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Introduction: On the basis of the results of recent clinical trials, histology-based decision-making for therapy of non-small-cell lung cancer has been advocated. We hypothesized associations of the biomarkers excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase M1 (RRM1), and thymidylate synthase (TS) with histology as a contributing factor to reported differences in chemotherapy outcomes between squamous cell carcinoma (SCCA) and adenocarcinoma (AC) subtypes. Here, we report analysis of the Response Genetics Inc., database and implications for histology-based therapy., Methods: RNA from microdissected formalin-fixed paraffin-embedded tumors was extracted and analyzed as previously described. Specimens from 2540 individual non-small-cell lung cancer patients were analyzed for one or more biomarkers, of which 1457 were categorized as AC or SCCA., Results: For each biomarker, gene expression was lower in AC compared with SCCA (<0.001), although there was a wide range between individual patients. Gene expression was higher in men versus women: ERCC1: 2.51 versus 2.22 (p = 0.005); RRM1: 1.41 versus 1.24 (p = 0.004); TS: 3.23 versus 2.83 (p < 0.001). However, SCCA was more frequent in men versus women (30%/19%; p < 0.001). When AC and SCCA were assessed separately, the statistical significance between gene expression and sex was lost (in SCCA: ERCC1, p = 0.14; RRM1, p = 0.26; TS, p = 0.11)., Conclusions: This analysis represents the largest data set for gene expression of these biomarkers reported so far. Significant histology-related associations for ERCC1, RRM1, and TS are seen. However, marked heterogeneity exists in individual patient tumor expression levels. Randomized phase III trials assessing the predictive value of these chemotherapy-related biomarkers are warranted.

Purpose: Pathologic complete response (pCR) after neoadjuvant therapy for locally advanced esophageal adenocarcinoma is associated with improved survival. The Southwest Oncology Group designed a trimodality, phase II, single-arm trial with objectives of achieving a pCR rate of 40% with prospective exploratory analyses of intratumoral molecular markers postulated to affect response and survival., Patients and Methods: Patients with clinically staged II or III esophageal adenocarcinoma received oxaliplatin 85 mg/m(2) on days 1, 15, and 29; protracted-infusion fluorouracil (PI-FU) 180 mg/m(2)/d on days 8 through 43; and external-beam radiation therapy (EBRT) 5 days a week at 1.8 Gy/d for 25 fractions; surgery was performed 28 to 42 days after neoadjuvant therapy. Chemotherapy was planned after surgery. Tumors were analyzed for mRNA expression and polymorphisms in genes involved in drug metabolism and DNA repair., Results: Ninety-three patients were evaluable. Two deaths (2.2%) were attributable to preoperative therapy, and two deaths (2.2%) were attributable to surgery. Grade 3 and 4 toxicities were recorded for 47.3% and 19.4% of patients, respectively. Seventy-nine patients (84.9%) underwent surgery; 67.7% of patients had R0 resections. Twenty-six patients (28.0%) had confirmed pCR (95% CI, 19.1% to 38.2%). At a median follow-up of 39.2 months, estimates of median and 3-year overall survival (OS) were 28.3 months and 45.1%, respectively. Intratumoral ERCC-1 gene expression was inversely related to progression-free survival and OS., Conclusion: Neoadjuvant oxaliplatin, PI-FU, and EBRT for esophageal adenocarcinoma is active and tolerable. Because the regimen failed to meet the primary end point, it does not define a new standard. However, future trials can be built on this platform to validate the role of ERCC-1 in determining the best systemic regimen for individual patients.

Background: The effect of ornithine decarboxylase (ODC) on the pathogenesis of non-small-cell lung cancer (NSCLC) remains poorly investigated. Hence, the aim of this study was to explore the potential role of ODC mRNA expression as a prognostic biomarker in patients with curatively resected NSCLC., Patients and Methods: A total of 91 tumor and matching nontumorous lung tissue samples from patients with NSCLC were analyzed using a quantitative real-time reverse-transcriptase polymerase chain reaction method. The relative ODC mRNA expression was measured in tumorous and nontumorous lung tissue using beta-actin as a reference gene. Squamous cell carcinoma was found in 43 patients (47%), adenocarcinoma in 33 (36%), and large-cell carcinoma in 15 of the patients (17%). All patients' disease was R0 resected., Results: Ornithine decarboxylase was detected in all 91 tumor and nontumorous lung tissue samples. The median tumorous expression of 9.11 (range, 0.92-155.35) was significantly elevated compared with the median ODC expression of 7.89 (range, 0.0-45.8) in nontumorous lung tissue. Ornithine decarboxylase expression levels were not associated with any clinicopathologic parameters. Using an ODC/beta-actin ratio of 10 as a cutoff, tumorous ODC (tODC) expression is a significant prognostic factor in NSCLC. The ODC ratio between tumorous and nontumorous expression was even more prognostic. Moreover, Cox proportional hazards model analysis showed ODC expression to be an independent prognostic factor., Conclusion: In this study, ODC is shown to have a prognostic potential in NSCLC. Low levels of tODC expression are associated with a more aggressive tumor biology. Also, an increase of ODC mRNA expression during carcinogenesis seems to have a favorable prognostic effect. Measuring the ODC expression in patients with NSCLC could aid in further chemotherapy decisions. Our results suggest that further investigation of ODC mRNA expression in NSCLC may be warranted.

Background: Thymidylate synthase (TS) is known to have a unique 28 bp tandemly repeated sequence in the promoter region, and the majorities of subjects have a heterozygous double repeat/triple repeat genotype in their non-cancerous tissue. Loss of heterozygosity (LOH) at the TS locus is known to occur in cancer patients, but there is no evidence that it is present in precancerous tissue. The aim of this study was to analyze the frequency and timing of LOH at the TS locus in Barrett-associated adenocarcinoma (BA) and its precursory lesions, such as intestinal metaplasia (IM) and dysplasia., Methods: One hundred twenty-three samples (including 37 with gastroesophageal reflux disease (GERD), 29 with IM, 13 with dysplasia, and 44 with BA) were obtained from 100 patients. Biopsies were obtained from the lower esophageal mucosa/IM/dysplasia/BA, when available. Normal squamous tissue from the upper esophagus was taken as a control. All tissues were analyzed for the TS genotype and TS mRNA expression using the real-time reverse-transcription polymerase chain reaction (RT-PCR) method after laser-capture microdissection., Results: Among the patients with informative heterozygous genotype in their control samples, no sample with LOH at the TS locus was observed in the lower esophageal mucosa in GERD patients (0/22 samples). However, 6 out of 21 samples (28.6%) had LOH in IM, 2 of 7 (28.6%) in dysplasia, and 10 of 25 (40.0%) in BA. No significant difference in TS mRNA expression levels was observed between TS genotypes., Conclusion: Our results demonstrate that LOH is a relatively frequent and early event in the IM-BA sequence.

Thymidylate synthase (TS) is known to have polymorphisms in the 5' and 3' untranslated region (UTR). These polymorphisms have been reported to be associated with high TS expression and chemoresistance to 5-FU. The aim of this study was to examine the prognostic roles of the 5'-UTR and 3'-UTR TS polymorphisms in esophageal adenocarcinoma patients, as well as their relation with TS mRNA expression. Eighty-three patients with esophageal adenocarcinoma were assessed. Thirty-four had received 5-FU containing chemotherapy and 49 were treated with surgery alone. Surgically resected tumor tissues were analyzed for TS genotype and TS mRNA expression using a quantitative real-time RT-PCR method. No survival difference was seen between the patients with 3RG allele (3RG group) and non-3RG group among surgery-alone patients. However, among patients with a history of 5-FU-based chemotherapy, the non-3RG group showed significantly better overall survival compared to the 3RG group (p=0.02). Moreover, whereas chemotherapy produced a significant increase in survival for the non-3RG group patients, those in the 3RG group obtained no survival benefit from chemotherapy. When patients were classified by low or high TS mRNA expression levels, low TS expressers obtained survival benefit from chemotherapy while high TS expressers did not, although there was no difference of median TS mRNA levels between 3RG and non-3RG group. The 3'-UTR polymorphism was not associated with overall survival. These results suggest that the status of the TS 5'-UTR polymorphism and TS mRNA expression are independent predictive markers for survival benefit from 5-FU-based therapy.

Hypothesis: Chronic inflammation of esophageal mucosa secondary to refluxed gastric juice increases gene expression of interleukin 8 (IL-8). Antireflux surgery can reduce this overexpression., Design: Prospective analysis of archival paraffin-embedded tissue., Setting: Academic tertiary medical center., Patients and Methods: One hundred eight patients with reflux symptoms were classified according to pH monitoring and endoscopic and histologic findings. Twenty patients did not have reflux or mucosal injury; 47 had reflux disease (16 esophagitis and 31 Barrett's esophagus), 20 had dysplasia, and 21 had adenocarcinoma. Microdissection was performed to exclude inflammatory cells and stromal tissue. After RNA isolation and reverse transcription, IL-8 messenger RNA expression was measured using quantitative real-time polymerase chain reaction. All patients with reflux disease had Nissen fundoplication with biopsies at matched levels within the esophagus preoperation and postoperation., Results: Expression of IL-8 was increased in patients with reflux compared with those without reflux. Patients with the highest IL-8 expression were those with Barrett's dysplasia and adenocarcinoma (P<.001). In patients with reflux, Nissen fundoplication led to significantly decreased IL-8 expression compared with preoperative levels in esophagitis (P = .01) and Barrett's esophagus (P = .03)., Conclusions: Interleukin 8 messenger RNA expression increases during the progression of reflux disease from normal squamous mucosa to esophageal adenocarcinoma. Elimination of reflux with Nissen fundoplication significantly reduces IL-8 expression in both squamous and Barrett's mucosa. These results demonstrate that effective antireflux surgery can modulate the gene expression of esophageal mucosa and may impact the natural history of reflux disease.

Objective: To identify a marker for completeness of resection and recurrent disease in patients with esophageal cancer., Design: Case series., Setting: Department of Surgery of the University of Southern California., Patients: Forty-four healthy subjects and 45 patients with esophageal cancer prior to esophagectomy. Six patients were unresectable and 39 had a complete resection., Main Outcome Measures: Plasma DNA levels were measured using polymerase chain reaction. Twenty resected patients had follow-up plasma DNA levels measured., Results: Preoperatively, plasma DNA levels exceeded the normal level in 38 (84%) of 45 patients. Preoperatively, 12 patients received neoadjuvant therapy and 11 had plasma DNA levels higher than normal. All 6 unresectable patients had DNA levels higher than normal. At initial follow-up, the plasma DNA levels remained higher than normal in 2 (10%) of 20 patients, and systemic disease was subsequently detected in each. Plasma DNA levels dropped lower than or remained normal in 18 (90%) of 20. In 14 of 18 patients, there was no evidence of recurrent disease at a median of 12 months (range, 3-20 months); in 4 patients, the plasma DNA level rose higher than normal on follow-up and all developed subsequent systemic disease on computed tomographic or positron emission tomographic scan. Six of the 20 patients developed systemic disease during the follow-up (2 had persistently elevated plasma DNA levels, and 4 developed elevated plasma DNA levels at subsequent follow-ups). In 4 of these 6 patients, elevated plasma DNA levels were detected prior to imaging evidence of disease., Conclusions: Plasma DNA levels are significantly elevated in patients with esophageal cancer and following complete resection should return to normal. Persistently elevated plasma DNA levels after resection or levels that rise on follow-up indicate residual or recurrent disease.

Previous studies from our laboratory and others have suggested that increased expression of cox-2 is important in the genesis of esophageal adenocarcinoma. In vitro studies suggest that cox-2 regulates expression of the anti-apoptotic protein bcl-2, thus possibly accounting for reduced apoptosis in carcinogenesis. The aim of this study was to investigate the relationship of these 2 genes in the development of Barrett's-associated adenocarcinoma. Histologic sections from endoscopic biopsies or esophagectomy specimens were classified as non-dysplastic Barrett's (n = 30), intraepithelial neoplasia (n = 12) and adenocarcinoma (n = 48). The desired tissue was isolated by laser capture microdissection and expression levels of cox-2 and bcl-2 were measured by quantitative real-time PCR (Taqman). Gene expression levels were compared to samples of the distal esophageal squamous epithelium (n = 55) and reflux-esophagitis (n = 25), without Barrett's or cancer. Expression of both bcl-2 and cox-2 were increased in non-dysplastic Barrett's (p = 0.0077, p = 0.0037), intraepithelial neoplasia (p = 0.0053, p = 0.0220) and adenocarcinoma (p < 0.0001, p < 0.0001) compared to squamous epithelium or reflux-esophagitis. Furthermore, there is a significant correlation between these two genes, especially in carcinoma (p < 0.0001)., (Copyright 2006 Wiley-Liss, Inc.)

Hypothesis: Cyclooxygenase 2 (COX-2), vascular endothelial growth factor (VEGF), and epidermal growth factor receptor (EGFR) are useful biological determinants in targeted therapy for esophageal adenocarcinoma., Design: Prospective analysis., Setting: University tertiary referral center., Patients: Sixteen patients with squamous mucosa and normal results of a pH study without mucosal injury (control group), 15 with Barrett esophagus (metaplasia group), and 44 with adenocarcinoma (carcinoma group)., Interventions: Biopsy specimens were obtained 3 cm above the gastroesophageal junction. Dysplastic tissue was additionally isolated from 9 of the patients in the carcinoma group. After laser-capture microdissection, quantitative real-time polymerase chain reaction was used to measure gene expression across the spectrum of the metaplasia-dysplasia-carcinoma sequence., Main Outcome Measures: Expression of COX-2, VEGF, and EGFR in each patient group., Results: Expression of both COX-2 and VEGF was significantly up-regulated in patients with metaplasia, dysplasia, and cancer compared with controls (P<.01). Expression levels of both were significantly higher in cancer than in the metaplasia group (P<.05) and increased sequentially from metaplasia to dysplasia to cancer. Expression of VEGF was significantly higher in the dysplastic tissue than in nondysplastic Barrett epithelium (P<.05). No change in expression levels of EGFR was seen in the histologic progression to esophageal adenocarcinoma., Conclusion: Gene expression data suggest that pharmacologic inhibition of COX-2 and VEGF may be useful adjuncts in targeted therapy for esophageal adenocarcinoma.

Background: Survivin, a member of the inhibitor-of-apoptosis family, is reported to be overexpressed in esophageal cancer but no data are available about its status in the metaplastic/dysplastic sequence. The aim of this study was to measure survivin gene expression in normal squamous/columnar epithelium and in the various stages of development of Barrett's adenocarcinoma., Methods: Endoscopic biopsy or operative specimen samples from 5 tissue types were analyzed: (1) squamous epithelium from 3 cm above the gastroesophageal junction in patients with a negative pH study and no histologic injury (n = 17, pH- control); (2) antral tissue from patients with no evidence of Barrett's, dysplasia, or cancer (n = 29, antral control); (3) specialized intestinal metaplasia from patients with Barrett's esophagus (n = 16; Barrett's group); (4) low- or high-grade dysplasia (n = 12, dysplasia group), and (5) adenocarcinoma (n = 45 cancer group). After laser-capture microdissection cellular RNA was extracted from each tissue and reverse transcribed to complementary DNA. Expression levels of survivin were measured by reverse-transcription polymerase chain reaction., Results: Survivin gene expression was greater in columnar (antral) compared with squamous (pH-) control tissues (P = .03). Expression in quiescent Barrett's epithelium was similar to both control tissues. Expression levels in dysplastic epithelium were greater than in squamous control (P = .01) and Barrett's tissues (P = .04), but not higher than columnar control tissues, whereas expression in adenocarcinoma was greater than all tissues except dysplasia (P < .001)., Conclusions: Survivin expression may be a biomarker in the development of Barrett's adenocarcinoma that is able to distinguish between quiescent Barrett's, dysplastic Barrett's, and Barrett's adenocarcinoma.

Background and Aim: Genetic alterations in the normal tissues surrounding various cancers have been described, but a comprehensive analysis of this carcinogenic field effect in Barrett's-associated adenocarcinoma of the esophagus disease has not been reported. The aim of this study was to analyze the gene expression profile of a panel of highly selected genes in the normal squamous esophagus epihelium of patients with Barrett's esophagus, patients with Barrett's-associated adenocarcinoma, and a healthy control group to define the existence of a carcinogenic field effect, and to investigate the clinical importance of such a field effect in the management of Barrett's disease., Methods: Forty-nine histologic normal squamous esophageal epithelia collected from 19 patients with Barrett's esophagus, 20 patients with Barrett's-associated esophageal adenocarcinoma, and a healthy control group of 10 patients were studied. A quantitative real-time reverse transcription-PCR method (TaqMan) was used to measure the expression of a panel of genes with known associations with gastrointestinal carcinogenesis., Results: A widespread carcinogenic field effect was detected for more than 50% of the genes analyzed including Bax, BFT, CDX2, COX2, DAPK, DNMT1, GSTP1, RARalpha, RARgamma, RXRalpha, RXRbeta, SPARC, TSPAN, and VEGF. Based on the expression signature of the normal appearing squamous esophagus, a linear discriminant analysis was able to distinguish between the three groups of patients with an error rate of 0%., Conclusion: This study provides the first comprehensive investigation of a carcinogenic field effect in Barrett's esophagus disease. Based on the gene expression signature of the normal esophagus, patients could be correctly characterized according to their pathologic classification by applying a linear discriminant analysis. Our results provide evidence that a molecular classification might have clinical importance for the diagnosis and treatment of patients with Barrett's esophagus disease.

Cyclooxygenase (Cox-2) is implicated in the pathogenesis of many cancers including esophageal adenocarcinoma (EAC), whereas the role of the isoform Cox-1 in carcinogenesis is not well understood. To further elucidate the role of these factors in the development of EAC, we measured the gene expressions (mRNA levels) of Cox-2 and Cox-1 by real-time quantitative polymerase chain reaction (QRT-PCR) in tissues from normal esophagus with and without erosive gastroesophageal reflux disease (GERD), Barrett's esophagus (BE), dysplasia, adenocarcinoma, and in healthy gastric antrum. All tissues were purified by laser capture microdissection from endoscopic or surgical resection specimens. Median Cox-2 gene expression did not differ significantly among the esophageal control groups but was elevated 5-fold in BE, 8-fold in dysplasia and 16-fold in EAC compared to normal esophageal controls with no erosive GERD. Erosive GERD tissue had slightly higher median Cox-2 expression but Cox-2 expression in normal antrum was much higher than that in a normal esophagus, close to that of dysplasia. In contrast to that of Cox-2, Cox-1 expression was significantly decreased in all neoplastic tissues compared to normal controls. Cox-1 and Cox-2 expression varied over a wide range in the neoplastic tissues but over a relatively narrow range in the esophageal normal tissues. The occurrence of substantial alterations in Cox-1 and Cox-2 expression at the BE stage indicates that these are early events in the development of EAC. These results confirm the important role of Cox-2 amplification in the pathogenesis of esophageal adenocarcinoma, but the unexpected down-regulation of Cox-1 raises questions about its role in carcinogenesis.

In order to identify genes or combination of genes that have the power to discriminate between premalignant Barrett's esophagus and Barrett's associated adenocarcinoma, we analysed a panel of 23 genes using quantitative real-time RT-PCR (qRT-PCR, Taqman and bioinformatic tools. The genes chosen were either known to be associated with Barrett's carcinogenesis or were filtered from a previous cDNA microarray study on Barrett's adenocarcinoma. A total of 98 tissues, obtained from 19 patients with Barrett's esophagus (BE group) and 20 patients with Barrett's associated esophageal adenocarcinoma (EA group), were studied. Triplicate analysis for the full 23 gene of interest panel, and analysis of an internal control gene, was performed for all samples, for a total of more than 9016 single PCR reactions. We found distinct classes of gene expression patterns in the different types of tissues. The most informative genes clustered in six different classes and had significantly different expression levels in Barrett's esophagus tissues compared to adenocarcinoma tissues. Linear discriminant analysis (LDA) distinguished four genetically different groups. The normal squamous esophagus tissues from patients with BE or EA were not distinguishable from one another, but Barrett's esophagus tissues could be distinguished from adenocarcinoma tissues. Using the most informative genes, obtained from a logistic regression analysis, we were able to completely distinguish between benign Barrett's and Barrett's adenocarcinomas. This study provides the first non-array parallel mRNA quantitation analysis of a panel of genes in the Barrett's esophagus model of multistage carcinogenesis. Our results suggest that mRNA expression quantitation of a panel of genes can discriminate between premalignant and malignant Barrett's disease. Logistic regression and LDAs can be used to further identify, from the complete panel, gene subsets with the power to make these diagnostic distinctions. Expression analysis of a limited number of highly selected genes may have clinical usefulness for the treatment of patients with this disease.

The HER-2/neu oncogene encodes a 185 kD protein that is phosphorylated upon ligand binding to other HER/erbB members and regulates cell growth and differentiation. Given that HER-2 receptor blockade can inhibit the growth of colon cancer cell lines and tumor xenografts, we investigated the frequency, localization and phosphorylation status of HER-2 in colon cancer cell lines and in human tumors. Protein expression was analyzed in relation to mRNA levels, HER-2 amplification, and clinicopathological variables. Colon cancer cell lines constitutively expressed HER-2 proteins and none showed HER-2 amplification by fluorescence in situ hybridization. Cell fractionation and immunoblotting showed HER-2 in both the membrane and cytosolic compartments. Primary colorectal carcinomas (n = 96) and their metastases (n = 25) were examined by immunohistochemistry. Strong membrane HER-2 staining was detected in 5 (5%) of primaries and in 3 (12%) metastases (p = 0.36). Membrane but not cytoplasmic localization was strongly associated with HER-2 gene amplification (p = 0.007). Cytoplasmic HER-2 staining was found in 61 (63.5%) of primary tumors and localization was confirmed by immunoelectron microscopy that also showed plasma membrane HER-2. Using real-time quantitative RT-PCR, HER-2 mRNA was increased in tumors with membrane compared to cytoplasmic staining (r = 0.66, p = 0.001). Cytoplasmic HER-2 was associated with tumor differentiation (p = 0.018), but not other clinicopathological variables. By immunoblotting, heterogeneity was seen in HER-2 levels with downregulation in 4 of 7 tumors relative to normal epithelia that uniformly expressed HER-2. Phosphorylated HER-2 was detected in approximately 50% of tumors and in normal mucosa. In conclusion, HER-2 is expressed constitutively in colon cancer cell lines and demonstrates relatively distinct localization patterns in human tumors. Strong membrane immunoreactivity is associated with high levels of HER-2 mRNA and gene amplification whereas cytoplasmic HER-2 is detected frequently and seems to be a marker of tumor differentiation., (Copyright 2003 Wiley-Liss, Inc.)

Objective: This study was undertaken to investigate the role of the angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor in the development and progression of Barrett esophagus and adenocarcinomas of the esophagus and gastroesophageal junction., Methods: Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels, relative to the control gene encoding beta-actin, were measured by using a quantitative reverse transcription-polymerase chain reaction method (ABI 7700 Sequence Detector system) in specimens of Barrett intestinal metaplasia (n = 16), dysplasia (n = 11), adenocarcinoma (n = l 5), and matching normal squamous esophageal tissues (n = 35). Vascular endothelial growth factor and basic fibroblast growth factor protein expression and CD31(+) microvessel density were assessed by means of immunohistochemistry in 25 tissue sections that included representative areas for each of these Barrett stages., Results: Expression levels were significantly increased in adenocarcinoma compared with in either normal squamous mucosa (P <.0001 for both genes) or intestinal metaplasia (vascular endothelial growth factor, P =.002; basic fibroblast growth factor, P <.0001). Vascular endothelial growth factor levels were also significantly higher in cancer tissues compared with dysplasia tissues (P =.024, Mann-Whitney U test). Basic fibroblast growth factor expression was also significantly increased in Barrett dysplastic mucosa compared with in intestinal metaplasia or normal esophageal mucosa. Microvessel density was generally higher in adenocarcinoma compared with in preneoplastic Barrett tissues. The pattern of vascular endothelial growth factor and basic fibroblast growth factor protein expression was similar to the messenger RNA expression pattern, with the exception that mucin-containing goblet cells stained intensely for vascular endothelial growth factor and only weak vascular endothelial growth factor staining was present in some adenocarcinomas., Conclusions: Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels are significantly upregulated in esophageal and gastroesophageal junction adenocarcinomas, suggesting a role for these angiogenic factors in the development of these cancers. Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels are also increased in some Barrett esophagus tissues, with this increase occurring at an earlier stage for basic fibroblast growth factor than for vascular endothelial growth factor. Basic fibroblast growth factor protein expression pattern is similar to the messenger RNA expression pattern, but unlike the messenger RNA findings, vascular endothelial growth factor protein expression is strongest in goblet cells.

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 beta-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.

Reliable quantification of gene expression offers the possibility of more accurate and prognostically relevant characterization of tissues than potentially subjective interpretations of histopathologists. We measured the expression of 18 selected genes and compared them to histological features in a spectrum of esophageal disease to evaluate the feasibility of molecular characterization of normal and pathologic esophageal epithelia. Esophageal tissue biopsies from 82 patients with foregut symptoms were laser capture microdissected, and the expression levels of 18 selected genes were measured by quantitative real-time polymerase chain reaction. Linear discriminant analysis, which uses combinations of genes to distinguish between histological groups, was performed to compare gene expression and the following five histological groups: (1) normal squamous epithelium (n = 35), (2) reflux esophagitis (n = 13), (3) non-dysplastic Barrett's (n = 33), (4) dysplastic Barrett's (n = 16), (5) adenocarcinoma (n = 31). A panel of seven genes had 90-94% predictive power to distinguish non-dysplastic and dysplastic Barrett's esophagus. Clustering analysis revealed structure in gene expression values even in the absence of histology. Expression levels in 17 genes differed significantly across histological groups. Classification based on gene expression agreed with histopathological assessment in the following percentage of cases: normal squamous epithelium = 53%, reflux esophagitis = 31%, non-dysplastic Barrett's = 76%, dysplastic Barrett's = 40%, and adenocarcinoma = 59%. Interestingly, predictive power improved markedly when inflammatory and dysplastic tissues were removed (77-94%). Gene expression classification agrees well with histopathological examination. When differences occur, it is unclear whether this effect is due to intraobserver variability in pathological diagnosis or to a genuine difference between gene expression and histopathology. [ABSTRACT FROM AUTHOR]

The Barrett''s multistage process is characterized histopathologically by progression from Barrett''s intestinal metaplasia to Barrett''s esophagus with dysplasia and ultimately adenocarcinoma. Understanding of the molecular alterations in this multistage process may contribute to improved diagnosis and treatment. Retinoid X receptors (RXR) play an important role in regulating the morphogenesis, development, growth, and differentiation of cells. Alterations in RXR expression have been observed in a variety of solid tumors; however, the role in Barrett''s esophagus disease has yet to be determined. The aim of this study was to assess the prevalence and timing of RXR messenger RNA expression in the Barrett''s metaplasia-dysplasia-adenocarcinoma sequence and to investigate its role in the development and progression of this disease. We analyzed the mRNA expression of all three RXR subtypes (RXR-alpha, RXR-beta, and RXR-gamma) by using a quantitative real-time reverse transcription–polymerase chain reaction method in 108 specimens from 19 patients with Barrett''s esophagus without carcinoma (BE group), 20 patients with Barrett''s-associated adenocarcinoma (EA group), and a control group of 10 patients without evidence of gastroesophageal reflux disease (CG). RXR-α mRNA expression was significantly decreased (P < 0.001; Kruskal-Wallis test), and RXR-γ was significantly increased (P < 0.001) at higher stages in Barrett''s esophagus. RXR-β expression was highest in Barrett''s tissues and was significantly increased compared to normal squamous tissues (P = 0.01; Wilcoxon test) and adenocarinoma tissues (P = 0.018, Mann-Whitney test). RXR-α and RXR-β mRNA expression was significantly associated in normal squamous esophagus tissues (r2 = 0.49; P < 0.001; Spearman test), Barrett''s tissues (r2 = 0.63; P < 0.001), and adenocarcinoma tissues (r2 = 0.68; P = 0.001). There were significant differences in RXR-α (P = 0.011) and RXR-β (P = 0.005) mRNA expression in histopathologically normal squamous esophagus tissues in patients with cancer and the control group without evidence of gastroesophageal reflux disease. These findings suggest that alterations in the mRNA expression of all three RXR subtypes are frequent events in the development and progression of Barrett''s esophagus and associated adenocarcinoma, that RXR mRNA expression levels may be useful biomarkers for this disease, and that a widespread “field-effect” is present in the normal esophagus of patients with esophageal adenocarcinoma. [Copyright &y& Elsevier]

These studies investigated the role of cyclooxygenase gene expression in esophageal diseases. The study from Germany by Zimmerman et al. detected cytoplasmic cyclooxygenase- 2 (COX-2) protein expression by immunohistochemistry in 156 of 172 (91%) esophageal squamous cell carcinomas (SCC) and in 21 of 27 (78%) Barrett's-associated esophageal adenocarcinomas. There was a wide range in COX-2 immunoreactivity in different cancer specimens, and also within individual specimens. Western blot analysis of COX-2 protein levels was performed for some of the SCC samples, confirming the immunohistochemistry results. No COX-2 protein expression was detected immunohistochemically in Barrett's intestinal metaplasia tissues. There were no significant associations between COX immunoreactivity and tumor stage for either cancer type, except for a higher frequency of local lymph node metastases in SCC patients with low COX-2 expression. In vitro studies using SCC cell lines showed that expression of COX-2 at the mRNA level was associated with production of both COX-2 protein and high prostaglandin (PGE2 and 6-keto-PGFlα) levels. Finally, this study showed that treatment of the COX-2-expressing SCC cells with the COX inhibitors flusolide and NS-398 inhibited prostaglandin synthesis, suppressed proliferative and mitotic activity, and induced widespread apoptosis (the apoptotic effect was assessed with flusolide only). In the non-COX- 2-expressing cell line, none of these effects were observed. The earlier publication, by Wilson et al., from the University of Maryland School of Medicine, used a reverse transcriptase-polymerase chain reaction method to detect elevated COX-2 mRNA levels in 15 of 19 (79%) Barrett's intestinal metaplasia specimens, 2 of 2 (100%) Barrett's dysplasia specimens, and 5 of 5 (100%) Barrett's-associated adenocarcinomas. Increased levels of COX-2 protein, inducible nitric oxide synthase mRNA, and transforming growth factor-α mRNA were also present in similar proportions in Barrett's esophagus and adenocarcinoma tissues. [ABSTRACT FROM AUTHOR]

PURPOSE: Pancreatic cancer still has one of the worst prognoses in gastrointestinal cancers with a 5-year survival rate of 5%, making it necessary to find markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens. In this study, we investigated the prognostic values of HIF1a, bFGF, VEGF, and PDGFA gene expressions as well as their interrelationships. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tissue samples were obtained from 41 patients with pancreatic adenocarcinoma (age, 65; range, 34-85 years). After laser capture microdissection, direct quantitative real-time reverse transcription-polymerase chain reaction assays were performed in triplicates to determine HIF1a, PDGFA, VEGF, and bFGF gene expression levels. Multivariate Cox proportional hazards regression analysis was used to assess the impact of HIF1a gene expression on prognosis. RESULTS: HIF1a was significantly correlated to every gene we tested: bFGF (P = .04), VEGF (P = .02), and PDGFA (P = .03). Tumor size, P = .04, and high HIF1a mRNA expression (cutoff, 75th percentile) had a significant impact on survival, P = .009 (overall model fit, P = .02). High HIF1a expression had a sensitivity of 87.1% and a specificity of 55.6% for the diagnosis short (<6 months) versus long (6-60 months) survival. CONCLUSIONS: Measuring PDGFA, bFGF, and HIF1a expression may contribute to a better understanding of the prognosis of patients with pancreatic cancer and may even play a crucial role for the distribution of patients to multimodal therapeutic regimens. Larger studies including patients treated with actual chemotherapeutics seem to be warranted. [ABSTRACT FROM AUTHOR]