Role of cytology in the diagnosis of Barrett's esophagus and associated neoplasia

We studied 327 consecutive paired esophageal biopsies and brushing specimens obtained during the same endoscopic session to evaluate the role of cytology for the diagnosis of Barrett's esophagus (BE) and/or surveillance for associated dysplasia. A diagnosis of BE was based on the cytologic presence of goblet cells. Cases were reviewed and categorized into: 1) benign esophageal lesions (125 cases), with 48 cases of Candida (32 cases diagnosed by both techniques and 16 diagnosed only by cytology), 3 cases of herpes simplex with only 1 case diagnosed by cytology, and 74 cases of inflammation and/or repair; 2) benign BE (141 cases), with 74 cases (52%) diagnosed by both techniques, 11 cases by cytology only (8%), and 56 cases (40%) by histology only; 3) low-grade dysplasia (LGD, 30 cases), with 5 cases (17%) diagnosed with both specimens, one case (3%) by cytology only, and 24 cases (80%) by histology only; 4) high-grade dysplasia (HGD, 10 cases), with 8 cases (80%) diagnosed with both specimens, 1 case (10%) by cytology, and 1 case (10%) by histology; and 5) carcinomas (23 cases), with 20 cases (87%) diagnosed with both specimens, 2 cases (9%) by cytology only, and 1 case (4%) by histology only. Our results support the high degree of diagnostic accuracy of cytology for the diagnosis of Barrett's-associated HGD and/or carcinoma, and moderate sensitivity for BE.     

Role of TFF3 as an adjunct in the diagnosis of Barrett's esophagus using a minimally invasive esophageal sampling device—The CytospongeTM

The incidence of esophageal carcinoma continues to increase whilst its prognosis remains poor. The most dramatic reduction in mortality is likely to follow early diagnosis of the preinvasive precursor lesion, Barrett's esophagus (BE), coupled with treatment of dysplastic lesions. The major risk factor for BE is gastroesophageal reflux disease, however this is highly prevalent and only a small proportion of individuals have BE, therefore an endoscopy‐based screening strategy to detect BE is unfeasible. Minimally invasive esophageal sampling devices offer an alternative, cost‐effective strategy which can be deployed within an at‐risk population in a primary care setting to identify individuals with probable BE who can then be referred for endoscopic confirmation. The device that has currently progressed furthest in clinical trials is the Cytosponge^TM which collects cells from the gastric cardia, gastroesophageal junction and along the whole esophageal length. The cell sample is processed into a formalin‐fixed paraffin‐embedded block and sections assessed for the presence of intestinal metaplasia. TFF3 immunohistochemistry has consistently been shown to be a valuable adjunct that increases the accuracy of the Cytosponge^TM test by highlighting early goblet cells which may be missed on morphological assessment and by allowing pseudogoblet cells to be differentiated from true goblet cells.     

Ultrasound-guided fine-needle aspiration diagnosis of adenocarcinoma of esophagus with signet-ring cell features arising in Barrett's esophagus: A case report

We report a rarely documented case of adenocarcinoma of esophagus with signet-ring cell features arising in a Barrett's esophagus. This was diagnosed by employing combined endosonography and fine-needle aspiration cytology. The patient had progressive dysphagia and a poorly circumscribed mass involving the distal esophagus and gastroesophageal junction. An incisional biopsy was performed which showed Barrett's esophagus with highly atypical glands suspicious for adenocarcinoma. Following that, an ultrasound-guided endoscopic fine-needle aspiration revealed a cellular specimen with multiple groups and singly dispersed atypical glandular cells with a predominance of signet-ring features. These findings represented an adenocarcinoma with signet-ring cell features arising in Barrett's esophagus. Diagn. Cytopathol. 1998;19:51–54. © 1998 Wiley-Liss, Inc.     

Role of epigenetic alterations in the pathogenesis of Barrett's esophagus and esophageal adenocarcinoma

Barrett's esophagus, a pre-malignant condition that can lead to esophageal adenocarcinoma, is characterized by histological changes in the normal squamous epithelium of the esophagus. Numerous molecular changes occur during the multistage conversion of Barrett's metaplasia to dysplasia and frank adenocarcinoma. Epigenetic changes, especially changes in DNA methylation are widespread during this process. Aberrant DNA methylation has been shown to occur at promoters of tumor suppressor genes, adhesion molecules and DNA repair genes during Barrett's esophagus. These epigenetic alterations can be used as molecular biomarkers for risk stratification and early detection of esophageal adenocarcinoma. We also show that genome wide analysis of methylation surprisingly reveals that global hypomethylation and not hypermethylation is the dominant change during Barrett's metaplasia. The transformation of Barrett's esophagus to frank adenocarcinoma is in turn characterized by much smaller wave of selective promoter hypermethylation. These studies reveal many novel, potential targets for new therapies and illustrate the utility of incorporating these epigenetic changes as biomarkers during endoscopic surveillance interval for patients with Barrett's esophagus.     

Esophageal adenocarcinoma arising in cervical inlet patch with synchronous Barrett's esophagus‐related dysplasia

Esophageal adenocarcinomas usually develop in Barrett's esophagus, typically through the metaplasia–dysplasia–carcinoma sequence, but adenocarcinomas can occur from heterotopic gastric mucosa in cervical esophagus (inlet patch). This report describes the first case of synchronous presentation of adenocarcinoma arising from cervical inlet patch and Barrett's esophagus‐related dysplasia in a 76‐year‐old man. Surveillance CT detected a 3‐cm polypoid mass in the cervical esophagus. Endoscopic biopsies confirmed a diagnosis of adenocarcinoma of the cervical esophagus. Barrett's esophagus was present also in the lower esophagus. Histologic examination of the surgically resected specimen revealed the polypoid mass as composed of tubular adenocarcinoma, and was associated with non‐neoplastic columnar mucosa representing pre‐existing inlet patch. Another isolated cervical inlet patch with intestinal metaplasia was also recognized. In the lower esophagus, high‐grade dysplasia was noted within the Barrett's esophagus. Immunohistochemically, the adenocarcinoma associated with inlet patch had intestinal immunophenotype (CDX2‐, CD10‐ and MUC2‐positive), whereas the Barrett's esophagus‐related high‐grade dysplasia showed mixed immunophenotype (MUC5AC‐ and MUC6‐positive, with scattered MUC2‐positive goblet cells). Previous studies and our findings suggest that intestinal metaplasia might predispose to the development of adenocarcinoma in the inlet patch. Therefore, endoscopists and pathologists should be aware of rare malignant transformation of inlet patches, especially those with intestinal metaplasia.     

p16 gene mutations in Barrett's esophagus in gastric metaplasia - intestinal metaplasia - dysplasia - adenocarcinoma sequence

PurposeBarrett's associated esophageal adenocarcinoma (ADC) is one of the malignancies of most rapidly increasing incidence. The aim of the study was to assess p16 tumor suppressor gene alterations in the ADC premalignant conditions.Material &; MethodsIn the present study two p16 gene mutations (A148T and I49S) analysis with PCR- RFLP method have been performed in oesophageal biopsy specimen in 33 patients with Barrett's gastric metaplasia (GM), 27 - with Barrett's intestinal metaplasia (IM), 8 - with dysplasia and 11 - with ADC.ResultsWe have detected the I49S mutation in 12% (4/33) patients with GM, 18% (5/27) with IM, 50% – with dysplasia (4/8) and in 27% (3/11) – with ADC. The A148T mutation were found in 3% (1/33) patients with GM, 22% (6/27) – IM, 25% (2/8) – dysplasia and 27% patients with ADC (3/11). The frequency of the A148S mutation was rising in GM – IM – dysplasia - ADC sequence and was significantly lower in GM compared to all other grades taken together (p=0.0256). The frequency of the I49S mutation was rising in GM – IM – dysplasia sequence, to drop in ADC cases. There were no significant differences in frequency of the I49S mutation between studied groups.ConclusionsThese findings are consistent with the hypothesis on the role of the p16 mutations in early phase of Barrett's epithelium progression to ADC. The presence of p16 mutations in esophageal metaplastic columnar epithelium without goblet cells suggest that this pathology may have malignancy potential.     

Sulindac prevents esophageal adenocarcinomas induced by gastroduodenal reflux in rats

PURPOSE: It is known that cyclooxygenase (COX)-2 expression is increased in Barrett's esophagus and esophageal adenocarcinomas. We studied COX-2 expression and the effect sulindac has on the genesis of Barrett's esophagus and adenocarcinoma in rats undergoing esophagogastroduodenal anastomosis (EGDA). MATERIALS AND METHODS: Fifty-one rats were divided into a control group (n=27), a 500 ppm sulindac-treated group (n=15) and 1000 ppm sulindac-treated group (n=9). Randomly selected rats were killed by diethyl ether inhalation at 20 and 40 weeks after surgery. RESULTS: At 40 weeks, rats treated with 1000 ppm sulindac showed narrower esophageal diameter and milder inflammation than the control rats. At 40 weeks, the incidence of Barrett's esophagus was similar between control and sulindac-treated groups, but the incidence of adenocarcinoma was significantly lower in the 1000 ppm sulindac-treated group than either the control or 500 ppm sulindac-treated groups. COX-2 was significantly increased in the lower esophagus of control rats killed at 40 weeks. Cyclin D1 expression was negligible in the sulindac- treated group compared with the control group. CONCLUSION: We suggest that the chemopreventive effect of sulindac is related to decreased COX-2 and cyclin D1 expression, which may be influenced by reduced inflammation.     

Barrett's oesophageal adenocarcinoma encompasses tumour‐initiating cells that do not express common cancer stem cell markers

Accumulating evidence has suggested that tumours have a hierarchical organization in which only the cancer stem cells (CSCs) have tumour‐initiating properties. Several surface antigens have been employed to isolate CSCs from various malignancies, although not from oesophageal adenocarcinoma (EA). We tested whether Barrett's oesophagus (BE) and EA might serve as a model for the CSC concept. In vivo assays were performed by transplantation of serially diluted bulk EA cells into NOD‐SCID mice to establish the presence and frequency of tumour‐initiating cells. These were found to be present as ca. 1 in 64 000 cells. The transplanted tumours fully recapitulated the primary lesions. Subsequently, a panel of previously established CSC markers was employed for immunohistochemistry. CD24, CD29 and CD44 showed heterogeneous staining in EA. Nuclear β‐catenin accumulation increased during progression from metaplasia to dysplasia and was often observed in the basal compartment with CD24 and CD29 staining. However, the overall staining patterns were not such to clearly point out specific candidate markers. Accordingly, all markers were employed to sort the corresponding subpopulations of cancer cells and transplant them at low multiplicities in NOD‐SCID mice. No increased tumour‐initiating capacity of sorted EA cells was observed upon transplantation. These results indicate that tumour‐initiating cells are present in EA, thus reflecting a hierarchical organization. However, antibodies directed against novel surface antigens are needed to detect subpopulations enriched for CSCs in EA by transplantation assays. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Analysis of NTRK expression in gastric and esophageal adenocarcinoma (AGE) with pan-TRK immunohistochemistry

Small molecule inhibitors such as Larotrectinib have been recently approved in the treatment of patients with fusions of the neurotrophic-tropomyosin receptor tyrosine kinase (NTRK) genes 1-3. These genomic rearrangements have been reported across different tumor subtypes with a high prevalence in rare tumors. However, in gastric and esophageal adenocarcinoma (AGE) NTRK fusions have also been described in a subset of Asian patients. In order to study the prevalence of this alteration in Caucasian patients with AGE we performed immunohistochemistry for pan-NTRK in 438 formalin-fixed paraffin embedded (FFPE) tumor samples. While we found NTRK expression in gastric glands and tumor adjacent nerve tissue, we did not detect this marker in the tumor compartment. Based on our findings NTRK fusions do not seem to play a role in the molecularpathology of Caucasian AEG patients, so that other treatment options are required.     

Nicotinic receptor‐associated modulation of stimulatory and inhibitory neurotransmitters in NNK‐induced adenocarcinoma of the lungs and pancreas

Small airway‐derived pulmonary adenocarcinoma (PAC) and pancreatic ductal adenocarcinoma (PDAC) are among the most common human cancers and smoking is a risk factor for both. Emerging research has identified cAMP signalling stimulated by the stress neurotransmitters adrenaline and noradrenaline as an important stimulator of adenocarcinomas, including PAC and PDAC. The nicotine‐derived nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is a potent mutagen and the most powerful tobacco carcinogen. NNK is also an agonist for nicotinic acetylcholine receptors (nAChRs). Using hamster models of NNK‐induced PAC and PDAC, we have tested the hypothesis that in analogy to chronic effects of nicotine in the brain, NNK may modulate the α₇‐ and α₄β₂nAChRs, causing an increase in stress neurotransmitters and a decrease in the inhibitory neurotransmitter γ‐aminobutyric acid (GABA). Immunoassays showed a significant increase in serum adrenaline/noradrenaline and increased intracellular cAMP in the cellular fraction of blood of NNK‐treated hamsters. Western blots on microdissected control small airway epithelia, alveolar epithelia, pancreatic islet and pancreatic duct epithelia, and from NNK‐induced PACs and PDACs showed that the GABA‐synthesizing enzyme glutamate decarboxylase 65 (GAD65) and GABA were suppressed in NNK‐induced PACs and PDACs. In contrast, protein expression of the α₇nAChR, α₄nAChR as well as p‐CREB and p‐ERK1/2 were up‐regulated. These findings suggest that NNK‐induced alterations in regulatory nAChRs may contribute to the development of smoking‐associated PAC and PDAC by disturbing the balance between cancer‐stimulating and ‐inhibiting neurotransmitters. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Attenuation of the exercise‐induced increase in skeletal muscle Flt‐1 mRNA by nitric oxide synthase inhibition

We investigated the vascular endothelial growth factor (VEGF) receptor [fms‐like‐tyrosine kinase (Flt‐1 and fetal liver kinase‐1 (Flk‐1)] response to acute exercise. In female Wistar rats, the VEGF receptor messenger RNA (mRNA) response to a single acute exercise bout was examined using semi‐quantitative Northern blot from the left gastrocnemius muscles at rest and post‐exercise at 0, 1, 2, 4, 8, 16, 24 and 48 h. Exercise altered both Flt‐1 and Flk‐1 mRNA, with significant increases in Flt‐1 mRNA at 1 and 24 h. However, post‐hoc analysis was unable to discern the time point where a significant increase in Flk‐1 mRNA occurred. To investigate the regulation of Flt‐1 mRNA by exercise we examined if nitric oxide synthase (NOS) inhibition alters the Flt‐1 mRNA response. Eight groups [Condition: Rest or Exercise; Drug: Saline, 30 mg kg^–1N^ω‐nitro‐L ‐arginine methyl ester (L ‐NAME), 300 mg kg^–1L ‐NAME or 300 mg kg^–1D ‐NAME] were used to determine the effect of NOS inhibition on the Flt‐1 mRNA response to exercise. L ‐NAME, a known NOS inhibitor, attenuated the exercise‐induced increase in Flt‐1 mRNA by ～50%. These findings suggest that: (1) exercise alters Flt‐1 and Flk‐1 gene expression; and (2) NO is important in the regulation of the Flt‐1 gene response to exercise.     

The expression profile and prognostic value of APE/Ref‐1 and NPM1 in high‐grade serous ovarian adenocarcinoma

To analyze the expression trends and clinical significance of Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1/Ref‐1) and Nucleophosmin (NPM1) proteins in high‐grade serous ovarian adenocarcinoma (HGSC). The expressions of APE1/Ref‐1 and NPM1 proteins in 94 patients with HGSC were determined using the immunohistochemical (IHC) method, and their relationships with clinicopathological features were analyzed by the χ² test or Fisher's exact test. The follow‐up data, Cox proportional hazards univariate and multivariate survival analyses were integrated to evaluate the prognostic factors affecting patients with HGSC. In the normal fallopian tubes, APE1/Ref‐1 and NPM1 protein were mainly distributed in the nuclear. The HGSC experienced changes in the cellular localization of APE1/Ref‐1 and NPM1 protein expressions, which were abnormally expressed in the cytoplasm. The rates of abnormal cytoplasmic expression of APE1/Ref‐1 and NPM1 proteins in 94 patients with HGSC were 69.1% and 73.4%, respectively, which were significantly higher than the normal fallopian tube tissues (p < 0.05). The abnormal cytoplasmic APE1/Ref‐1 and NPM1 are significantly correlated with the lymph node metastasis, chemosensitivity, FIGO staging, and prognosis. The COX multivariate survival analysis showed that the abnormal expression of APE1/Ref‐1 protein, FIGO staging, and lymph node metastasis are independent prognostic factors. Collectively, the abnormal cytoplasmic APE1/Ref‐1 and NPM1 proteins are associated with the oncogenic progression and chemoresistance of HGSC, and predict a poor prognosis.     

Anti‐interleukin‐6 receptor antibody prevents systemic bone mass loss via reducing the number of osteoclast precursors in bone marrow in a collagen‐induced arthritis model

Systemic bone loss is a hallmark of rheumatoid arthritis (RA). Inflammatory cytokines such as interleukin (IL)‐6 promote bone resorption by osteoclasts. Sphingosine‐1‐phosphate (S1P) controls the migration of osteoclast precursor cells (OCPs) between the blood and bone marrow, in part via S1P receptors (S1PR1 and S1PR2) expressed on the surface of OCPs. OCPs (CD11b⁺Gr‐1^low+med) isolated from bone marrow of DBA/1J mice were stimulated with IL‐6. S1P‐directed chemotaxis of OCPs was evaluated using a transwell plate. mRNA expression of S1PR1 and S1PR2 was measured. DBA/1J mice were immunized with bovine type II collagen (days 0 and 21) and anti‐mouse IL‐6 receptor antibody (MR16‐1) was administered on days 0 and/or 21. Trabecular bone volume was analysed using micro‐computed tomography. The percentage of OCPs in tibial bone marrow and S1PR1 and S1PR2 mRNA expression in OCPs were measured. IL‐6 stimulation significantly decreased S1P‐directed chemotaxis of OCPs. IL‐6 induced S1PR2 mRNA expression, but not S1PR1 mRNA expression, in OCPs. Bone volume was significantly lower in arthritic mice than in non‐arthritic control mice on day 35. Treatment of immunized mice with MR16‐1 significantly inhibited bone loss. In MR16‐1‐treated mice, the percentage of OCPs and expression of S1PR2 mRNA was each decreased compared with arthritic mice on day 14, but not on day 35. IL‐6 increased the number of OCPs in tibial bone marrow via up‐regulating S1PR2, thus playing a crucial role in systemic bone loss induced by inflammation.     

Calorie restriction increases insulin‐stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate‐1 in rat skeletal muscle

A moderate reduction in calorie intake (calorie restriction, CR) improves insulin‐stimulated glucose transport in skeletal muscle. Therefore, we studied muscle insulin signalling in ad libitum (AL) and CR (~60% AL intake for 20 days) fed rats, which received a control injection (sterile water) or an insulin injection (30 U kg^–1 body weight). In control (not insulin‐treated) rats, there was no detectable tyrosine phosphorylation of insulin receptor (IR), regardless of diet; no diet effect on tyrosine phosphorylation of insulin receptor substrate‐1 (IRS1) or IRS1‐associated phosphatidylinositol 3‐kinase (PI3K) protein and 21% higher IRS1‐associated PI3K activity in AL vs. CR. In insulin‐treated rats, tyrosine‐phosphorylated IR was 79% higher for CR vs. AL; tyrosine‐phosphorylated IRS1 was 109% higher for CR vs. AL; IRS1‐associated PI3K protein and IRS1‐associated PI3K activity were unaffected by diet. Calorie restriction amplifies early insulin signalling steps without changing IRS1‐associated PI3K, suggesting enhanced glucose transport is mediated by altering: IRS1‐PI3K localization, PI3K associated with proteins other than IRS1 or post‐PI3K events.     

Tau‐Tubulin Kinase 1 Expression,Phosphorylation and Co‐Localization with Phospho‐Ser422 Tau in the Alzheimer's Disease Brain

Recent reports have implicated tau‐tubulin kinase 1 (TTBK1) in the pathological phosphorylation of tau that occurs in Alzheimer's disease (AD). The present study was undertaken to provide an extensive characterization of TTBK1 mRNA and protein expression in human brain from AD cases and non‐demented controls so as to better understand the disease relevance of this novel kinase. In situ hybridization and immunohistochemistry revealed abundant expression of TTBK1 in the somatodendritic compartment of cortical and hippocampal neurons of both AD cases and controls. TTBK1 immunoreactivity appeared to vary with the level of phospho‐tau staining, and was strong in the somatodendritic compartment of apparently healthy hippocampal neurons as well as in pre‐tangle neurons where it co‐localized with diffuse phospho‐Ser422 tau staining. Ser422 was confirmed as a TTBK1 substrate in vitro, and an antibody towards the site, in addition to labeling AT8‐positive neurofibrillary tangles (NFTs), neuritic plaques and neuropil threads, also labeled a small population of neurons that were unlabeled with AT8. These data suggest a role for TTBK1 in pre‐tangle formation prior to the formation of fibrillar tau and strengthen the idea that tau is phosphorylated at Ser422 at an early/intermediate stage in NFT formation.