Heme oxygenase-1 promoter polymorphism is a predictor of disease relapse in pancreatic neuroendocrine tumors.

BACKGROUND: Current available preoperative diagnostic workup is insufficient to differentiate between benign and malignant pancreatic neuroendocrine tumors (PNET). The aim of the present study was to evaluate the potential prognostic role of the promoter GTn repeat polymorphism (GTn) of the heme oxygenase-1 gene in PNET. METHODS: Tumor, metastasis, corresponding healthy tissue, and peripheral blood leukocyte DNA of 46 patients who underwent surgical resection for PNET were analyzed for GTn by PCR, capillary electrophoresis, and DNA-sequencing. The GTn was correlated to clinicopathologic parameters and clinical outcome. RESULTS: GTn was classified into short (<25) and long (≥ 25) alleles and three (SS, SL, and LL) genotypes were defined. There was no difference in GTn genotype among tumor, healthy tissue, metastasis, and peripheral blood leukocyte DNA. The SS and SL genotype displayed significantly more poor differentiated tumors (P = 0.001) and higher tumor recurrence rate (P = 0.0001) compared with LL patients. The LL genotype patients presented predominantly benign PNET (P < 0.001). The LL genotype had the longest disease-free (P < 0.001) and overall survival (P = 0.006). Besides the WHO classification the GTn was identified as a strong predictor of tumor recurrence (hazard ratio 3.1, 95% confidence interval 1.3-7.3) in PNET. CONCLUSION: GTn differentiates between benign and malignant PNET and is a strong predictor of tumor recurrence.

Heme oxygenase-1 germ line GTn promoter polymorphism is an independent prognosticator of tumor recurrence and survival in pancreatic cancer.

BACKGROUND: Heme oxygenase-1 (HO-1) correlates with aggressive tumor behavior and chemotherapy resistance in pancreatic cancer (PC). We evaluated the prognostic value of the basal transcription controlling germ line GTn repeat polymorphism (GTn) in the promoter region of the HO-1 gene in PC. PATIENTS AND METHODS: We determined the GTn in 100 controls and 150 PC patients. DNA was extracted from blood leukocytes and GTn determined by PCR, electrophoresis, and sequencing. Clinicopathological parameters, disease-free, and overall survival (DFS, OS) were correlated with GTn. RESULTS: Three genotypes were defined based on short (S) <25 and long (L) ≥25 GTn repeat alleles. In PC patients, a steadily increasing risk was evident between LL, SL, and SS genotype patients for larger tumor size, presence of lymph node metastasis, poor tumor differentiation and higher recurrence rate (P < 0.001 each). The SS genotype displayed the most aggressive tumor biology. The LL genotype had the best and the SS genotype the worst DFS and OS (P < 0.001 each). The GTn genotype was the strongest prognostic factor for recurrence and survival (P < 0.001 each). CONCLUSION: The GTn repeat polymorphism is a strong prognostic marker for recurrence and survival in PC patients.

Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung.

BACKGROUND: Pancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line. METHODS: The newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-ras, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp-/-/rag2-/- mice. Sensitivity towards chemotherapy was analysed by MTT assay. RESULTS: PaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with multiple numerical and unbalanced structural aberrations. Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3). Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6. Subcutaneous transplantation of PaCa 5061 into pfp-/-/rag2-/- mice resulted in formation of primary tumors and spontaneous lung metastasis. CONCLUSION: The established PaCa 5061 cell line and its injection into pfp-/-/rag2-/- mice can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.

Short tandem repeat polymorphism in exon 4 of esophageal cancer related gene 2 predicts relapse of oral squamous cell carcinoma.

Short tandem repeat (STR) polymorphisms in exon 4 of esophageal cancer related gene 2 (ECRG2) are a prognostic marker for squamous cell carcinoma (SCC) of the esophagus. The aim of the present study was to correlate these STRs with clinical outcome of the similar tumor type oral squamous cell carcinoma (OSCC). DNA of 81 patients that underwent complete surgical resection of OSCC was analyzed for STRs TCA3/TCA3, TCA3/TCA4 and TCA4/TCA4 in exon 4 of ECRG2 by PCR, capillary electrophoresis and DNA sequencing. ECRG2 STR TCA3/TCA3 were found in 45 (56%), TCA3/TCA4 in 33 (41%) and TCA4/TCA4 in 3 (3%) patients. TCA3/TCA3 was significantly associated with reduced relapse-free survival of OSCC, compared with TCA3/TCA4 and TCA4/TCA4 genotypes (P<0.05; log-rank test). TCA3/TCA3 STR was independent prognostic factor determined by multivariate Cox regression analysis (p<0.05). STR polymorphism TCA3/TCA3 in exon 4 of ECRG2 is associated with poor relapse-free survival in surgically completely resected OSCC patients and might be a potential prognostic marker.

Short tandem repeat polymorphisms of exon 4 in Kazal-type gene ECRG2 in pancreatic carcinoma and chronic pancreatitis.

BACKGROUND: Short tandem repeat (STR) polymorphisms in exon 4 of the Kazal-type esophageal cancer related gene (ECRG2) have been reported to be associated with esophageal carcinoma. Kazal-type genes are associated with cancer and pancreatic disease. The aim of the present study was to examine whether ECRG2 STR polymorphisms are associated with pancreatic carcinoma and chronic pancreatitis. MATERIALS AND METHODS: A total of 209 surgically treated patients were analyzed, 92 with pancreatic adenocarcinoma and 117 with chronic pancreatitis. We retrospectively analyzed genomic DNA from peripheral blood leukocytes for STR TCA3/TCA3, TCA3/TCA4 and TCA4/TCA4 in the noncoding region of exon 4 of ECRG2. Associations between STRs and survival of cancer patients were investigated using log-rank test. RESULTS: ECRG2 STR of highest incidence was TCA3/TCA3 [47 (51%) in pancreatic carcinoma; 59 (50%) in pancreatitis patients], followed by the TCA3/TCA4 [37 (40%); 54 (46%)] and TCA4/TCA4 [8 (9%); 4 (4%)] genotypes. No correlation in frequency of STRs comparing chronic pancreatitis and pancreatic cancer was determined using the Chi-squared test (p = 0.23). STR polymorphisms were not significantly associated with reduced tumor-specific or overall survival (p > 0.05; log-rank test). CONCLUSION: The data show that ECRG2 STR polymorphism TCA3/TCA3 in exon 4 is the most prevalent polymorphism found in pancreatic adenocarcinoma and chronic pancreatitis detected in peripheral blood. None of the polymorphisms was associated with poor clinical outcome in pancreatic cancer patients.

Microsatellite DNA alterations of gastrointestinal stromal tumors are predictive for outcome.

PURPOSE: In gastrointestinal stromal tumors (GIST), loss of heterozygosity (LOH) on chromosome 22 and its presumptive biological function has been described. The prognostic value of these and other DNA regions for patient survival remains unclear. EXPERIMENTAL DESIGN: Sixty patients who underwent surgery at our institution between 1992 and 2003 for GIST were histopathologically reclassified by immunohistochemistry and the GIST consensus group criteria 2001. Twenty-one microsatellite loci on chromosomes 3, 9, 13, 17, 18, and 22 were screened for alterations in tumor and healthy DNA. Survival was calculated by Kaplan-Meier plots. RESULTS: Eleven (18.3%) of 60 patients showed metastases at presentation. Thirteen (21.7%) of 60 were high-risk GISTs. LOH was found in all tumors. Twenty-eight (46.7%) of 60 showed more than two LOH in 21 microsatellite marker sites. The frequency of single marker LOH varied from 1.7% to 28.3% among tumors. Frequent LOH was found on chromosomes 22 and 17. The correlation of LOH positivity and the consensus scoring was significant (P=0.005, chi2 test). After a median observation time of 33.3 months (95% confidence interval, 23.9-42.6), overall survival was best for patients with tumors of very low, low, and intermediate risks with only 6 of 36 death events, whereas 14 of 24 high-risk and metastasized patients had died (P<0.001, log-rank test). Likewise, LOH significantly predicted survival (P=0.013) and the effect was particularly detrimental for LOH on chromosome 17 (P<0.001). CONCLUSIONS: LOH is a useful phenomenon for the prognosis of GIST. Rather than chromosome 22 markers, chromosome 17 markers independently predict survival.

Allelic loss of Hox11L1 gene locus predicts outcome of gastrointestinal stromal tumors.

Loss of heterozygosity (LOH) in tumors has been described to have prognostic impact. Hox11L1 gene, located on chromosome 2, has a role in proliferation of neuronal myenteric Cajal cells being the progenitor cells of GISTs. The aim was to examine the frequency and prognostic value of allelic loss of Hox11L1 gene locus in GISTs. Tumor and control DNA of 72 GIST patients was extracted after microdissection from tissue sections. Patients underwent surgery between 1992 and 2003 and were histopathologically reclassified. Microsatellite marker D2S286 on chromosomes 2 near Hox11L1 gene locus was used for detection of LOH by PCR and capillary electrophoresis. Survival was calculated by Kaplan-Meier plots. LOH was found in 7 (10%) of 72 GISTs. Fifty-four (75%) cases did not show LOH. Eleven (15%) were homozygous and consequently non-informative. Survival analysis (n=59) revealed a significantly worse tumor-specific and relapse-free survival for GIST patients with LOH in the tumor by univariate analysis (p<0.05 by log-rank test; median follow-up time 37 months). LOH of Hox11L1 gene locus is a useful parameter for prognosis of GIST. The data propose that Hox11L1 has a role in tumorigenesis in GISTs.

Association of rare SPINK1 gene mutation with another base substitution in chronic pancreatitis patients.

AIM: To verify and expand the known spectrum of serine protease inhibitor Kazal type 1 (SPINK1) gene mutations in chronic pancreatitis. METHODS: DNA extracted from 172 chronic pancreatitis patients was assayed for SPINK1 gene mutations by PCR and DNA sequencing. A control cohort of 90 unrelated healthy individuals was analysed by the same methods for presence of common populational polymorphisms, and frequency of five-loci haplotypes was calculated. Linkages of gene aberrations in single SPINK1 gene copies were analysed by long-distance PCR followed by allele-specific PCR and DNA sequencing. RESULTS: The most frequent SPINK1 gene mutation N34S was found at a frequency of 6%. Furthermore, we detected the heterozygous intervening sequence (IVS) 3 + 2 T > C mutated gene in 2 German patients and 1 Macedonian chronic pancreatitis patient. In all three SPINK1 gene copies an additional rare base substitution was found: 5'untranslated region (UTR)-215 G > A. Polymorphism analysis revealed that all three affected genes carried the same five-loci haplotype. DNA sequencing of another chronic pancreatitis-related gene PRSS1 (cationic trypsinogen) did not reveal any mutations in these 3 patients. CONCLUSION: We found in 3 (2%) of 172 chronic pancreatitis patients an IVS3 + 2 T > C SPINK1 gene mutation and a base substitution 5'UTR-215 G > A in the same gene copy. Most probably the 5'UTR-215 G > A represents a rare polymorphism and not a mutation as previously concluded. Haplotype analysis suggests a common origin of the IVS3 + 2 T > C mutation in these patients.

EGFR intron-1 CA repeat polymorphism is a predictor of relapse and survival in complete resected only surgically treated esophageal cancer.

Basal transcription regulation of the epidermal growth factor receptor is dependent upon a CA simple sequence repeat polymorphism in the intron-1 (CA-SSR-1). Here, we evaluate the role of CA-SSR-1 in complete resected esophageal cancer (EC) patients without neoadjuvant or adjuvant treatment. Genomic DNA was extracted from peripheral blood leukocytes of 241 patients. To determine the number of the CA repeats in the CA-SSR-1, DNA was amplified by polymerase chain reaction and sequenced. The results were correlated with clinicopathological parameters and clinical outcome. Three genotypes were defined based on cut-off points for short allele (S) with ≤18 and long allele (L) >18 CA repeats. A steadily increasing risk was evident between LL, SL, and SS genotype for larger tumor size, presence of lymph node metastases, and disseminated tumor cells in bone marrow as well as tumor recurrence (P < 0.001, chi-square test). A gradual decrease in disease-free and overall survival (OS) was present among LL, SL, and SS patients (P < 0.001, log-rank test). The different outcomes were also evident in nodal status and histological type adjusted subgroup analyses. CA-SSR-1 was identified as the strongest independent prognosticator of tumor recurrence and OS (P < 0.001, Cox regression analysis). CA-SSR-1 is a strong predictive factor for tumor recurrence and overall survival in patients with complete resected esophageal cancer without neoadjuvant or adjuvant therapy.

The GNAS1 T393C single nucleotide polymorphism predicts the natural postoperative course of complete resected esophageal cancer.

BACKGROUND: Genetic variations in cancer patients may serve as important prognostic indicators of clinical outcome. The GNAS1 T393C single nucleotide polymorphism (SNP) diversely correlates with the clinical outcome in cancer. The aim of this study was to evaluate the potential prognostic value of T393C-SNP in complete resected only surgically treated esophageal cancer (EC). METHODS: Genomic DNA was extracted from peripheral blood leucocytes of 190 patients who underwent only complete surgical resection for EC. T393C-SNP was correlated with clinic-pathological parameters, tumor cell dissemination in bone marrow (DTC) and clinical outcome. RESULTS: T-allele carriers had more advanced disease due to presence of lymph node metastasis (P < 0.0001) and DTC (P = 0.01) and higher recurrence rate (P = 0.01) compared to CC genotype. The disease-free (P < 0.001) and overall survival (P < 0.001) was better in CC compared to TT and TC patients. In the multivariate Cox regression disease-stage adjusted analysis the T393C-SNP was identified as a strong independent prognostic factor for recurrence (hazard ratio 1.8, P = 0.01) and survival (hazard ratio 2.5, P < 0.001) in EC patients. CONCLUSION: Determination of T393C-SNP preoperatively will allow allocation of EC patients into different risk profiles which may help to stratify patients eligible for neoadjuvant and or adjuvant therapy.

Polymorphism Arg290Arg in esophageal-cancer-related gene 1 (ECRG1) is a prognostic factor for survival in esophageal cancer.

BACKGROUND: Esophageal cancer is one of the most frequent cancers worldwide and is associated with poor outcome. Besides clinicopathological data, few prognostic molecular markers exist. Esophageal-cancer-related gene1 (ECRG1) short tandem repeats are associated with higher risk for developing esophageal squamous cell carcinoma. The aim of the present study was to evaluate the impact of DNA polymorphisms in the coding region of ECRG1 in esophageal carcinoma. METHODS: Genomic DNA of 107 patients with esophageal cancer that underwent complete surgical resection between 1997 and 2005 was extracted. DNA was analyzed for ECRG1 polymorphisms Arg290Arg, Arg290Gln, and Gln290Gln by PCR and gel electrophoresis. Polymorphisms were correlated with survival data by the Kaplan-Meier method, multivariate Cox regression analysis, and odds ratio were determined. For all variables, cross tables were generated, followed by calculation of the p value by using the chi-square test/Fisher-exact test. RESULTS: Follow-up data of 102 patients with esophageal cancer were available after complete surgical resection for a median follow-up time of 24.3 months. Polymorphism Arg290Arg was found in 47 patients (46.1%), Arg290Gln in 48 patients (47.0%), and Gln290Gln in seven cases (6.9%). Arg290Arg polymorphism was significantly associated with reduced overall survival (p = 0.01) and tumor-free survival (p = 0.01) by the log-rank test. Multivariate regression analysis by Cox revealed polymorphism Arg290Arg to be a significant prognostic factor for survival (p = 0.012). CONCLUSIONS: Polymorphism Arg290Arg in ECRG1 is associated with poor clinical outcome after complete surgical resection in patients with esophageal cancer.

Short tandem repeat polymorphism in exon 4 of esophageal cancer-related gene 2 detected in genomic DNA is a prognostic marker for esophageal cancer.

BACKGROUND: Short tandem repeat (STR) polymorphisms in exon 4 of the esophageal cancer-related gene 2 (ECRG2) are a risk marker for esophageal carcinoma. The aim of the present study was to correlate these STRs with clinical outcome. METHODS: Genomic DNA of 86 patients who underwent complete surgical resection was analyzed for STRs TCA3/TCA3, TCA3/TCA4, and TCA4/TCA4 in exon 4 of ECRG2 by polymerase chain reaction and DNA sequencing. RESULTS: ECRG2 STR TCA3/TCA3 and TCA3/TCA4 were found in 40 (47%) patients, respectively, and TCA4/TCA4 in 6 (7%) cases. TCA3/TCA3 genotype was significantly associated with reduced survival (P < .05, log-rank test). TCA3/TCA3 STR was the strongest prognostic factor determined by multivariate Cox regression analysis. CONCLUSIONS: Genetically fixed STR polymorphism TCA3/TCA3 in exon 4 of ECRG2 is associated with poor clinical outcome in surgically treated esophageal cancer patients and might be a potential prognostic marker. The usefulness of these genetic markers to predict responsiveness toward neoadjuvant treatment of esophageal cancer patients would be of high clinical interest and should be examined in future studies.

10A1-MuLV but not the related amphotropic 4070A MuLV is highly neurovirulent: importance of sequences upstream of the structural Gag coding region.

Recombinants of Moloney murine leukemia virus (MoMuLV) with either an amphotropic (MoAmphoV) or 10A1-tropic host range (Mo10A1V) induce a spongiform neurodegenerative disease in susceptible mice. To test whether MoMuLV -derived sequences are required for induction of neuropathology, mice were inoculated with either the original 10A1 or the amphotropic (4070A) MuLV isolate. Strikingly, wild-type 10A1 was more neurovirulent than Mo10A1V, inducing severe neurological clinical symptoms with a median latency of 99 days in 100% of infected mice. In contrast, no motor disturbances were detected in any of the 4070A-infected mice, although limited central nervous system lesions were observed. A viral determinant conferring high neurovirulence to 10A1 was mapped to a region encompassing the first 676 bases of the viral genome, including the U5 LTR and encoding the amino-terminus of glycosylated Gag (glycoGag). In contrast to studies with the highly neurovirulent CasFr(KP) virus, an inverse correlation between surface expression levels of glycoGag and neurovirulence was not observed; however, this does not rule out a common underlying mechanism regulating virus pathogenicity.