Ulrich H. Frey

The T393C polymorphism of the Gαs gene (GNAS1) is a novel prognostic marker in bladder cancer

The G protein Gαs pathway is linked to proapoptotic signaling in cancer cell lines. To assess the role of the GNAS1 locus encoding Gαs as a genetic factor for disease progression of transitional cell carcinoma (TCC) of the bladder, we genotyped the synonymous T393C polymorphism in 254 patients with TCC (minor allele frequency: 0.43) to examine a potential association between genotypes and disease progression. Using Kaplan-Meier estimates to calculate 5-year probabilities of follow-up, we could show that progression-free survival, metastasis-free survival, and cancer-specific survival was significantly increased in TT genotypes (56%, 84%, 82%) compared with CC genotypes (35%, 53%, 58%). In multivariate Cox proportional hazard analysis, the T393C polymorphism was an independent prognostic factor for clinical outcome. Homozygous CC patients were at highest risk for progression [odds ratio (OR), 1.94; P = 0.020], metastasis (OR, 3.49; P = 0.005), and tumor-related death (OR, 2.49; P = 0.031) compared with TT genotypes. Heterozygous patients had an intermediate risk compatible with a gene-dose effect. Real-time PCR analysis of urothelial tumor tissue as well as adipose and heart tissue revealed that Gαs mRNA expression was highest in TT genotypes, indicating a proapoptotic effect in these genotypes. In conclusion, the GNAS1 T393C status associated with differential Gαs mRNA expression is a novel independent prognostic marker for clinical outcome supporting a functional role of Gαs in bladder cancer progression.

GNAS1 T393C Polymorphism and Survival in Patients with Sporadic Colorectal Cancer

Purpose: Signaling via the G protein Gαs pathway is linked to proapoptotic processes in cancer cell lines. We have recently shown an association between the GNAS1 T393C polymorphism and disease progression in patients with bladder cancer with homozygous TT genotypes displaying increased transcription of Gαs and a more favorable clinical course compared with C-allele carriers. Experimental Design: In the present study, 151 patients with sporadic colorectal cancer were retrospectively genotyped to examine a potential association between T393C genotypes and survival. Moreover, two other single-nucleotide polymorphisms in common haplotype blocks within the gene GNAS1 and their interaction with the T393C polymorphism were investigated. Results: The allele frequency in the patients group was not significantly different from that of healthy blood donors. Kaplan-Meier curves for overall survival (mean follow-up, 43 months) showed that in International Union Against Cancer (UICC) stages I to II, the 5-year survival rate was significantly higher in TT genotypes (87.8%) compared with TC (71.0%) and CC genotypes (50.0%; P = 0.009), whereas no genotype effect could be observed for UICC stages III to IV. In multivariate Cox proportional analysis the T393C polymorphism was an independent prognostic factor for survival. Homozygous CC patients were at highest risk for death (hazard ratio, 12.1; P = 0.006) compared with TT genotypes. Heterozygous patients had an intermediate risk compatible with a gene-dose effect. The two haplotype blocks investigated were not associated with clinical outcome. Conclusions: The results support the role of the T393C polymorphism as a marker for survival in patients with colorectal cancer stages I to II and in the identification of patients who may benefit from adjuvant chemotherapy.

The GNAS1 T393C Polymorphism Predicts Survival in Patients with Clear Cell Renal Cell Carcinoma

Purpose: G proteins mediate signaling from cell surface receptors to specific intracellular proteins. In vitro cancer cell line studies revealed a link between the Gαs protein and proapoptotic processes. We have recently shown that TT genotypes of the GNAS1 T393C polymorphism display increased transcription of Gαs and a more favorable clinical course in bladder and colorectal cancer patients compared both with TC or CC genotypes. Experimental Design: In the present study, 150 patients with clear cell renal cell carcinoma surgically treated by nephrectomy with curative intent were retrospectively genotyped to elucidate a potential association between T393C genotypes and clinical outcome. Results: The C-allele frequency in the renal cell carcinoma patient group was 0.51, which is not significantly different from that of a healthy blood donor group. Kaplan-Meier curves for tumor progression, development of metastasis, and tumor-related death showed a significant association of the T393C polymorphism with outcome (5-year cancer-specific survival rates: TT, 91%; TC, 81%; CC, 69%; P = 0.015). Multivariate Cox proportional analysis of a 10-year follow-up confirmed the T393C polymorphism as an independent prognostic factor in clear cell renal cell carcinoma. Homozygous CC patients were at highest risk for progression (hazard ratio, 2.48; P = 0.009) or tumor-related death (hazard ratio, 3.15; P = 0.018) compared with T-allele carriers. Conclusion: Our results show that besides tumor stage, lymph node status, and tumor grade, the GNAS1 T393C status is a novel independent host factor for disease progression in patients with clear cell renal cell carcinoma and provides further evidence for the T393C polymorphism as a general prognostic tumor marker.

Comparison of thromboelastometry with procalcitonin, interleukin 6, and C-reactive protein as diagnostic tests for severe sepsis in critically ill adults

IntroductionEstablished biomarkers for the diagnosis of sepsis are procalcitonin, interleukin 6, and C-reactive protein. Although sepsis evokes changes of coagulation and fibrinolysis, it is unknown whether thromboelastometry can detect these alterations. We investigated whether thromboelastometry variables are suitable as biomarkers for severe sepsis in critically ill adults.MethodsIn the observational cohort study, blood samples were obtained from patients on the day of diagnosis of severe sepsis (n = 56) and from postoperative patients (n = 52), and clotting time, clot formation time, maximum clot firmness, alpha angle, and lysis index were measured with thromboelastometry. In addition, procalcitonin, interleukin 6, and C-reactive protein levels were determined. For comparison of biomarkers, receiver operating characteristic (ROC) curves were used, and the optimal cut-offs and odds ratios were calculated.ResultsIn comparison with postoperative controls, patients with sepsis showed an increase in lysis index (97% ± 0.3 versus 92 ± 0.5; P < 0.001; mean and SEM) and procalcitonin (2.5 ng/ml ± 0.5 versus 30.6 ± 8.7; P < 0.001). Clot-formation time, alpha angle, maximum clot firmness, as well as interleukin 6 and C-reactive protein concentrations were not different between groups; clotting time was slightly prolonged. ROC analysis demonstrated an area under the curve (AUC) of 0.901 (CI 0.838 - 0.964) for the lysis index, and 0.756 (CI 0.666 - 0.846) for procalcitonin. The calculated cut-off for the lysis index was > 96.5%, resulting in a sensitivity of 84.2%, and a specificity of 94.2%, with an odds ratio of 85.3 (CI 21.7 - 334.5).ConclusionsThe thromboelastometry lysis index proved to be a more reliable biomarker of severe sepsis in critically ill adults than were procalcitonin, interleukin 6, and C-reactive protein. The results also demonstrate that early involvement of the hemostatic system is a common event in severe sepsis.

The GNAS1 T393C Polymorphism Is Associated with Disease Progression and Survival in Chronic Lymphocytic Leukemia

Purpose: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal mature B cells. The G protein Gαs subunit has been linked to proapoptotic processes in cancer cell lines. The TT genotype of the GNAS1 T393C polymorphism is associated with increased Gαs transcript levels and a more favorable clinical course in different solid cancers. Experimental Design: We retrospectively genotyped 144 patients with B-CLL to examine a potential association between T393C genotypes with progression-free survival (time from diagnosis to initiation of chemotherapy) and overall survival. Results: The C-allele frequency in the patient group was 0.57 and not significantly different from that of healthy blood donors. Median progression-free survival was significantly different between genotypes (TT 130 months; TC 100 months; CC 31 months; P = 0.0066). Multivariable analysis showed that besides of ZAP-70 (P = 0.005) and Binet stage (P < 0.001), the T393C polymorphism was an independent prognostic factor for progression-free survival [hazard ratio (HR) CC versus TT 2.7; P = 0.010]. In Binet A stages, ZAP-70–positive patients with CC genotypes had a HR of 4.4 to receive first therapy compared with ZAP-70–negative patients with T-alleles (P = 0.0001). Regarding overall survival, CC genotypes (median overall survival, 197 months) were at highest risk for death compared with T-alleles (median overall survival, 310 months) in both univariate (HR, 4.8; P < 0.0001) and multivariable analysis (HR, 5.6; P = 0.002). Conclusions: Here, we show that the GNAS1 T393C status is a novel independent prognostic marker in patients with B-CLL. These results could help to define patients who could benefit from an early individualized therapy.

Comparison of thrombelastometry with simplified acute physiology score II and sequential organ failure assessment scores for the prediction of 30-day survival: a cohort study.

Disseminated intravascular coagulation contributes to mortality of sepsis. The study was performed to investigate thromboelastometry as a potential predictor of 30-day survival in severe sepsis and to compare thromboelastometry to Simplified Acute Physiology Score II (SAPS II) and Sequential Organ Failure Assessment (SOFA) scores. Ninety-eight patients with severe sepsis were included in the cohort study. Thromboelastometry clotting time, clot formation time (CFT), maximum clot firmness (MCF), and &agr; angle as well as SAPS II and SOFA scores were determined at the day of diagnosis. Thromboelastometry variables differed in survivors and nonsurvivors. Mean CFT was prolonged (276 ± 194 vs. 194 ± 109 s, P = 0.021; mean ± SD), and both MCF (52.7 ± 12.1 mm vs. 57.3 ± 11.5 mm, P = 0.042) and &agr; angle (53.4 ± 12.8 degrees vs. 58.9 ± 11.8 degrees, P = 0.028) were reduced in nonsurvivors. Clotting time and SAPS II and SOFA scores were not different. Thromboelastometry values were classified as normal and pathological, respectively, using the median of the variables as the cutoff. Thromboelastometry values were normal if CFT was less than 185 s, MCF was greater than 55 mm, and &agr; was greater than 57.5 degrees. Thirty-day survival was 85.7% when all thromboelastometry variables were normal, but 58.7% when at least one variable was pathological (P = 0.005). Multivariate analysis revealed that the absence or presence of at least one pathological thromboelastometry variable allows for better prediction of 30-day survival in severe sepsis than the SAPS II and SOFA scores (P = 0.01; odds ratio, 4.1), respectively, emphasizing the importance of the coagulation system in sepsis.

Myocardial protection by remote ischaemic pre‐conditioning is abolished in sulphonylurea‐treated diabetics undergoing coronary revascularisation

Remote ischaemic pre‐conditioning attenuates myocardial injury. Because sulphonylurea drugs interfere with ischaemic and anaesthetic pre‐conditioning, we assessed whether remote ischaemic pre‐conditioning effects are altered in sulphonylurea‐treated diabetics.

Quantification of allele-specific G-protein β3 subunit mRNA transcripts in different human cells and tissues by Pyrosequencing

The G-protein 825T allele is associated with altered drug responses while the underlying mechanism is not fully understood. Differential expression of transcripts from the C and T alleles could contribute to this process. The C825T polymorphism located in exon 10 is in close linkage disequilibrium with the A(−350)G promoter single nucleotide polymorphism (SNP) and the C1429T SNP and could therefore serve as a marker for allele-specific expression resulting from the promoter SNP. However, alternative splicing of exon 10 in 825T allele carriers may result in under-represented mRNA transcripts. We, therefore, established a novel method based on the Pyrosequencing technology to quantify allele-specific transcript expression and quantified the allelic variance of the C1429T polymorphism located in the 3′-untranslated region of GNB3. Validation of the method was performed using linear regression analysis of measured versus expected ratios of DNA mixed at different known concentrations as well as determining allele-specific mRNA expression of the partially imprinted IGF-2 gene. We genotyped the C1429T polymorphism of 83 samples derived from six different human tissues and cell lines and quantified mRNA transcripts from different alleles using heterozygous samples. No significantly different transcript amounts from the two alleles were found. There were also no significantly different transcript amounts associated with different G(−350)A genotypes (P>0.05). As a result, we could show that Pyrosequencing provides a sensitive tool to quantify allele-specific transcript expression. Our data do not support the hypothesis that differential G-protein activity associated with the C825T SNP results from different transcript amounts associated with specific GNB3 genotypes.

1513A/C polymorphism in the P2X7 receptor gene in chronic lymphocytic leukemia: absence of correlation with clinical outcome

Purinergic P2X7 receptors are ligand‐gated cation channels expressed on the cells of the immune and hemopoietic system which have been shown to mediate the ATP‐induced apoptotic death of monocytes, macrophages and lymphocytes. A common single nucleotide polymorphism within the P2X7 gene has been described in exon 13 (1513A/C), the gene products encoding fully active and non‐functional proteins. We genotyped the P2X7 1513A/C polymorphism using DNA from 111 patients with chronic lymphocytic leukemia (CLL) and 97 healthy controls using polymerase chain reaction (PCR) amplification followed by Hha1 restriction analysis. We found no significant difference in allele frequency between CLL patients and controls. Time periods from diagnosis to initiation of chemotherapy, a surrogate marker for disease progression, were not different in patients displaying the combined 1513A/C and C/C or the 1513A/A genotype (P = 0.97). Similar results were observed in a subgroup analysis of prognostically more favorable CD38‐negative and ZAP‐70‐negative CLL patients. In conclusion, our data do not support a role of the P2X7 genotype as a prognostic marker in B‐cell CLL.

Characterization of the GNAQ promoter and association of increased Gq expression with cardiac hypertrophy in humans

AIMS Transgenic mice with cardiac overexpression of Gq develop cardiac hypertrophy, apoptosis, and heart failure. Similar mechanisms may contribute to human left ventricular hypertrophy (LVH). However, mechanisms regulating transcription of the human GNAQ gene encoding the Gq protein are unknown and single-nucleotide polymorphisms have not been reported. METHODS AND RESULTS We delineated essential elements for transcription in the human GNAQ promoter using reporter assays and showed promoter induction by serum and angiotensin II. Sequencing of the whole promoter revealed a common (minor allele frequency 0.48) dinucleotide polymorphism at position -694/-695, resulting in an exchange of two adjacent nucleotides (TT > GC). The GC allele had increased transcription factor binding and was associated with enhanced transcriptional activation by serum or angiotensin II, resulting in enhanced Gq expression and intracellular signalling. Genotyping a population-based survey (n = 1204) revealed a higher prevalence of LVH in individuals with the GC/GC genotype [odds ratio (OR) 4.07; 95% CI 1.63-10.16; P = 0.003], this effect being more pronounced in women (OR 5.52; P = 0.005). CONCLUSION A novel polymorphism in the Gq promoter region is associated with enhanced promoter activity, Gq expression, intracellular signal transduction, and increased prevalence of LVH, particularly in women.