Viacheslav Kalinin

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

BackgroundPancreatic 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.MethodsThe 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.ResultsPaCa 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.ConclusionThe 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.

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, χ2 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.

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

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.

Microsatellite GTn‐repeat polymorphism in the promoter of heme oxygenase‐1 gene is an independent predictor of tumor recurrence in male oral squamous cell carcinoma patients

BACKGROUND Transcriptional activity of the heme oxygenase-1 gene (HMOX-1) is modulated by a GTn-repeat promoter polymorphism. The long GTn-repeat allele has been previously reported to be associated with increased risk of oral squamous cell carcinoma (OSCC) in male areca chewer and short GTn-repeat allele has been proposed to have protective properties in OSCC patients. The aim of the present study was to correlate the GTn-repeat genotypes with clinicopathological characteristics along with clinical outcome of non-areca chewer OSCC patients. METHODS DNA of 99 patients that underwent complete surgical resection of OSCC was analyzed for GTn-repeat polymorphism in the HMOX-1 promoter by polymerase chain reaction, capillary electrophoresis and DNA sequencing. RESULTS Seven SS (7.1%), 51 SL (51.5%) and 41 LL (41.4%) genotypes were found. In a total of 14 (14.1%) patients, tumor recurrence (TR) was observed. There was no TR in the SS allele carriers. In SL carriers three and in LL 11 TR occurred (P = 0.009, chi-squared test). Mean relapse-free survival was 109.2 months in SL allele carriers compared with 72.3 months in LL allele carriers (P = 0.01, log-rank test). Multivariate Cox regression modeling identified GTn-repeat genotype as an independent prognostic factor (P = 0.03; relative risk (RR) 4.1; 95% CI 1.1-14.6). CONCLUSION Presence of S allele was associated with a lower TR rate and better relapse-free survival in OSCC patients. HMOX-1 promoter polymorphism might be considered as a potential prognostic marker in OSCC patients.

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.

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

BackgroundGenetic 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).MethodsGenomic 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.ResultsT-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.ConclusionDetermination 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.

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.

Single nucleotide polymorphism in esophageal cancer related gene 1: an analysis in resected oral squamous cell carcinoma patients

Esophageal cancer related gene 1 (ECRG1) is a novel candidate tumor suppressor gene in human esophageal squamous cells. Overexpression of ECRG1 protein inhibits tumor cell proliferation. Genetic polymorphisms in coding sequences of the gene may cause functional alterations of the gene product and be associated with higher cancer risk and disease phenotypes. A single nucleotide polymorphism (SNP) (Arg290Gln) found in the coding region of ECRG1 might play a role in susceptibility to esophageal squamous cell carcinoma. This study examined SNPs in ECRG1 in a similar tumor type (oral squamous cell carcinoma; OSCC) and investigated the relationship between SNPs in ECRG1 and the clinical outcome of patients with OSCC. DNA samples of 137 OSCC patients were analyzed for SNP genotypes Arg/Arg, Arg/Gln and Gln/Gln in the coding region (exon 8) of ECRG1. SNP genotypes Arg/Arg were found in 70 (51%), Arg/Gln in 60 (43%) and Gln/Gln in 7 (5%) patients. There was no significant association between genotypes and survival (p=0.77) or relapse free survival (p=0.32). The Gln/Gln genotype had the best survival (not significant) probably due to rare cases of SNP Gln/Gln genotype. Genotype Arg/Arg might be a potential negative prognostic marker in OSCC, but more studies with higher patient numbers are required.

Diverse prognostic value of the GTn promoter polymorphism in squamous cell and adeno carcinoma of the oesophagus.

The basal transcription of heme oxygenase‐1 (HO‐1) regulation is dependent upon a GT repeat germ line polymorphism (GTn) in the promoter of the HO‐1 gene. We determined the prognostic value of HO‐1 promoter polymorphism on the natural postoperative course of complete resected oesophageal cancer. Genomic DNA from 297 patients was amplified by polymerase chain reaction and sequenced. The results were correlated with clinicopathological parameters, disseminated tumour cells in bone marrow (DTC) and clinical outcome. Depending on short allele with <25 and long allele with ≥25, GTn repeats three genotypes (SS, SL and LL) were defined. A diverse role of GTn was evident in squamous cell carcinoma (SCC) and adenocarcinoma (AC). In SCC, the SS genotype presented less advanced tumours with lower rate DTC in bone marrow and relapse compared with L‐allele carriers. In contrast, AC patients with the SS genotype displayed a complete opposing tumour characteristic. The disease‐free (DFS) and overall survival (OS) in SCC patients was markedly reduced in LL genotypes (p < 0.001). In AC contrarily the SS genotype patients displayed the worst DFS and OS (p < 0.001). GTn is a strong prognostic factor with diverse prognostic value for recurrence and survival in AC and SCC.

Abstract B1: Heme oxygenase-1 transcription controlling GTn repeat polymorphism has contrary prognostic value in squamous cell carcinoma and adenocarcinoma of the esophagus

Purpose: The regulation of basal transcription of heme oxygenase-1 (HO-1) is dependent upon a GTn repeat polymorphism (GTn) in the promoter of the heme oxygenase-1 gene (HMOX-1). Here, we evaluate the role of GTn in surgically resected esophageal cancer patients without neoadjuvant or adjuvant treatment. Patients and Methods: Genomic DNA was extracted from peripheral blood leucocytes of 297 patients. To determine the number of the GTn repeats DNA was amplified by RT-PCR and sequenced. The results were correlated with clinicopathological parameters, disseminated tumor cells (DTC) and clinical outcome. Results: Three genotypes (SS, SL and LL) were defined based on cut-off points for short allele (“S”) with GTn repeats Conclusion: GTn was identified as an independent prognostic factor with contrary prognostic value for tumor recurrence and death in the two subtypes of esophageal cancer, SCC and AC. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B1