Kumari Wickramasinghe

Adenomatous polyposis coli gene promoter hypermethylation in non-small cell lung cancer is associated with survival

Methylation of 5′ CpG islands in promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of this study was to determine whether hypermethylation of the adenomatous polyposis coli (APC) gene promoter occurs in primary non-small cell lung cancer (NSCLC), and whether hypermethylated APC has any relationship with survival. APC promoter 1A methylation was determined in normal and corresponding tumor tissue from 91 NSCLC patients and in a control group of 10 patients without cancer, using a quantitative fluorogenic real-time PCR (Taqman®) system. APC promoter methylation was detectable in 86 (95%) of 91 tumor samples, but also in 80 (88%) of 91 normal samples of NSCLC patients, and in only two (20%) of 10 normal lung tissues of the control group. The median level of APC promoter methylation was 4.75 in tumor compared to 1.57 in normal lung tissue (P<0.001). Patients with low methylation status showed significantly longer survival than did patients with high methylation status (P=0.041). In a multivariate analysis of prognostic factors, APC methylation was a significant independent prognostic factor (P=0.044), as were pT (P=0.050) and pN (P<0.001) classifications. This investigation shows that APC gene promoter methylation occurs in the majority of primary NSCLCs. High APC promoter methylation is significantly associated with inferior survival, showing promise as a biomarker of biologically aggressive disease in NSCLC.

Cytokeratin and DAS-1 immunostaining reveal similarities among cardiac mucosa, CIM, and Barrett’s esophagus

OBJECTIVE:The normal histology at the gastroesophageal junction, and in particular the nature of cardiac mucosa, remains in dispute. Likewise, the relationship of intestinal metaplasia at the gastroesophageal junction (CIM) to Barretts and intestinal metaplasia of the stomach (GIM) is unclear. The aim of this study was to assess the immunostaining characteristics of cardiac mucosa and CIM and compare their staining pattern with that of other foregut mucosal types. We hypothesized that the immunostaining patterns of these foregut tissues would provide insight into the nature and etiology of cardiac mucosa and CIM.METHODS:Paraffin-embedded biopsy specimens from 50 patients with normal antral or fundic mucosa, cardiac mucosa, squamous mucosa, CIM, GIM, or Barretts were obtained and immunostained with a panel of monoclonal antibodies including those for cytokeratins 7 and 20 (CK7/CK20) and DAS-1.RESULTS:Biopsies from normal gastric antral and fundic mucosa and squamous esophageal mucosa all showed a non-Barretts type CK7/CK20 immunostaining pattern, whereas in 85% of patients, cardiac mucosa had a Barretts type CK7/CK20 pattern (p <0.001). A Barretts type CK7/CK20 staining pattern was seen in 100% of Barretts, 78% of CIM, and 0% of GIM patients. Likewise, DAS-1 staining was similar in patients with CIM and Barretts and significantly different in patients with GIM.CONCLUSIONS:Cytokeratin immunostaining of cardiac mucosa demonstrates significant differences from recognized normal gastric and esophageal mucosa but a similarity to Barretts. This suggests that cardiac mucosa, like Barretts, may be acquired. Likewise, immunostaining similarities between CIM and Barretts biopsies point to the possibility of a reflux etiology for CIM in some patients.

Adenocarcinomas of the distal esophagus and gastric cardia are predominantly esophageal carcinomas

BackgroundAdenocarcinoma of the distal esophagus and gastric cardia are defined by the relationship of its epicenter to the gastro-esophageal junction, which is presently defined as the end of the tubular esophagus. We have recently suggested that the true gastro-esophageal junction is best defined by the proximal limit of gastric oxyntic mucosa. AimTo reclassify adenocarcinomas of this region by the relationship of the tumor to the proximal limit of gastric oxyntic mucosa. MethodsSeventy-four patients who had esophago-gastrectomy for adenocarcinomas in this region were classified as adenocarcinoma of distal esophagus (38 patients) and gastric cardia (36 patients) by present criteria. The epithelial type at the epicenter and distal edge of these tumors was assessed. ResultsThe epicenter of the tumor in 64 patients with noncircumferential tumors had squamous (5 cases), cardiac (21 cases), oxynto-cardiac (4 cases), and intestinal (Barrett-type) (34 cases) epithelia. None had gastric oxyntic mucosa. Of the 10 patients with circumferential tumors, 7 had cardiac or oxynto-cardiac epithelium at the distal tumor edge. ConclusionsIf the gastro-esophageal junction is defined histologically as the proximal limit of oxyntic mucosa, 71/74 patients would be classified as adenocarcinoma of the distal esophagus. The other 3 patients were questionable as to gastric or esophageal origin. We suggest that this reclassification based on the proposed new definition of the gastro-esophageal junction provides an explanation for the epidemiologic relationship that exists between adenocarcinoma of the “gastric cardia” and gastro-esophageal reflux disease.

Glutathione S–Transferase-Pi Expression Is Downregulated in Patients With Barrett's Esophagus and Esophageal Adenocarcinoma

The glutathione S-transferases (GSTs) are a family of enzymes that play an important role in the prevention of cancer by detoxifying numerous potentially carcinogenic compounds. GSTs conjugate reduced glutathione to a variety of electrophilic and hydrophobic compounds, converting them into more soluble, more easily excretable compounds. Decreased glutathione S-transferase-pi (GSTPI) enzyme activity has been reported in Barrett’s esophagus, and an inverse correlation was demonstrated between GST enzyme activity and tumor incidence in the gastrointestinal tract, but the role of GSTPI messengerRNA (mRNA) expression in Barrett’s esophagus and associated adenocarcinomas is uncertain. The purpose of this study was to investigate the role of GSTPI mRNA and protein expression in the development and progression of the Barrett’s metaplasia-dysplasia-adenocarcinoma sequence, and to investigate the potential of GSTPI quantitation as a biomarker in the clinical management of this disease. GSTPI mRNA expression levels, in relation to the housekeeping gene β-actin, were analyzed using a quantitative real-time reverse transcription-polymerase chain reaction method (TaqMan) in 111 specimens from 19 patients with Barrett’s esophagus without carcinoma (BE group), 21 patients with Barrett’s-associated adenocarcinoma (EA group), and a control group of 10 patients without evidence of Barrett’s esophagus or chronic gastroesophageal reflux disease. GSTPI mRNA expression was detectable in all 111 samples investigated. Analyzed according to histopathologic group, the median GSTPI mRNA expression was highest in normal squamous esophagus epithelium, intermediate in Barrett’s esophagus, and lowest in adenocarcinoma tissues (P < 0.001). The median GSTPI expression was significantly decreased in Barrett’s esophagus tissues compared to matching normal squamous esophagus from either the BE group (P = 0.001) or the EA group (P = 0.023). GSTPI expression levels in adenocarcinoma tissues were decreased compared to matching normal esophagus tissues from the patients with adenocarcinoma (P = 0.011). Furthermore, GSTPI mRNA expression values were significantly different between metaplastic, dysplastic, and adenocarcinoma tissues (P = 0.026). GSTPI expression levels were also significantly lower in histologically normal squamous esophagus tissues from patients with cancer (EA group) compared to both normal esophagus tissues from patients without cancer (BE group; P = 0.007) and normal esophagus tissues from the control group with no esophageal abnormality (P = 0.002). GSTPI protein expression was generally highest in the basal layer of normal squamous esophagus epithelium and lowest in adenocarcinoma cells, with Barrett’s cells showing intermediate staining intensity. Our results show that downregulation of GSTPI expression is an early event in the development of Barrett’s esophagus and esophageal adenocarcinoma. Loss of GSTPI expression may have an important role in the development and progression of this disease.

Etiology of intestinal metaplasia at the gastroesophageal junction

Background: Intestinal metaplasia occurs in the esophagus as a consequence of gastroesophageal reflux disease and in the stomach secondary to H. pylori infection. The etiology of intestinal metaplasia limited to the gastroesophageal junction or cardia (CIM) is disputed. We hypothesized that CIM has dual etiologies: gastroesophageal reflux in some, H. pylori infection in others, and that cytokeratin immunostaining can help to differentiate between these two etiologies. Methods: We defined CIM as the presence of intestinal metaplasia within cardiac mucosa on biopsy from an endoscopically normal-appearing gastroesophageal junction. Thirty patients with CIM who had multiple biopsy specimens taken from the esophagus, gastroesophageal junction, and stomach were identified. Tissue blocks from biopsy specimens taken at the gastroesophageal junction were sectioned and immunostained for cytokeratins 7 and 20. The cytokeratin 7/20 staining of the CIM in each patient was determined to be either a Barretts or non-Barretts pattern. H. pylori infection was assessed by Giemsa staining of antral biopsy specimens. Results: H. pylori infection was present in 16 patients. A Barretts cytokeratin 7/20 staining pattern in the CIM was present in only 46% of the H. pylori–positive patients, as compared to 86% in the 14 patients with CIM and no H. pylori (p = 0.025). Objective evidence of reflux disease was present in 71% of patients with CIM and no H. pylori, as compared to 31% of patients with H. pylori. Conclusions: The two different patterns of cytokeratin 7/20 staining found in patients with CIM support the concept of dual etiologies for CIM. A Barretts staining pattern was associated with objective evidence of gastroesophageal reflux and the absence of H. pylori, suggesting that cytokeratin 7/20 immunostaining is useful to determine the likely etiology of CIM.

Detection of Barrett's epithelium by acoustic microscopy.

Barretts esophagus is associated with increased risk of adenocarcinoma of the gastroesophageal junctional region. The presence of goblet cells (intestinal metaplasia) in columnar cell-lined esophageal mucosa defines Barretts change. The diagnosis of Barretts esophagus is based on the presence of intestinal metaplasia in a biopsy from an endoscopically visualized abnormal columnar epithelium. In this pilot study, acoustic microscopy was used to identify the mucosal structure of 10 distal esophageal biopsies. Sections cut at 5 microm of archival paraffin blocks on glass slides were used for this study. Acoustic microscopy permitted the identification of low- and high-power images of epithelial architecture and cellular detail, including Barretts epithelium. This modality of visualization has the potential to detect lesions such as Barretts metaplasia, low- and high-grade dysplasia and early carcinoma. If it can be applied to in vivo endoscopy, acoustic microscopy has the potential to increase the accuracy of the diagnosis of Barretts esophagus, dysplasia and malignancy by providing a method of accurately directing biopsies at endoscopy.