Helene Geddert

Hypermethylation of tumor suppressor genes (p16INK4A, p14ARF and APC) in adenocarcinomas of the upper gastrointestinal tract

Aberrant promoter methylation is an important mechanism for gene silencing. In the present study, 50 Barretts esophagus‐associated esophageal adenocarcinomas (ADC), 50 cardiac ADC and 50 gastric ADC were investigated by means of methylation‐specific real‐time PCR for hypermethylation in the tumor suppressor genes APC, p16INk4A and p14ARF. Additionally, expression of p16INK4A protein in the carcinomas was assessed using immunohistochemistry. Marked differences in hypermethylation were found between esophageal, cardiac and gastric ADC in the APC gene (78% vs. 32% vs. 84%) and in the p16INK4A gene (54% vs. 36% vs. 10%). Hypermethylation of p14ARF was absent from esophageal ADC and present infrequently in cardiac (2%) and gastric ADC (10%). Complete loss of p16INK4A protein expression was detectable in 45% of all tumors and was significantly associated with hypermethylation of the p16INK4A gene (p<0.0001, χ2‐test). Our results suggest that hypermethylation of p16INK4A and APC are frequent findings in esophageal, cardiac and gastric ADC. Additionally, the data point to a tumor specific methylation pattern in upper gastrointestinal ADC.

Association between NAD(P)H: Quinone oxidoreductase 1 (NQ01) inactivating C609T polymorphism and adenocarcinoma of the upper gastrointestinal tract

NQO1 is an antioxidant enzyme, important in the detoxification of environmental carcinogens. A single nucleotide polymorphism (C→T) at position 609 of the NQO1 cDNA has been associated with susceptibility to tumours induced by chemical carcinogens. In our case‐control study, we determined the prevalence of the C609T NQO1 polymorphism by PCR‐RFLP analysis in Caucasian patients with oesophageal adenocarcinoma (OAC; n=61), cardiac adenocarcinoma (CAC; n=120) or gastric adenocarcinoma (GAC; n=203) vs. a control group that consisted of 252 healthy blood donors. Additionally, NQO1 mRNA expression and NQO1 protein expression were determined by RT‐PCR and immunohistochemistry in a subset of cases. The NQO1 C609T genotype distribution was significantly different among controls (C/C, 73.4%; C/T, 25.0%; T/T, 1.6%) as compared to OAC patients (C/C, 49.2%; C/T, 47.5%; T/T, 3.3%; p=0.0004), CAC patients (C/C, 55.8%; C/T, 40.0%; T/T, 4.2%; p=0.0005) and with GAC patients (C/C, 65.5%; C/T; 30.6%, T/T; 3.9%; p=0.0377). The 609T allele overall frequency was 0.141 in controls, 0.270 in OAC patients, 0.241 in CAC patients and 0.192 in GAC patients. Individuals carrying 1 or 2 609T alleles had a 2.85‐fold higher risk (95% CI: 1.61–5.07; p=0.0003) for the development of OAC and a 2.18‐fold higher risk (95% CI: 1.38–3.44; p=0.0007) for the development of CAC than wild‐type gene homozygotes. Immunohistochemical analysis showed NQO1 protein expression in 133 carcinomas, whereas 17 carcinomas were negative. Negativity for NQO1 protein expression correlated strongly with the NQO1 genotype being present in 3.9% of cases with C/C, 13.9% of cases with C/T and 62.5% of cases with T/T genotype (p<0.001). In contrast, NQO1 mRNA expression was detectable irrespective of underlying genotype. In conclusion, determination of the NQO1 genotype may gain importance as a stratification marker in future prevention trials for adenocarcinoma of upper gastrointestinal tract.

Gene Amplification and Protein Overexpression of c-erb-b2 in Barrett Carcinoma and Its Precursor Lesions

We examined 39 samples of metaplastic specialized epithelium (SE), 27 of low-grade dysplasia (LGD), 27 of high-grade dysplasia (HGD), and 46 of adenocarcinoma (CA) derived from Barrett esophagus for c-erb-b2 gene amplification using differential polymerase chain reaction and for overexpression of c-erb-b2 protein using immunohistochemical analysis. Amplification of the c-erb-b2 gene was as follows: SE, 0.0%; LGD, 0.0%; HGD, 11.1%; and CA, 13.6%; and protein overexpression was as follows: SE, 0.0%; LGD, 7.4%; HGD, 18.5%; and CA, 21.7%. In 8 (89%) of 9 samples, c-erb-b2 gene amplification correlated with protein overexpression. The reverse was true in 8 (47%) of 17 samples: c-erb-b2 protein overexpression was proved with simultaneous gene amplification. Amplification of c-erb-b2 is a late event in the carcinogenesis of Barrett esophagus. In contrast, protein overexpression appears more often and earlier Besides gene amplification, other mechanisms to induce protein overexpression must exist.

The role of p63 and ΔNp63 (p40) protein expression and gene amplification in esophageal carcinogenesis

p63, a member of the p53 gene family, is known to encode functionally antagonistic protein isoforms. Although transactivating protein isoforms display p53-like functions, deltaNp63 isoforms act toward p53 in a dominant negative way. Using immunohistochemistry, we examined the expression of pan-p63 and deltaNp63 in 50 esophageal squamous cell carcinomas (SCCs) as well as in squamous low-grade intraepithelial neoplasias (S-LGINs; n = 4) and high-grade intraepithelial neoplasias (S-HGINs; n = 18). Additionally, 50 esophageal adenocarcinomas (ADCs) that arose in Barretts esophagus (BE) as well as adjacent specialized metaplastic epithelium (SE; n = 41), low-grade intraepithelial neoplasias (B-LGINs; n = 27), and high-grade intraepithelial neoplasias (B-HGINs; n = 21) in BE were investigated. Furthermore, p63 gene amplification was determined by fluorescent differential polymerase chain reaction in a subset of 10 SCCs and 10 ADCs. Whereas in normal esophageal epithelium, expression of pan-p63 is invariably restricted to the basal cell layer, in 100% of S-LGINs, 94.4% of S-HGINs, and 88.0% of SCCs, expression of p63 was found in >75% of the cells. Concerning BE, only in a small subset of SEs (7.3%), B-LGINs (14.8%), B-HGINs (14.3%) and ADCs (16.0%) was a weak p63 protein expression (<10% positive cells) detectable, whereas the rest of the samples were completely negative. Expression of deltaNp63 was identical to expression of pan-p63 in the vast majority of samples. p63 gene amplification was found in 2 of 10 (20.0%) investigated SCCs and in 1 of 10 (10.0%) ADCs. In summary, strong expression of p63, especially its deltaNp63 isoforms, is a frequent finding in esophageal precancerous and cancerous squamous lesions, whereas this is not the case in carcinogenesis of BE. p63 gene amplification is an infrequent finding in esophageal SCCs and ADCs and does not correlate with protein overexpression.

Aberrant promoter methylation of p16INK4a, RARB2 and SEMA3B in bronchial aspirates from patients with suspected lung cancer

Aberrant promoter methylation of normally unmethylated CpG‐islands offers a promising tool for the development of molecular biomarkers. We investigated bronchial aspirates of patients admitted for suspected lung cancer with regard to the prevalence of aberrant methylation of potential marker genes. Applying quantitative methylation specific PCR (QMSP) we analyzed bronchial aspirates from 75 patients with primary lung cancer and 64 bronchial aspirates of patients diagnosed with benign lung disease for promoter methylation of 3 candidate marker genes (p16INK4a, RARB2 and SEMA3B). Hypermethylation of p16INK4a detected 18/75 (24%) cases with primary lung cancer and was present predominantly in squamous cell carcinomas (14/25; 56%). RARB2 QMSP at an assay threshold greater than 30 was found in 42/75 (56%) patients with lung cancer without relation to histological subtype. Patients with benign lung disease showed methylation of p16INK4a and a RARB2 QMSP at an assay threshold greater than 30 in 0/64 (0%) and 8/64 (13%) cases, respectively. Combining the 2 methylation markers, p16INK4a and RARB2, yielded a sensitivity of 69% and a specificity of 87% for the diagnosis of pulmonary malignancy. In contrast, SEMA3B displayed frequent promoter methylation (around 90%) both in bronchial aspirates of tumor and nontumor cases and thus was not suited as a biomarker. The results of this study indicate that QMSP analysis of p16INK4a and RARB2 may aid the diagnosis of primary lung cancer in bronchial aspirates. In particular, detection of p16INK4a methylation by QMSP may serve as a highly specific marker of pulmonary squamous cell carcinoma.

Gene expression profiling for molecular distinction and characterization of laser captured primary lung cancers

MethodsWe examined gene expression profiles of tumor cells from 29 untreated patients with lung cancer (10 adenocarcinomas (AC), 10 squamous cell carcinomas (SCC), and 9 small cell lung cancer (SCLC)) in comparison to 5 samples of normal lung tissue (NT). The European and American methodological quality guidelines for microarray experiments were followed, including the stipulated use of laser capture microdissection for separation and purification of the lung cancer tumor cells from surrounding tissue.ResultsBased on differentially expressed genes, different lung cancer samples could be distinguished from each other and from normal lung tissue using hierarchical clustering. Comparing AC, SCC and SCLC with NT, we found 205, 335 and 404 genes, respectively, that were at least 2-fold differentially expressed (estimated false discovery rate: < 2.6%). Different lung cancer subtypes had distinct molecular phenotypes, which also reflected their biological characteristics. Differentially expressed genes in human lung tumors which may be of relevance in the respective lung cancer subtypes were corroborated by quantitative real-time PCR.Genetic programming (GP) was performed to construct a classifier for distinguishing between AC, SCC, SCLC, and NT. Forty genes, that could be used to correctly classify the tumor or NT samples, have been identified. In addition, all samples from an independent test set of 13 further tumors (AC or SCC) were also correctly classified.ConclusionThe data from this research identified potential candidate genes which could be used as the basis for the development of diagnostic tools and lung tumor type-specific targeted therapies.

Polymorphism of p16INK4A and cyclin D1 in adenocarcinomas of the upper gastrointestinal tract

Purpose: We investigated the prevalence of single nucleotide polymorphisms in the p16 gene (C540G) and the cyclin D1 gene (G870A), both known to regulate function in G1 arrest and therefore, may play an important role in carcinogenesis. Methods: Using PCR based restriction fragment length polymorphism and single strand conformational polymorphism, we determined single nucleotide exchanges in the p16 and cyclin D1 genes among 56 esophageal adenocarcinomas (ADC) arising in Barrett’s esophagus, 95 cardiac gastric ADC, and in 191 distal gastric ADC. The allelic frequencies were compared to a control group of 253 healthy blood donors. Results: The C/G genotype of p16 was identified in 10.4% of esophageal carcinomas, 13.3% of cardiac carcinomas, and in 14.1% of gastric carcinomas, compared to 17.4% in the healthy control group. All other cases showed the C/C wildtype, as no homozygous G/G nucleotide exchange was detected in the group of cancer patients or in the control group. In esophageal cancer, cyclin D1 G/G genotype was found 28.6%, A/G in 46.4%, and A/A in 25.0%. In cardiac carcinoma, frequency of cyclin D1 genotype was 27.4% for G/G, 57.9% for A/G, and 14.7% for AA. In distal gastric carcinoma, both homozygous genotypes (G/G and A/A) had a frequency of 15.2% each, while the heterozygous A/G genotype occurred in 69.6% of patients. The control group displayed 24.9% G/G, 53.8% A/G, and 21.3% A/A genotype. Conclusions: Our results show that frequencies of p16 or cyclin D1 polymorphisms in gastric and esophageal ADC do not differ significantly from the healthy control group. Therefore, these polymorphisms are unlikely to be associated with risk of ADC of the upper gastrointestinal tract.

Expression of the tyrosine kinase c‐kit is an independent prognostic factor in patients with small cell lung cancer

In a retrospective analysis of 203 patients with small cell lung cancer (SCLC), we examined the prognostic value of c‐kit expression on survival. Expression of c‐kit was examined immunohistochemically in formalin‐fixed, paraffin‐embedded tissue sections. c‐kit was observed in 87.7% of SCLC tumors. Using the Kaplan‐Meier model, we found that lack of c‐kit expression was associated with significantly shorter survival time compared to the presence of c‐kit expression (mean survival 151 ± 27 vs. 358 ± 49 days, p = 0.0084). Moreover, the proportion of c‐kit+ cells within the tumor was also related to survival time. Patients with tumors in which >75% of cells stained positive for c‐kit had a mean overall survival time of 424 (±72) compared to 295 (±67) days for patients with 25–75% c‐kit+ tumor cells. Patients with tumors containing <25% c‐kit+ cells had the worst survival, with 164 (±24) days (p = 0.0033). Further parameters associated with short survival times were low performance status, elevated levels of lactate dehydrogenase and higher stage according to the TNM classification. Multivariate analysis using the Cox regression model showed that the proportion of c‐kit+ cells within the tumor specimen was one of 3 independent prognostic parameters (p = 0.004) for overall survival next to TNM classification (p = 0.001) and performance status (p < 0.001).

Hypermethylation of hMLH1, HPP1, p14 ARF , p16 INK4A and APC in primary adenocarcinomas of the small bowel

Small bowel adenocarcinoma (SB‐AC) is a very rare tumor entity. Epigenetic alterations, including hypermethylation of DNA mismatch repair genes and tumor suppressor genes, seem to be important for carcinogenesis in tumors of the gastrointestinal tract, but have not yet been investigated in SB‐AC. In the current study, the prevalence of hypermethylation in a panel of genes involved in gastrointestinal carcinogenesis (hMLH1, HPP1, p14ARF, p16INK4A, APC) was determined in a series of SB‐AC. Paraffin‐embedded tumor samples from 56 patients with SB‐AC who underwent surgical resection between January 1985 and December 2003 were investigated for hypermethylation by means of methylation‐specific real‐time PCR, and compared with our findings in a previously investigated series of 50 gastric adenocarcinomas. In comparison with adenocarcinomas of the stomach, SB‐AC revealed a significantly higher rate of hypermethylation of HPP1 (86% versus 54%, p = 0.0003), p16INK4A (32% versus 10%, p = 0.0006), and a significantly lower rate of hypermethylation of APC (48% versus 84%, p = 0.0001). Hypermethylation of hMLH1 and p14ARF was present in 23% and 9% of SB‐AC, respectively. Locally advanced tumor categories (pT3/4) showed a higher rate of hypermethylation of HPP1 (90%) than did early tumor categories (pT1/2 categories, 40%; p = 0.0036). This was also reflected by the correlation between the HPP1 hypermethylation and high UICC stage (p = 0.02). No correlation was found between hypermethylation and other clinicopathologic parameters such as age, tumor grade and nodal status. Our findings suggest that hypermethylation of hMLH1, HPP1, p16INK4A and APC is frequent in primary adenocarcinomas of the small bowel. The differences in the hypermethylation spectrum of small bowel and stomach cancer indicate significant epigenetic differences between these tumors.

Polymorphisms in methionine synthase (A2756G) and cystathionine β-synthase (844ins68) and susceptibility to carcinomas of the upper gastrointestinal tract

PurposeFolate deficiency is considered to increase the risk for the development of malignant tumors such as prostate and colorectal cancer. Methionine synthase (MTR) and cystathionine ß-synthase (CBS) are enzymes that play a central role in folate metabolism, thereby affecting DNA methylation and synthesis. A single A→G substitution at nucleotide 2756 of the MTR and a 68xa0bp CBS insertion polymorphism in exon 8 have been associated with decreased enzyme activity. The purpose of this study is to compare the association of the MTR A2756G polymorphism and CBS insertion polymorphism with susceptibility to carcinomas of the upper gastrointestinal tract.MethodsUsing the restriction fragment length polymorphism (RFLP)-PCR, the prevalence of MTR A2756G and CBS insertion polymorphism was determined in healthy controls (nxa0=xa0257) and in patients with esophageal squamous cell carcinoma (ESCC) (nxa0=xa0263), Barrett’s esophagus-associated esophageal adenocarcinoma (BC) (nxa0=xa089), cardiac carcinoma (CC) (nxa0=xa0144), or gastric carcinoma (GC) (nxa0=xa0221) from German Caucasian subjects.ResultsNo significant difference in MTR A2756G genotype distribution was observed between controls (A/A 66.9%, A/G 29.8%, G/G 3.3%) and patients with ESCC (A/A 61.7%, A/G 36.3%, G/G 2.1%), BC (A/A 69.2%, A/G 26.9%, G/G 3.9%), CC (A/A 51.8%, A/G 44.6%, G/G 3.6%), or GC (A/A 73.4%, A/G 20.9%, G/G 5.7%). Similarly, the CBS genotype (I: allele with 68xa0bp insertion; N: allele without insertion) distribution among German patients with ESCC (N/N 86.8%, I/N 13.2%), BC (N/N 90.2%, I/N 9.8%), CC (N/N 90.1%, I/N 9.9%) or GC (N/N 91.3%, I/N 8.7%) was not different from healthy controls (N/N 90.4%, I/N 9.6%). The gene allele constellation I/I was not present.ConclusionsThe current study suggests that there is no association between MTR A2756G polymorphism and the CBS (844ins68) insertion polymorphism and cancer of the upper gastrointestinal tract.