Molecular phenotyping reveals the identity of Barrett's esophagus and its malignant transition.

The origin of human metaplastic states and their propensity for cancer is poorly understood. Barrett's esophagus is a common metaplastic condition that increases the risk for esophageal adenocarcinoma, and its cellular origin is enigmatic. To address this, we harvested tissues spanning the gastroesophageal junction from healthy and diseased donors, including isolation of esophageal submucosal glands. A combination of single-cell transcriptomic profiling, in silico lineage tracing from methylation, open chromatin and somatic mutation analyses, and functional studies in organoid models showed that Barrett's esophagus originates from gastric cardia through c-MYC and HNF4A-driven transcriptional programs. Furthermore, our data indicate that esophageal adenocarcinoma likely arises from undifferentiated Barrett's esophagus cell types even in the absence of a pathologically identifiable metaplastic precursor, illuminating early detection strategies.

Significance of Fas and FasL protein expression in cardiac carcinoma and local lymph node tissues.

OBJECTIVE: To investigate the relation of Fas and Fas ligand (FasL) protein expression with carcinogenesis and metastasis of cardiac carcinoma. METHODS: Immunohistochemistry was used to detect Fas and FasL protein expression in 64 cardiac carcinoma tissue samples and 20 normal gastric tissue samples. Relation between FasL and Fas expression, age and gender of gastric cancer patients, and pathological subtype and lymph node metastasis of gastric cancer was analyzed. RESULTS: The Fas expression level was significantly higher in normal gastric tissue samples than in cardiac carcinoma tissue samples (85.0% vs. 25.0%, P<0.001), while the FasL expression level was significantly lower in normal gastric tissue samples than in cardiac carcinoma tissue samples (30.0% vs. 81.3%, P<0.001). The Fas expression level was significantly higher in invasive lymph nodes than in non-invasive lymph nodes (82.9% vs. 56.5%, P<0.003) and in well-differentiated gastric carcinoma tissue samples than in poorly-differentiated cardiac carcinoma tissue samples (50.0% vs. 18.0%, P=0.015). The FasL expression level was significantly lower in well-differentiated cardiac carcinoma tissue samples than in poorly- differentiated cardiac carcinoma tissue samples (42.9% vs. 84.0%, P=0.021). The Fas and FasL expression levels (25.0% and 81.3%) were significantly different in cardiac carcinoma tissue samples (P<0.001), but had a non-linear correlation (P=0.575). CONCLUSION: Abnormal Fas and FasL expressions in cardiac carcinoma and lymph node tissues are involved in carcinogenesis and metastasis of gastric cancer.

Identification of a novel gene fusion (BMX-ARHGAP) in gastric cardia adenocarcinoma.

BACKGROUND: Gastric cardia adenocarcinoma (GCA) is one of the major causes of cancer related mortality worldwide. We aim to provide new understanding in the pathogenesis of GCA through investigations on gene expression alterations. METHODS: We preformed RNA-Seq for one pair of GCA and matched non-tumor tissues. Differentially expressed genes (DEGs) and fusion genes were acquired. PCR and gel analysis in additional 14 pairs of samples were performed to validate the chimeric transcripts. RESULTS: 1590 up-regulated and 709 down-regulated genes were detected. Functional analysis revealed that these DEGs were significantly overrepresented in gene ontology items of cell cycle, tumor invasion and proliferation. Moreover, we firstly discovered 3 fusion genes in GCA, including BMX-ARHGAP, LRP5- LITAF and CBX3-C15orf57. The chimeric transcript BMX-ARHGAP was validated and recurrently occurred in 4/15 independent tumor tissues. CONCLUSIONS: Our results may provide new understanding of GCA and biomarkers for further therapeutic studies. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_218.

MCM2 expression levels predict diagnosis and prognosis in gastric cardiac cancer.

BACKGROUND: Gastric Cardiac Cancer (GCC) has high incidence and poor prognosis requiring early screening of high-risk populations. Minichromosome maintenance (MCM) proteins are used as diagnostic-biomarkers in many cancers but not validated for GCC. We evaluate MCM protein 2 (MCM2), comparing it with the validated markers Ki67 and PCNA. METHODS: GCC and corresponding cardiac precancerous samples were immunostained with Ki67, MCM2 and PCNA antibodies. RESULTS: 90% of dysplasia samples expressed MCM2, whereas Ki67 and PCNA were expressed in 67% and 80% respectively. The sensitivity and negative predictive values of MCM2 were also superior at 90% and 87%, respectively. Ki67 and PCNA expression was correlated with MCM2, but their expressions seldom reached surface layers, whereas MCM2 manifested mostly in easily accessible superficial layers. Labeling indices (LI) of Ki67 and PCNA were also lower. Significant associations between LI (MCM2), LI (PCNA), and TNM-stages, lymph node metastases and GCC grade were found (P<0.05). Increased protein expressions were associated with reduced overall and disease-free survival (P<0.05). Although Ki67 and PCNA were significant prognostic factors, there was no significant improvement in multivariate statistical analyses, in contrast to LI (MCM2) findings. CONCLUSIONS: MCM2 is a sensitive, specific and efficient biomarker of GCC having potential use in clinic.

Human papillomavirus and the risk of Barrett's esophagus.

Human papillomavirus (HPV) is strongly associated with squamous esophageal cancer. The potential role of HPV in Barrett's esophagus (BE) has been examined but remains unclear. The aim of the study was to determine the prevalence of HPV in esophageal and gastric tissues obtained from patients with and without BE. We designed a cross-sectional study was conducted with prospective enrollment of eligible patients scheduled for esophagogastroduodenoscopy (EGD). All participants had biopsies of endoscopic BE, squamous-lined esophagus, and stomach. Immunohistochemistry (IHC) on formalin-fixed and paraffin-embedded tissue was conducted using monoclonal antibodies. Polymerase chain reaction (PCR) for HPV was performed on DNA extracted from esophageal biopsies snapped frozen within 30 minutes after endoscopic capture. The Roche HPV Linear Array Assay with PGMY primers that has high sensitivity for detecting 37 types of HPV was used. A total of 127 subjects were included: 39 with definitive BE had IHC done on samples from non-dysplastic BE, squamous esophagus, gastric cardia, and gastric body; and 88 control patients without BE had IHC done on squamous esophageal samples, gastric cardia, and gastric body. HPV was not detected in any of the samples in either group. For confirmation, HPV DNA PCR was performed on randomly selected samples from 66 patients (both esophagus and BE from 13 patients with BE, and 53 esophagus from patients without BE); no sample had HPV DNA detected via PCR in the presence of adequate quality control. HPV infection does not play a role in the formation of non-dysplastic Barrett's esophagus in men in the United States.

Gastric adenocarcinoma with chief cell differentiation: a proposal for reclassification as oxyntic gland polyp/adenoma.

Gastric adenocarcinoma with chief cell differentiation (GA-CCD) has been reported as a new, rare variant of gastric adenocarcinoma. Only 12 cases in Japanese patients have been described to date, but they demonstrate distinct clinicopathologic features. To further characterize these lesions, we have collected 10 additional cases. Patients ranged in age from 44 to 79 years (mean, 64.2 y) with a relatively equal sex distribution (6 women and 4 men). Stratified by race, 4 patients were Hispanic, 2 were White, 2 were African American, 1 was Asian (Chinese), and the race was unknown for 1 patient. All patients presented with gastroesophageal reflux that prompted an endoscopic examination. The majority of GA-CCDs were identified in the fundus (7 of 10, 70%) and the remaining in the cardia (n=3). Grossly, they were solitary and polypoid, ranging in size from 0.2 to 0.8 cm (mean, 0.4 cm). Histologically, all cases were centered in the deep mucosa, with focal involvement of surface foveolar epithelium in 3 (30%) cases but not the submucosa. The tumors consisted of clustered glands and irregular branching cords of oxyntic epithelium. Thin wisps of radiating smooth muscle separated the epithelium, but desmoplasia was distinctly absent in all cases. The oxyntic mucosa was 1 to 2 cells thick and composed of a mixture of mucous neck, parietal, and chief cells. In 7 of 10 (70%) cases, chief cells were the predominant cell type, whereas the remaining 3 cases consisted primarily of mucous neck cells. The nuclei were mildly enlarged with slight nuclear pleomorphism, but no mitotic figures were identified. In addition, necrosis, lymphovascular invasion, and perineural invasion were absent. Immunohistochemically, GA-CCDs were diffusely positive for MUC6 (10 of 10, 100%) and negative for MUC5AC (0%) and MUC2 (0%). Ki-67 immunolabeling demonstrated variable expression, with the highest areas ranging from 0.2% to 10%. Clinical follow-up was available for 9 of 10 (90%) patients and ranged from 6 to 39 months. One patient had persistence of lesion at 6 months because of incomplete removal, whereas the other 8 were disease free. In summary, GA-CCDs are solitary, mucosal lesions of the gastric cardia/fundus that arise in patients from multiple ethnic backgrounds. Considering that patients within this study and those reported previously have had neither true recurrence nor progression of disease, these lesions are best regarded as benign. Consequently, the term GA-CCD is contradictory and we prefer the descriptive term "oxyntic gland polyp/adenoma" until further studies can clarify the pathogenesis of these lesions and their natural history.

Aberrant CpG island hypermethylation of RASSF1A in gastric cardia adenocarcinoma.

Ras-association domain family 1A (RASSF1A) gene, a candidate tumor suppressor gene, is inactivated in several human tumors, usually by hypermethylation of its promoter region. RASSF1A induces cell cycle arrest through inhibition of cyclin D1 accumulation. In this work, the promoter methylation status of the RASSF1A in 92 gastric cardia adenocarcinoma (GCA) and corresponding normal tissues were investigated using Methylation-specific PCR (MSP) approach, immunohistochemistry method and RT-PCR were used respectively to examine the protein expression and mRNA expression of RASSF1A in tumors and corresponding normal tissues. Cyclin D1 expression was examined by immunohistochemistry. RASSF1A was methylated in 54/92 (58.7%) tumor specimens, which was significantly higher than that in corresponding normal tissues (p <.001). Methylation frequencies of stage III and IV tumor tissues were significantly higher than that in stage I and II tumor tissues (p <.05). By immunostaining, 43/92 (46.7%) tumor tissues demonstrated heterogeneous, positive immunostaining of tumor tissues was significantly reduced with comparison to matched normal tissues (p <.001). mRNA expressions of RASSF1A in GCA tumor tissues were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2376 +/- 0.2315 vs 0.6874 +/- 0.2668, p <.001). RASSF1A mRNA expression in methylation group of GCA was significantly different from that in unmethylation group (p <.001). Cyclin D1 hyper-expression was found in 72/92 (78.3%) cases and correlated with RASSF1A methylation (p <.05). Our data suggested that epigenetic silencing of RASSF1A gene expression by promoter hypermethylation may play an important role in GCA.

Expression of multidrug resistance-related proteins p-glycoprotein, glutathione-s-transferases, topoisomerase-II and lung resistance protein in primary gastric cardiac adenocarcinoma.

BACKGROUND/AIMS: Multidrug resistance (MDR) is closely correlated to an unfavorable prognosis in various human cancers. However, the clinical significance of the expression of MDR-related proteins p-glycoprotein (PGP), glutathione-s-transferases (GST-pi), topoisomerase-II (Topo-II) and lung resistance protein (LRP) in primary gastric cardiac adenocarcinoma (PGCA) remains unclear. In this study, the total of the four kinds of MDR-related proteins mentioned above were detected by using immunohistochemistry and their clinical significance in chemoresistance were also investigated. METHODOLOGY: This retrospective study included 69 resected specimens from patients with PGCA. The expression of PGP, GST-pi, Topo-II and LRP in formalin-fixed paraffin-embedded tissue sections was determined by a labelled streptavidin-biotin immunohistochemical technique, and the results were analyzed in correlation with clinicopathological data. None of these patients received chemotherapy prior to surgery. RESULTS: The positive rates of expression of PGP, GST-pi, Topo-II and LRP in malignant tissues (49.2%, 75.4%, 68.1% and 58%, respectively) were all higher than that of the normal tissues (0, 30%, 20% and 0, respectively, P<0.01). PGP expression in tumors that had metastasized was significantly more frequent than in tumors that had not metastasized (67.5% vs. 24.1%, P<0.01).The expression of PGP was closely related with clinicopathologic staging (staging 1/2 vs. 3/4, 28.6% vs. 58.3%, P<0.05). No significant correlation was shown between PGP and increasing differentiated degree (40%, 42.4% and 61.5%, P>0.05). GST-pi expression status progressively increased with increasing differentiated degree (40%, 75.8% and 88.5%, P<0.05) and clinico pathologic stage (staging 1/2 vs. 3/4, 57.1% vs. 83.3%, P<0.05). In addition, a significant positive correlation was also observed between GST-pi and lymphatic metastasis (with vs. without metastasis, 87.5% vs. 58.6%, P<0.05). The expression of Topo-II was associated with increasing differentiated degree (33.3%, 69.7% and 80.7%, P<0.01). No significant differences with Topo-II expression was found in relation to the clinicopathologic stage (staging 1/2 vs. 3/4, 57.1% vs. 72.9%, P>0.05) and lymphatic metastasis (with vs. without metastasis, 65.0% vs. 72.4%, P>0.05). Moreover, a significant difference with the expression of LRP was found in relation to the clinicopathologic stage (staging 1/2 vs. 3/4, 38% vs. 66.6%, P<0.05), and lymphatic metastasis (with vs. without metastasis, 70.0% vs. 41.4%, P<0.05). Comparing the well, moderately and poorly differentiated cohort, a non-statistical increasing trend towards LRP expression status was noted (50.0%, 54.5% and 65.3%, respectively, P>0.05). Besides, the co-expression of all four tested MDR-related proteins also existed. The positive rates of co-expression of PGP and GST-pi, PGP and Topo-II, PGP and LRP, GST-pi and Topo-II, LRP and GST-pi, LRP and Topo-II, PGP, GST-pi, Topo-II and LRP in malignant cells were 23.2%, 15.9%, 11.6%, 13.0%, 26.1%, 7.24%, 5.8%, respectively. CONCLUSIONS: MDR-related proteins PGP, GST-pi, Topo-II and LRP are involved in multiple mechanisms of drug resistance in PGCA. Combined determination of PGP, GST-pi, Topo-II and LRP may be prospectively valuable for optimizing the chemotherapy regimes, developing high quality anti-cancer drugs, and further predicting the outcomes of those patients with PGCA.

Expression of multidrug-resistance-related proteins P-glycoprotein, glutathione-S-transferases, topoisomerase-II and lung resistance protein in primary gastric cardiac adenocarcinoma.

AIM: Multidrug resistance (MDR) is closely correlated to an unfavorable prognosis in various human cancers. However, the clinical significance of the expression of MDR-related proteins p-glycoprotein (PGP), glutathione-s-transferases (GST-pi), topoisomerase-II (Topo-II) and lung resistance protein (LRP) in primary gastric cardiac adenocarcinoma (PGCA) remains unclear. In this present study, the total of the four kinds of MDR-related proteins mentioned above were detected by using immunohistochemistry, and their clinical significance in chemoresistance were also investigated. METHODS: This retrospective study included 69 resected specimens from patients with PGCA. The expression of PGP, GST-pi, Topo-II and LRP in formalin-fixed paraffin-embedded tissue sections was determined by a labelled streptavidin-biotin immunohistochemical technique, and the results were analyzed in correlation with clinicopathological data. None of these patients received chemotherapy prior to surgery. RESULTS: The positive rates of expression of PGP, GST-pi, Topo-II and LRP in malignant tissues (49.2%, 75.4%, 68.1% and 58%, respectively) were all higher than that of the normal tissues(0, 30%, 20% and 0, respectively, P < 0.01). PGP expression in tumors that had metastasized was significantly more frequent than in tumors that had not metastasized (67.5% vs 24.1%, P < 0.01). The expression of PGP was closely related with clinicopathologic staging (staging 1/2 vs 3/4, 28.6% vs 58.3%, P < 0.05). No significant correlation was shown between PGP and increasing differentiated degree (40%, 42.4% and 61.5%, P > 0.05). GST-pi expression status progressively increased with increasing differentiated degree (40%, 75.8% and 88.5%, P < 0.05) and clinicopathologic stage (staging 1/2 vs 3/4, 57.1% vs 83.3%, P < 0.05). In addition, a significant positive correlation was also observed between GST-pi and lymphatic metastasis (with vs. without metastasis, 87.5% vs 58.6%, P < 0.05). The expression of Topo-II was associated with increasing differentiated degree (33.3%, 69.7 and 80.7%, P < 0.01). No significant differences with Topo-II expression were found in relation to the clinicopathologic stage (staging 1/2 vs 3/4, 57.1% vs 72.9%, P > 0.05) and lymphatic metastasis (with vs. without metastasis, 65.0% vs 72.4%, P > 0.05). Moreover, a significant difference with the expression of LRP was found in relation to the clinicopathologic stage (staging 1/2 vs 3/4, 38% vs 66.6%, P < 0.05), and lymphatic metastasis (with vs without metastasis, 70.0% vs 41.4%, P < 0.05). Comparing the well, moderately and poorly differentiated cohort, a non-statistical increasing trend towards LRP expression status was noted (50.0%, 54.5% and 65.3%, respectively, P > 0.05). Besides, the co-expression of all four tested MDR-related proteins also existed. The positive rates of co-expression of PGP and GST-pi, PGP and Topo-II, PGP and LRP, GST-pi and Topo-II, LRP and GST-pi, LRP and Topo-II, PGP, GST-pi, Topo-II and LRP in malignant cells were 23.2%, 15.9%, 11.6%, 13.0, 26.1, 7.24, 5.8, respectively. CONCLUSIONS: MDR-related proteins PGP, GST-pi, Topo-II alpha and LRP are involved in multiple mechanisms of drug resistance in PGCA. Combined determination of PGP, GST-pi, Topo-II and LRP may be prospectively valuable for optimizing the chemotherapy regimes, developing high quality anti-cancer drugs, and further predicting the outcomes of those patients with PGCA.

The use of cytokeratin stain to distinguish Barrett's esophagus from contiguous tissues: a systematic review.

Our objective was to systematically review the existing literature regarding the use of cytokeratin (CK) stain in differentiating Barrett's esophagus (BE) from tissues of the gastric cardia, corpus, or antrum, with or without intestinal metaplasia (IM). Pubmed was searched for full publications in English (1983-2005) addressing the use of CK for differentiation of BE from contiguous tissues. Information was collected on the study sample, blinding, the methods used for CK staining, and for defining and applying the gold standard tests. Test characteristics were obtained or calculated. Sixteen studies (containing 46 comparisons) met the inclusion and exclusion criteria. Immunostaining for CK 7 and 20 was generally highly specific in distinguishing long-segment BE from antrum IM, fundus IM, or noncardiac gastric IM; 27 comparisons showed statistically significant differences. However, only 8 of 15 comparisons (6 of 12 studies) reported significant differences in CK staining patterns between BE and gastric cardia IM with a high sensitivity (89%-100%) and specificity (83%-100%) for long-segment BE and lower estimates for short-segment BE, while the other seven comparisons showed no significant differences and a very low sensitivity. Examination by a blinded pathologist was reported in five of six positive studies and in only one of six of the negative studies. In addition, variation in the patient populations, use of surgical resection versus endoscopic biopsies, and biopsy sampling technique in endoscopic studies may have accounted for these differences. Finally, two studies did not find significant differences in CK staining patterns between BE and normal cardiac mucosa. In conclusions, CK immunostaining has not performed well in differentiating BE, especially short-segment BE, from cardia IM. There seems to be a spectrum bias where the accuracy varies with different tested populations. CK immunostaining distinguished well between BE and IM in noncardiac segments of the stomach; however, these comparisons are not clinically relevant.

The effect of rabeprazole on regional gastric acidity and the postprandial cardia/gastro-oesophageal junction acid layer in normal subjects: a randomized, double-blind, placebo-controlled study.

BACKGROUND: Postprandial intragastric acidity is not uniform. Postprandial proximal gastric acid pockets have been described in the present study. AIM: To determine the effects of rabeprazole on regional intragastric acidity and proximal acid pockets. METHODS: Ten normal subjects underwent two 8-day oral dosing regimens with placebo or rabeprazole 20 mg each morning in a randomized, double-blind protocol. Oesophago-gastric pH monitoring was performed on days 1 and 8. RESULTS: Rabeprazole increased fasting and postprandial gastric pH to above 4 in each area of the stomach on days 1 and 8. With placebo, acid pockets were identified at the cardia/gastro-oesophageal junction in 62 and 50 of 150 pull-throughs on days 1 and 8, respectively. Acid pockets were detected postprandially 3.1 +/- 0.2-5.8 +/- 0.1 cm below the proximal border of the lower oesophageal sphincter with a mean pH drop from 4.6 +/- 0.1 to 1.5 +/- 0.1. Rabeprazole decreased the number of acid pockets to 30 and 27 on days 1 and 8, respectively. Rabeprazole also decreased their length and magnitude of the pH drop. CONCLUSIONS: Rabeprazole increased intragastric pH on day 1 and 8 and maintained an elevated pH during and after meals. Postprandial acid pockets, identified in the region of the cardia/gastro-oesophageal junction area postprandially, were decreased in number, length and magnitude by rabeprazole.

Is cytokeratin immunoreactivity useful in the diagnosis of short-segment Barrett's oesophagus in Korea?

BACKGROUND: Cytokeratin 7/20 staining has been reported to be helpful in diagnosing Barrett's oesophagus and gastric intestinal metaplasia. However, this is still a matter of some controversy. OBJECTIVE: To determine the diagnostic usefulness of cytokeratin 7/20 immunostaining for short-segment Barrett's oesophagus in Korea. METHODS: In patients with Barrett's oesophagus, diagnosed endoscopically, at least two biopsy specimens were taken from just below the squamocolumnar junction. If goblet cells were found histologically with alcian blue staining, cytokeratin 7/20 immunohistochemical stains were performed. Intestinal metaplasia at the cardia was diagnosed whenever biopsy specimens taken from within 2 cm below the oesophagogastric junction revealed intestinal metaplasia. Barrett's cytokeratin 7/20 pattern was defined as cytokeratin 20 positivity in only the superficial gland, combined with cytokeratin 7 positivity in both the superficial and deep glands. RESULTS: Barrett's cytokeratin 7/20 pattern was observed in 28 out of 36 cases (77.8%) with short-segment Barrett's oesophagus, 11 out of 28 cases (39.3%) with intestinal metaplasia at the cardia, and nine out of 61 cases (14.8%) with gastric intestinal metaplasia. The sensitivity and specificity of Barrett's cytokeratin 7/20 pattern were 77.8 and 77.5%, respectively. CONCLUSION: Barrett's cytokeratin 7/20 pattern can be a useful marker for the diagnosis of short-segment Barrett's oesophagus, although the false positive or false negative rate is approximately 25%.

Identical cytokeratin expression pattern CK7+/CK20- in esophageal and cardiac cancer: etiopathological and clinical implications.

Surgical treatment and prognosis is different in esophageal, cardiac and distal gastric adenocarcinomas. Determination of the origin, in particular of adenocarcinomas situated at the gastroesophageal junction, may be difficult. It has been suggested that esophageal adenocarcinomas are characterized by a specific cytokeratin pattern, namely the CK7+/CK20- pattern. According to the same authors, this cytokeratin pattern is absent in gastric adenocarcinomas. The aim of our study is to evaluate if this cytokeratin pattern CK7+/CK20- is absent in cardiac and distal gastric adenocarcinomas. Therefore, we evaluated the combined immunohistochemical expression of CK7 and CK20 on paraffin-embedded material of 214 resection specimens for adenocarcinoma, comprising 66 esophageal, 73 cardiac and 75 distal gastric adenocarcinomas (UICC-classification). The adenocarcinomas were subtyped into intestinal- and diffuse-type according to the Lauren classification. The immunohistochemical staining was considered as positive if 50% or more of the tumor cells were stained. Statistical analysis has been performed applying the chi2-test. The tumors situated at the gastroesophageal junction, esophageal as well as cardiac adenocarcinomas, showed predominantly a CK7+/CK20- expression pattern (67 vs 68%), whereas this cytokeratin pattern is rather uncommon in distal gastric adenocarcinomas (31%, P<4 x 10(-5)). Independent of their localization, intestinal- as well as diffuse-type adenocarcinomas have a similar cytokeratin pattern. Our data show that the combined expression of CK7 and CK20 is different for the adenocarcinomas situated on both sides of the gastroesophageal junction compared to the distal gastric adenocarcinomas. However, in contrast to data in the literature, the combined expression of CK7 and CK20 has a low specificity in the distinction between esophageal and cardiac adenocarcinomas. This may suggest a similar origin (cell lineage) and thus may have an impact on therapeutic strategies.

Conditions for acid catalysed luminal nitrosation are maximal at the gastric cardia.

BACKGROUND: Saliva has a high nitrite concentration, derived from the enterosalivary recirculation of dietary nitrate, and is the main source of nitrite entering the acidic stomach. Acidification of nitrite in the presence of secondary amines or amides generates potentially carcinogenic N-nitroso compounds. The reaction is inhibited by ascorbic acid and catalysed by thiocyanate. AIM: To determine whether there is intragastric regional variation in the chemical conditions promoting luminal nitrosation following nitrate ingestion. METHODS: Using microdialysis probes, we measured concentrations of nitrite, ascorbic acid, total vitamin C, and thiocyanate simultaneously in saliva, the distal oesophagus, cardia, and the proximal and distal stomach of 17 healthy volunteers before and following intragastric nitrate (2 mmol) administration. RESULTS: The median pH in the distal oesophagus, cardia, and proximal and distal stomach were 7, 2.6, 1.9, and 1.7, respectively, before, and were similar following nitrate administration. Mean nitrite concentration in the distal oesophagus was similar to that of saliva, being 29.1 micro M and 36.7 micro M, respectively, before nitrate and increasing to 181.6 micro M and 203.3 micro M after nitrate ingestion. Within the stomach, mean (SEM) nitrite concentration following nitrate was higher in the cardia (45.5 (12.7) micro M) than in the mid (7.8 (3.1)) (p<0.01) or distal (0.8 (0.6)) (p<0.1) stomach, and ascorbic acid concentration was lower at the cardia (13.0 (6.1)) than in the mid (51 (19.2)) (p<0.02) or distal (86 (29)) (p<0.01) stomach. Consequently, the median ascorbic acid to nitrite ratio was lowest at the cardia (0.3) (p<0.01) versus the mid (7.8) or distal (40) stomach. Thiocyanate concentration was similar throughout the stomach. CONCLUSIONS: The conditions favouring luminal generation of N-nitroso compounds from dietary nitrate are maximal at the most proximal cardia region of the acidic stomach and may contribute to the high incidence of mutagenesis at this site.

Immunohistochemical study of neuroendocrine cells at the gastric cardia mucosa.

BACKGROUND/AIMS: The gastric cardia mucosa is a narrow band of tissue between the oesophagus and the stomach. The physiological role of this tissue is unknown. This study examined the presence and characteristics of neuroendocrine cells at this site. METHODS: Biopsy samples were obtained from across normal appearing squamocolumnar junctions. The cardiac mucosa was defined as the presence of special type mucosa composed of mucous secreting glands in the immediate vicinity of oesophageal squamous epithelium. Biopsy specimens were stained with haematoxylin and eosin, alcian blue (pH 2.5) periodic acid Schiff, and modified Giemsa. The chromogranin A and Fontana-Masson stains were used to identify neuroendocrine cells, which were also stained immunohistochemically for gastrin, serotonin, glucagon, pancreatic polypeptide, somatostatin, and vasoactive intestinal peptide. RESULTS: Chromogranin positive cells were seen in 18 cases with adequate biopsy specimens from the gastric cardia mucosa. These cells were all serotonin positive, but stains for other peptide hormones remained negative. Serotonin positive cells were detected only at the base of foveolae at the periphery of mucous secreting cardiac glands, giving a microscopic appearance resembling that of endocrine cells at the gastric antrum. The presence and numbers of serotonin positive cells did not correlate with chronic inflammation or intestinal metaplasia of the cardiac mucosa. These cells were seen both in Helicobacter pylori positive and negative patients. CONCLUSIONS: Serotonin positive cells appear to be the sole neuroendocrine cell type at the gastric cardia mucosa. These cells may have a role in regulating the physiology of the gastric cardia mucosa and the lower oesophageal sphincter.

Utilization of cytokeratins 7 and 20 does not differentiate between Barrett's esophagus and gastric cardiac intestinal metaplasia.

Long segment Barrett's esophagus (LSBE) is a recognized risk factor for the development of esophageal dysplasia and carcinoma. However, the risk of dysplasia arising within intestinal metaplasia below a normal-appearing Z-line (i.e., in native cardiac mucosa) is unknown. Regular endoscopic surveillance is required in patients with LSBE and is frequently performed in short segment BE (SSBE), but the need for surveillance in cardiac intestinal metaplasia (CIM) is unknown. Unfortunately IM arising in SSBE and immediately below a normal Z-line can be indistinguishable histologically on H&E stains. Previous reports suggest that the appearance of superficial CK20 immunohistochemical staining accompanied by intermediate and deep CK7 positivity is characteristic of BE, whereas CIM specimens show superficial and deep CK20 positivity and weak to absent CK7 staining. We hypothesized that CK7/20 immunostaining of metaplastic biopsies from the esophagus and stomach would allow complete differentiation of these two entities when correlated with the endoscopic appearance. We undertook an evaluation of gastric and esophageal specimens to determine whether these characteristics were valid. Cases of both BE (long and short segment) and CIM, as well as cases of gastric cardiac biopsies lacking IM, were evaluated for CK7 and CK20 and correlated with the endoscopic appearance. We observed that, although the "Barrett's" pattern of CK7/20 was maintained for many cases of BE, the sensitivity and specificity were only moderate (65% and 56%, respectively). The pattern of staining for the CIM was variable, i.e., some cases showed a CK7/20 Barrett's pattern despite a normal appearance at endoscopy. The differences between this and previous studies may be due to inaccurate visualization of SSBE on endoscopy, the development of very early SSBE cases, inter-observer variability, fixation differences, or antibody differences. Whatever the cause of the differences, if results between laboratories are not comparable, CK7/20 immunostaining cannot be used to differentiate reliably between IM present in biopsy specimens taken from above versus below the Z-line. However, further studies should be performed to determine whether the presence or absence of a Barrett's pattern of CK7/20 immunostaining could predict progression to dysplasia or carcinoma.

Molecular and clinical differences between adenocarcinomas of the esophagus and of the gastric cardia.

Adenocarcinoma of the esophagus (ADCE) with Barrett's mucosa and adenocarcinoma of the cardia (ADCC) are often reported as a single pathological entity. In this study we have used strict anatomical-pathological criteria to distinguish between these two lesions and we have investigated their differences in TP53 mutations, MDM2 gene amplification, and cytokeratin expression. DNA was extracted from the tumor areas of formalin-fixed, paraffin-embedded sections in 26 ADCC and 28 ADCE patients. TP53 mutations were detected by temporal temperature gradient electrophoresis and identified by sequencing. MDM2 amplification was assessed by differential polymerase chain reaction. The expression of cytokeratins 4, 7, and 13 was examined by immunohistochemistry. In ADCC, the male to female ratio was 1.8:1, compared to 27:1 in ADCE. Five ADCC patients had a history of other neoplasms, compared to only one ADCE patient. The two types of tumor differed in the prevalence of TP53 mutations (31% in ADCC and 50% in ADCE) and of MDM2 gene amplification (19% in ADCC and 4% in ADCE), and in the pattern of expression of cytokeratin 7 (positive in 100% of ADCE and in 41% of ADCC) and cytokeratin 13 (positive in 81% of ADCE and in 36.5% of ADCC). ADCE and ADCC differ in their clinical characteristics, in the prevalence of TP53 mutations and MDM2 amplifications, and in the patterns of cytokeratin expression. These results support the notion that ADCC and ADCE are distinct pathological entities.

Human gastric intrinsic factor expression is not restricted to parietal cells.

Gastric parietal cells have been accepted as the only site of intrinsic factor production in the human stomach. In animals, however, intrinsic factor has been localised to various other cell types of foregut origin, including chief and enteroendocrine cells in gastric mucosa, and duct cells from salivary glands and pancreas. The availability of recombinant human intrinsic factor has led to production of high titre, monospecific antiserum which was used to reexamine the distribution and subcellular localisation of intrinsic factor in the human stomach. Immunolight microscopy revealed that most positively stained cells were gastric parietal cells, but at the margins of the anatomical regions (e.g. cardia/fundus, body/antrum) clusters of gastric chief cells and individual enteroendocrine cells were found to contain intrinsic factor. Immunoelectron microscopy demonstrated the highest antigen density on endocytic and apical membranes of parietal cells. Exocrine secretory granules of a subpopulation of chief cells, the secretory granules of some enteroendocrine cells, and the plasma membranes and smooth vesicles of endothelial cells of the lamina propria capillaries underlying enteroendocrine cells were also positive for the antigen. Labelling in all cells was specific, as it was abolished by preabsorption of the antisera with purified recombinant human intrinsic factor. These findings demonstrate a potential for cellular expression of human intrinsic factor in nonparietal cells. Because such expression occurs normally at the margins of anatomical gastric regions, it suggests that local factors may influence expression of intrinsic factor.

Accumulation of p53 protein in normal, dysplastic, and neoplastic Barrett's oesophagus.

Accumulation of p53 protein was determined by immunohistochemistry in archival material of biopsy specimens from 102 patients with Barrett's oesophagus with different grades of dysplasia, in 24 oesophageal adenocarcinomas associated with Barrett's oesophagus, and in 23 cases of metaplastic epithelium adjacent to these carcinomas. Immunostaining for the p53 protein was found in 23/102 (23 per cent) cases of the Barrett's oesophagus biopsies and in 12/23 (52 per cent) cases of Barrett's oesophagus adjacent to adenocarcinoma. Significant correlations were found between the grade of dysplasia and p53 immunoreactivity in both Barrett's biopsies without adenocarcinoma (P < 0.001) and Barrett's oesophagus adjacent to adenocarcinoma (P < 0.05). In the adenocarcinomas, intense nuclear immunohistochemical staining for p53 was diffusely or focally present in 20/24 (83 per cent) of the specimens. In Barrett's oesophagus, p53 is a progression marker with high expression in high-grade dysplasia (89 per cent) and adenocarcinoma (83 per cent).

Changes in p53 and cyclin D1 protein levels and cell proliferation in different stages of human esophageal and gastric-cardia carcinogenesis.

The objective of this study was to quantify the changes in p53 and cyclin D1 protein levels in different stages of human esophageal and gastric cardia carcinogenesis in a high-risk population in Henan, China. Immunoreactivity of p53, cyclin D1 and proliferating-cell nuclear antigen (PCNA) was observed in the cell nuclei of esophageal and gastric cardia biopsies. The number of p53-immunostaining-positive cells was low in normal epithelia, slightly increased in basal-cell hyperplasia (BCH), markedly increased in dysplasia (DYS) (10-fold), and further increased in squamous-cell carcinoma (SCC) (40-fold). This pattern of change was similar to that of cell proliferation as indicated by PCNA immunostaining. On the other hand, the number of cyclin D1-immunostaining-positive cells did not increase from BCH to DYS, although a slight increase from DYS to SCC was noted. In the gastric cardia, again, the pattern of change of p53-positive cells in different stages of lesions paralleled the pattern of cell proliferation. The number of p53-positive cells was very low, much lower than that of PCNA-positive cells, in normal, chronic superficial gastritis (CSG) and chronic atrophic gastritis (CAG); therefore, the increase of p53-positive cells from CAG to DYS was more dramatic (100-fold). From DYS to adenocarcinoma (AC), the p53-positive and the PCNA-positive cells increased 4-fold. On the other hand, the number of cyclin D1-positive cells did not increase in pre-cancerous lesions, but increased slightly in AC. This study demonstrates that p53 protein accumulation increased with the progression of pre-cancerous lesions, especially in the genesis of dysplasia, both in the esophagus and in the gastric cardia. Our approach of quantitative immunohistochemistry sheds light on the mechanisms of genesis of esophageal and gastric-cardia cancers, which frequently occur together in many high-incidence areas.