Sylke Schneider

Loss of Heterozygosity at the Thymidylate Synthase (TS) Locus on Chromosome 18 Affects Tumor Response and Survival in Individuals Heterozygous for a 28-bp Polymorphism in the TS Gene

Thymidylate synthase (TS), the target enzyme of the fluoropyrimidine class of drugs, has a 28-bp repeat polymorphism in the promoter region that has been associated with response of tumors to 5-fluorouracil-based therapy. Patients homozygous for the double repeat (2R/2R) in the TS gene have an overall better outcome from treatment than patients homozygous for the triple repeat (3R/3R). However, due to loss of heterozygosity at the TS locus on chromosome 18 in cancer cells, heterozygous 2R/3R individuals can acquire the 2R/loss or the 3R/loss genotype in their tumors. The purpose of this study was to determine whether the response of colorectal cancer to fluoropyrimidine therapy is associated with the resulting tumor TS genotype when loss of heterozygosity occurs in tumor DNA. A total of 30 colorectal cancer patients treated with the fluoropyrimidine-based combination S-1, all of whom had stage IV disease, were studied. The response rate to S-1 in this group of patients was 13 of 30 (43%). The heterozygous 2R/3R genotype was found in 22 of 30 normal tissues, whereas 10 (45%) of the matched cancer tissues showed only the 2R-sequence band (2R/loss), and 7 cancer tissues (32%) showed only the 3R-sequence band (3R/loss). The response rate of the 2R/loss tumor genotype patients was 80% (8 of 10) compared with 14% (1 of 7) in the 3R/loss genotype group (P = 0.029). Patients with tumor 3R/loss genotypes had significantly lower survival than 2R/loss genotypes. Heterozygous patients with a 2R/loss tumor genotype had the same survival as 2R/2R patients, whereas patients with a 2R/3R tumor genotype had a short survival similar to homozygous 3R/3R genotypes. These results show that: (a) response to 5-fluorouracil-based therapy is determined by tumor genotype; and (b) the 3R repeat is a direct negative determinant of outcome.

Osteopontin but not osteonectin messenger RNA expression is a prognostic marker in curatively resected non-small cell lung cancer

Purpose: The purpose of this study was to better define the role of osteopontin (OPN) and osteonectin [also known as secreted protein acidic and rich in cysteine (SPARC)] in lung tumorigenesis by comparing the expressions of these genes in lung tumor tissue and matched normal tissue and by determining the prognostic significance of the gene expressions. Experimental Design: Quantitative real-time reverse transcription-PCR was used to analyze OPN and SPARC mRNA expression in normal lung tissue and matching tumor samples from 82 patients with non-small cell lung cancer. Gene expression data for each patient were matched to survival data. Results: The overall median mRNA expression level of OPN was about 20-fold higher in tumor tissues than in matching normal lung tissues (P < 0.001), whereas SPARC gene expression was not significantly different in both tissue types. Forty of 82 patients had high (≥4.1) intratumoral OPN expression, and 15 of 82 patients had high (≥15.5) SPARC expression. High OPN expression in the tumor tissue was associated with inferior survival (P = 0.014), whereas high SPARC expression showed a trend toward longer survival (P = 0.095). The impact of high OPN and low SPARC expression on patient survival was additive (P = 0.001). Conclusions: The large increase in OPN expression in tumors compared with normal tissue and its association with survival suggest a role for OPN in lung tumorigenesis.

A multigene expression panel for the molecular diagnosis of Barrett's esophagus and Barrett's adenocarcinoma of the esophagus.

In order to identify genes or combination of genes that have the power to discriminate between premalignant Barretts esophagus and Barretts 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 Barretts carcinogenesis or were filtered from a previous cDNA microarray study on Barretts adenocarcinoma. A total of 98 tissues, obtained from 19 patients with Barretts esophagus (BE group) and 20 patients with Barretts 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 Barretts 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 Barretts 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 Barretts and Barretts adenocarcinomas. This study provides the first non-array parallel mRNA quantitation analysis of a panel of genes in the Barretts esophagus model of multistage carcinogenesis. Our results suggest that mRNA expression quantitation of a panel of genes can discriminate between premalignant and malignant Barretts 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.

Genomic profiling associated with recurrence in patients with rectal cancer treated with chemoradiation

PURPOSE Stage II and III adenocarcinoma of the rectum has an overall 5-year survival rate of approximately 50%, and tumor recurrence remains a major problem despite an improvement in local control through chemotherapy and radiation. The efficacy of chemoradiation therapy may be significantly compromised as a result of interindividual variations in clinical response and host toxicity. Therefore, it is imperative to identify those patients who will benefit from chemoradiation therapy and those who will develop recurrent disease. In this study, we tested whether a specific pattern of 21 polymorphisms in 18 genes involved in the critical pathways of cancer progression (i.e., drug metabolism, tumor microenvironment, cell cycle regulation, and DNA repair) will predict the risk of tumor recurrence in rectal cancer patients treated with chemoradiation. PATIENTS AND METHODS A total of 90 patients with Stage II or III rectal cancer treated with chemoradiation were genotyped using polymerase chain reaction (PCR)-based techniques for 21 polymorphisms. RESULTS A polymorphism in interleukin (IL)-8 was individually associated with risk of recurrence. Classification and regression tree analysis of all polymorphisms and clinical variables developed a risk tree including the following variables: node status, IL-8, intracellular adhesion molecule-1, transforming growth factor-beta, and fibroblast growth factor receptor 4. CONCLUSION Genomic profiling may help to identify patients who are at high risk for developing tumor recurrence, and those who are more likely to benefit from chemoradiation therapy. A larger prospective study is needed to validate these preliminary data using germline polymorphisms on tumor recurrences in rectal cancer patients treated with chemoradiation.

Quantitative, tissue-specific analysis of cyclooxygenase gene expression in the pathogenesis of Barrett's adenocarcinoma.

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 (QRTPCR) 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.

Intratumoral COX-2 Gene Expression Is a Predictive Factor for Colorectal Cancer Response to Fluoropyrimidine-Based Chemotherapy

Purpose: Cyclooxygenase-2 (COX-2) is generally elevated in tumors compared with normal tissue and apparently has an important role in tumor development. A number of studies have found high expression of COX-2 to be an unfavorable prognostic factor for overall survival in several cancers. However, the influence of COX-2 expression levels on tumor response to chemotherapy has been relatively little studied. The purpose of this study was to ascertain if COX-2 gene expression is associated with tumor response in the clinical treatment of colorectal cancer with the fluoropyrimidine-based therapy S-1. Experimental Design: Patients with advanced (stage IV) colorectal cancer were treated with S-1 twice daily based on the patients body surface area (BSA; BSA < 1.25 m2, 80 mg/d; 1.25 m2 ≤ BSA < 1.5 m2, 100 mg/d; BSA ≥ 1.5 m2, 120 mg/d) for 28 days followed by a 2-week period rest. mRNA was isolated from paraffin-embedded pretreatment primary tumor specimens and expression levels of COX-2 relative to β-actin as the internal reference gene were measured using a quantitative reverse transcription-PCR (Taqman) system. Results: The overall response rate in a group of 44 patients treated with S-1 was 40.9%. Sufficient tumor tissue was available from 40 of these patients for COX-2 mRNA quantitation. COX-2 gene expression was significantly lower in the responding tumors compared with the nonresponders (P = 0.012, Wilcoxon test). Patients with COX-2 values above the cutoff value of 3.28 × 10−3 had a significantly shorter survival than those with COX-2 gene expressions below the cutoff value (adjusted P = 0.031). Conclusions: Intratumoral COX-2 gene expression is associated with likelihood of response to chemotherapy with S-1 and is a prognostic factor for survival of patients after the start of S-1 chemotherapy.

Increasing cyclooxygenase-2 (cox-2) gene expression in the progression of Barrett's esophagus to adenocarcinoma correlates with that of Bcl-2.

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 Barretts‐associated adenocarcinoma. Histologic sections from endoscopic biopsies or esophagectomy specimens were classified as non‐dysplastic Barretts (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 Barretts or cancer. Expression of both bcl‐2 and cox‐2 were increased in non‐dysplastic Barretts (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).

Loss of heterozygosity at thymidylate synthase locus in Barrett's metaplasia, dysplasia, and carcinoma sequences

BackgroundThymidylate 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.MethodsOne 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.ResultsAmong 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.ConclusionOur results demonstrate that LOH is a relatively frequent and early event in the IM-BA sequence.

Quantitative Determination of p16 Gene Expression by RT-PCR

In recent years, gene expression quantitation of tumor cells has become of principal importance to analyze gene patterns responsible for cancer development, progression, and resistance to treatment. Whereas semi-quantitative methods, such as Northern blotting analysis, allow only a dichotomous differentiation between positive and negative gene expressions, the real-time quantitative polymerase chain reaction (qRT-PCR) combines a large range of results with an accurate and highly reproducible quantitation of genes. In addition to forward and reverse primers, as used in a conventional PCR, the qRT-PCR system utilizes a probe that is labeled with a fluorescent dye. The probe is an oligonucleotide, homologous to a DNA sequence between the two flanking PCR primers. Degradation of the probe by the activity of the Taq DNA polymerase generates a fluorescent signal that will be detected by means of a laser integrated in the sequence detector. This chapter will cover the isolation of total RNA from frozen tissue, the transcription of RNA to cDNA, and the analysis of the relative gene expression by qRT-PCR. Although in principle applicable to any gene of interest, we will use as an example the qRT-PCR analysis of p16INK4a, whose gene product is involved in cell cycle and senescence checkpoint control.

The role of retinoid X receptor mRNA expression in Barrett's esophagus and Barrett's associated adenocarcinoma of the esophagus

Objective The Barretts multistage process is characterized histopathologically by progression from Barretts intestinal metaplasia to Barretts 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 m 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 Barretts esophagus d ~ . s e has yet to be determined. Aim of this study was to assess the prevalence and timing of RXR mRNA expression in Barretts metaplasia-dysplasiaadenocarcinoma sequence and to determine its role for the development and progression of this disease. Methods We analyzed the mRNA expression of all 3 RXR subtypes (RXRalpha, RXR-beta, RXR-gamma) by using a quantitative real-time RT-PCR method (Taqman) in 108 specimens from 19 patients with Barretts esophagus without carcinoma (BE-group), 20 patients with Barretts associated adenocarcinoma (F.A-group), and a control group of 10 patients without evidence of gastro-esophageal reflux disease (CG). Results RXR-alpha mRNA expression was significantly decreased (p<0.001; Kruskal-Walhs test), and RXRgamma (p<0.001) was significantly increased at higher stages in Barretts esophgns disease. gXR-beta expression was highest in Barretts tissues and significantly increased compared to normal squamous tissues (p = 0.01, Wilcoxon test) and adenocarinoma tissues (p = 0.018, Mann-Whitney test). RXR-alpha and RXR-beta mRNA expression were significantly associated in normal sqnamons esophagus tissues (r2 = 0.49; p<0.001; Spearman test), Barratts tissues (r2 ~ 0.63; p<0.001), and adenocarcinoma tissues (r2 = 0.68; p = 0.001). There were significant differences in RXR-alpha (p~0.011) and RXR-beta (p=O.O05) mRNA expression in histopathologically normal squamons esophagus tissues in patients with cancer and the normal control group(CG). Conclusions These findings suggest that alterations in the mRNA expression of all 3 RXR subtypes are frequent events in the development and progression of Barretts esophagus and associated adenocarcinoma, that qnantitation of RXR mRNA expression may be useful biomarkers for this disease, and that a widespread held-effect is present in the normal esophagus of patients with esophageal adenocarcinoma.