Diagnostic Concordance of Cytology and Histology in Samples Obtained via Endoscopic Ultrasound-Guided Fine-Needle Biopsy (EUS-FNB).

Introduction Endoscopic ultrasound (EUS)-guided fine-needle aspiration and biopsy (FNA/FNB) to obtain cytological aspirates and histological core samples, respectively, are the standard of care for diagnosing lesions in/adjacent to the upper/lower gastrointestinal tract. Due to the lack of standardization of tissue processing, it is unclear whether core samples should be sent only for histology (formalin) or cytology (CytoLyt), or both. The aim of this study was to investigate the diagnostic concordance rates between cytology and histology on EUS-FNB core samples. Methods A total of 227 patients underwent EUS-FNB between October-2017 and February-2019 by a single therapeutic endoscopist; 44 core-tissue samples (41 patients) were placed alternately in CytoLyt (cytology) and formalin (histology), with equal passes into each, to best achieve a proportionate sample amount. The patient's demographics, medical history, pertinent imaging, EUS indication/findings were reviewed. Main outcomes included concordance rates between cytology-histology and diagnostic accuracy for malignancy. Results Cytology and histology were discordant in five cases (11.5%); four with negative cytology but a definite diagnosis of malignancy achieved with histology. One case was suspected as neoplasm on cytology but further characterized as benign on histology. Cytology failed to sub-characterize an additional four mass-like pancreatic benign entities, due to inadequate tissue architecture assessment in the CytoLyt sample. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of cytology for diagnosis of malignancy were 87.88% (95%CI: 71.8-96.6), 90.91% (95%CI: 58.7-99.7), 96.67% (95%CI: 81.6-99.4), and 71.43% (95%CI: 49.4-86.4). Discussion We observed 11.5% diagnostic discordance between cytology and histology on EUS-FNB core samples, with histology being superior. Future multicenter prospective randomized studies are needed to establish an accurate and cost-effective diagnostic process.

Fine-needle aspiration biopsy of lytic bone lesions: An institution's experience.

OBJECTIVES: Fine needle aspiration (FNAB) is an effective, minimally-invasive, inexpensive, diagnostic technique. The objective of this study was to evaluate the accuracy of FNAB in the diagnosis of bone lesions. METHODS: FNABs of bone lesions diagnosed at our institution over a 2-year period were retrospectively analyzed. RESULTS: 241 samples were reviewed. Patients included 121 males and 120 females, with ages ranging from 4-95 years (mean = 66 years). Of these 241 cases, 43.2% had FNAB and 56.8% had FNAB with core needle biopsy (CNB). The cytologic diagnoses were categorized as nondiagnostic, benign, atypical, suspicious, and positive for malignant cells. Total of 84.3% of FNABs were diagnostic. Of the malignant cases, 78.5% were metastases from nonosseous primary sites, 17.1% were lymphoproliferative lesions, and 4.4% were primary bone tumors. The most common site of metastasis was the pelvic bones (43.5%) followed by the vertebral column (38.7%). Breast (21%), lung (12.7%), and prostate (11.3%) were the most common identifiable primary site in metastatic cases. FNA smears and cell blocks allowed identification of metastatic lesions in 94.3% cases with immunohistochemistry (IHC). Obtaining a concomitant CNB did not result in a statistically significant increase in overall diagnostic yields (P = .20), ascertaining presence of metastatic lesion (P = .96) or ability to identify site of primary tumor in cases of metastasis (P = .53) compared to FNAB alone. Diagnostic accuracy was improved by reviewing clinical history, performing cell block, and IHC. CONCLUSIONS: FNAB is a reliable tool for diagnosis of bone lesions with comparable diagnostic sensitivity to CNB.

Quality assessment and improvement of "Unsatisfactory" liquid-based cervicovaginal papanicolaou smears.

OBJECTIVES: The quality of cervicovaginal smears determines the success of cytology in screening programs for cervical cancer. Bethesda 2014 revisited the adequacy criteria for atrophic smears and redefined the squamous cell count in the "unsatisfactory" category. In this study, we evaluated the smear quality of Thinprep liquid-based cervicovaginal Papanicolaou cytology slides (TPS) that were previously deemed unsatisfactory, to determine reasons for such categorization. In addition, we attempted to establish the impact of the new adequacy criteria on the rate and management of unsatisfactory diagnosis. METHODS: About 234 unsatisfactory TPS were examined. The reasons for unsatisfactory were noted. The number of squamous cells was recounted, as per the new Bethesda criteria, in borderline adequacy cases that showed an atrophic pattern. RESULTS: The leading cause for unsatisfactory TPS was lubricating gel, followed by blood, as observed in older and younger age groups, respectively (Figure 1). Eleven borderline cases were reclassified as "satisfactory" using the new Bethesda cell count, with 27% above 60 years of age. About 82% of these borderline cases were negative for intraepithelial lesion or malignancy on repeat testing. CONCLUSIONS: There was no difference of management or change in rate of unsatisfactory when patients above 60 were reclassified into the satisfactory category using the new Bethesda count. However, a larger study is needed to evaluate whether the new recommendation for minimum cellularity can be implemented in patients above a certain age cut-off. The study highlights the need for improvement in collection practices and education of practitioners.

FGF21 promotes endothelial cell angiogenesis through a dynamin-2 and Rab5 dependent pathway.

Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK) is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A) attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and ß-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling.

Neuropilin-1 stimulates tumor growth by increasing fibronectin fibril assembly in the tumor microenvironment.

The tumor microenvironment, including stromal myofibroblasts and associated matrix proteins, regulates cancer cell invasion and proliferation. Here, we report that neuropilin-1 (NRP-1) orchestrates communications between myofibroblasts and soluble fibronectin that promote α5ß1 integrin-dependent fibronectin fibril assembly, matrix stiffness, and tumor growth. Tumor growth and fibronectin fibril assembly were reduced by genetic depletion or antibody neutralization of NRP-1 from stromal myofibroblasts in vivo. Mechanistically, the increase in fibronectin fibril assembly required glycosylation of serine 612 of the extracellular domain of NRP-1, an intact intracellular NRP-1 SEA domain, and intracellular associations between NRP-1, the scaffold protein GIPC, and the nonreceptor tyrosine kinase c-Abl that augmented α5ß1 fibronectin fibril assembly activity. Analysis of human cancer specimens established an association between tumoral NRP-1 levels and clinical outcome. Our findings indicate that NRP-1 activates the tumor microenvironment, thereby promoting tumor growth. These results not only identify new molecular mechanisms of fibronectin fibril assembly but also have important implications for therapeutic targeting of the myofibroblast in the tumor microenvironment.

Complementary vascular and matrix regulatory pathways underlie the beneficial mechanism of action of sorafenib in liver fibrosis.

UNLABELLED: Paracrine signaling between hepatic stellate cells (HSCs) and liver endothelial cells (LECs) modulates fibrogenesis, angiogenesis, and portal hypertension. However, mechanisms regulating these processes are not fully defined. Sorafenib is a receptor tyrosine kinase inhibitor that blocks growth factor signaling in tumor cells but also displays important and not yet fully characterized effects on liver nonparenchymal cells including HSCs and LECs. The aim of this study was to test the hypothesis that sorafenib influences paracrine signaling between HSCs and LECs and thereby regulates matrix and vascular changes associated with chronic liver injury. Complementary magnetic resonance elastography, micro-computed tomography, and histochemical analyses indicate that sorafenib attenuates the changes in both matrix and vascular compartments that occur in response to bile duct ligation-induced liver injury in rats. Cell biology studies demonstrate that sorafenib markedly reduces cell-cell apposition and junctional complexes, thus reducing the proximity typically observed between these sinusoidal barrier cells. At the molecular level, sorafenib down-regulates angiopoietin-1 and fibronectin, both released by HSCs in a manner dependent on the transcription factor Kruppel-like factor 6 , suggesting that this pathway underlies both matrix and vascular changes associated with chronic liver disease. CONCLUSION: Collectively, the results of this study demonstrate that sorafenib inhibits both matrix restructuring and vascular remodeling that accompany chronic liver diseases and characterize cell and molecular mechanisms underlying this effect. These data may help to refine future therapies for advanced gastrointestinal and liver diseases characterized by abundant fibrosis and neovascularization.

Increased expression of HIF-1alpha, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency.

PURPOSE: A mouse model of renal insufficiency with arteriovenous fistula (AVF) and venous stenosis was created. The authors tested the hypothesis that there is increased gene expression of hypoxia-inducible factor-1 alpha (HIF-1alpha); vascular endothelial growth factor-A (VEGF-A) and its receptors (VEGFR-1, -2); matrix metalloproteinase-2 (MMP-2), -9 (MMP-9); tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, -2); and a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1) at the venous stenosis. MATERIALS AND METHODS: Nineteen male C57BL/6 mice underwent a left nephrectomy and a surgical occlusion of the right upper pole to induce renal function characterized in eight animals. Twenty eight days later, an AVF (n = 11) was created from the right carotid artery to ipsilateral jugular vein, and the mice were killed at day 7 (n = 4) and day 14 (n = 4). The outflow and control veins were removed for gene expression. Three mice were killed at day 28 for histologic analysis. RESULTS: The mean serum blood urea nitrogen level remained significantly elevated for 8 weeks when compared with baseline (P < .05). By day seven, there was a significant increase in the expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein, with HIF-1alpha and TIMP-1 levels significantly elevated at day 14 (P < .05). By day 28, the venous stenosis was characterized by a thickened vein wall and neointima. CONCLUSIONS: A mouse model of renal insufficiency with AVF was developed that had increased expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein with venous stenosis by day 28.

Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment.

Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 microg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase (P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 microM), an inhibitor of MEK, which is a kinase upstream from Erk (P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity (P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation (P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.

The mouse arteriovenous fistula model.

PURPOSE: The first aim of the present study was to create a mouse carotid artery-to-jugular vein arteriovenous (AV) fistula model. This model was used to test the hypothesis that there is increased gene expression of matrix metalloproteinase (MMP)-2 and MMP-9 at the venous stenosis. MATERIALS AND METHODS: Ten male FVB/NJ mice underwent the creation of an AV fistula between the left carotid artery and ipsilateral jugular vein, with the contralateral vessels serving as controls. Two mice died 1 day after surgery and the other eight were euthanized at day 28. Reverse transcriptase polymerase chain reaction was performed in five mice, with the grafted vein and control vein tissue used to determine the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2. Immunohistochemical analysis of the grafted vein and control vein was performed in three mice. RESULTS: Venous stenosis formed at the outflow vein, characterized by a thickened neointima with cells staining positive for alpha-smooth muscle actin. There was increased expression of MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 by day 28 at the venous stenosis compared with control vein. CONCLUSIONS: A mouse carotid artery-to-jugular vein AV fistula model was developed and used to demonstrate increased expression of several markers known to be associated with AV fistula stenosis. The model may be useful in investigating mechanisms responsible for AV fistula venous stenoses.