of guiding surgeries for head and neck cancers and potentially other cancers with challenging imaging conditions, increasing the probability of complete resections and improving oncologic outcomes. Cancer Res; 78(17); 5144-54. ©2018 AACR.

Clinicopathologic Threshold of Acute Colorectal Graft-versus-Host Disease.

CONTEXT: -Colon biopsies are often used to determine the presence and severity of acute gastrointestinal graft-versus-host disease following bone marrow transplant. OBJECTIVE: -To establish a threshold consensus within our institution on the number of crypt apoptotic bodies (CAB) indicative of grade 1 acute colorectal graft-versus-host disease, we retrospectively reviewed colon biopsies from posttransplant patients and incorporated clinical and endoscopic findings to validate recently proposed minimum criteria for grade 1 graft-versus-host disease as 7 or more CAB per 10 contiguous crypts. DESIGN: -Eighty-one biopsies performed for suspected graft-versus-host disease from 74 individual patients were initially stratified based on their prior (prestudy) diagnoses: no significant abnormality, grade 1 graft-versus-host disease, and descriptive diagnoses mentioning increased apoptosis. A chart review was performed to assess the clinical and endoscopic impression at the time of biopsy and to determine the subsequent management and outcome. RESULTS: -Twenty-six biopsies with an average of 3 CAB were considered true-negative cases, and 32 biopsies with an average of 9.75 CAB were considered true-positive cases (t = 3.95999, P < .001). True-negative cases had an average density of 1.36 CAB per crypt, and true-positive cases had an average density of 2.97 CAB per crypt (t = 3.950178, P < .001). CONCLUSIONS: -A threshold of 7 or more CAB per 10 contiguous crypts promotes appropriate treatment of grade 1 acute graft-versus-host disease after other diagnostic entities are excluded. Although this threshold is 100% specific to grade 1 acute colorectal graft-versus-host disease after other histologic mimics are excluded, this threshold has a low sensitivity (59.4%) as patients with less than 7 CAB per 10 contiguous crypts constitute a heterogeneous group.