Brigitte Peters

High Prognostic Value of p16INK4 Alterations in Gastrointestinal Stromal Tumors

PURPOSE Gastrointestinal stromal tumors (GISTs) represent a distinctive (but histologically heterogeneous) group of neoplasms, the malignant potential of which is often uncertain. To determine the prognostic relevance of p16INK4 alterations in GISTs, we investigated a larger group of GISTs and correlated the genetic findings with clinicopathological factors and patient survival. MATERIAL AND METHODS We evaluated the methylation status of the promotor by methylation-specific polymerase chain reaction (PCR), the presence of mutations by PCR-SSCP-sequencing, the loss of heterozygosity at the p16INK4 locus (using the c5.1 marker), and the immunohistochemical expression of p16INK4 protein in 43 GISTs in 39 patients. RESULTS p16INK4 alterations were found in 25 of 43 GISTs (58.1%), with benign, borderline, or malignant GISTs showing no differences in the type and frequency of alteration. p16INK4 alterations were correlated with a loss of p16INK4 protein expression (P <.01). Patients who had tumors with p16INK4 alterations had a poorer prognosis than patients with tumors without such alterations (P =.02). There was a high predictive value for p16INK4 alterations only in the group of benign and borderline GISTs (P <.01) with regard to clinical outcome. Univariate Coxs proportional hazard regression analysis revealed a strong correlation between p16INK4 alterations, tumor size, mitotic index, and overall survival (P <.02), whereas multivariate Coxs analysis confirmed only p16INK4 alterations as an independent prognostic factor. CONCLUSION We believe that the evaluation of p16INK4 alteration status is a helpful prognosticator, particularly in the benign and borderline groups of GISTs.

Determinants and consequences of atrial fibrosis in patients undergoing open heart surgery

OBJECTIVE Atrial fibrillation (AF) is a frequent complication following open-heart surgery (OHS). Increased atrial fibrosis may indicate the presence of an intrinsic arrhythmogenic substrate. The aim of this prospective study was to determine whether atrial fibrosis is associated with increased prevalence of AF after OHS. METHODS Right atrial appendages were obtained from 259 patients undergoing OHS; none of the patients had a history of AF. Atrial fibrosis was quantitatively analyzed with point counting. All patients were followed prospectively until hospital discharge. None of the patients received anti-arrhythmic prophylaxis. Post-operative AF was defined as an episode of AF lasting > or = 5 min. RESULTS Quantitation of atrial fibrosis yielded a mean volume percentage of 15.8 +/- 4.3% (V%; range 4.6-32.4%). Patient age was found to correlate with the amount of atrial fibrosis (r = 0.165; P < 0.01) and surface P-wave duration (r = 0.249; P < 0.01). The degree of fibrosis combined with P-wave duration predicted post-operative AF (P < 0.01). Age (> 60 years) and P-wave duration (> or = 100 ms) were independent predictors of post-operative AF (age: relative risk 2.20; P-wave: relative risk 2.69; P < 0.05). The patients were divided into three groups: group 1, V% = 4.6-13.8%; group 2, V% = 13.9-23.1%; group 3, V% = 23.2-32.4%. A total of 52 patients (20.1%) developed AF, which occurred least commonly in group 1 (16.3%) and group 2 (21.2%) as compared with group 3 (33.3%). CONCLUSIONS Atrial fibrosis provides a pathophysiological substrate for post-operative AF. The results support the importance of P-wave duration as a predictor of post-operative AF, and explain the increased prevalence of AF in elderly patients after OHS.

Sex-Specific Pediatric Percentiles for Ventricular Size and Mass as Reference Values for Cardiac MRI Assessment by Steady-State Free-Precession and Phase-Contrast MRI Flow

Background— Cardiac MRI is important in the treatment of children with congenital heart disease, but sufficient normative data are lacking. For ventricular volumes and mass, we sought to deliver reference centiles and to investigate sex effects. Methods and Results— We included 114 healthy children and adolescents, uniformly distributed spanning an age range of 4 to 20 years, as required by the Lambda-Mu-Sigma method to achieve a percentile distribution, thus avoiding arbitrary age categories. Subjects underwent axial volumetry (1.5-T scanner) using standardized 2D steady-state free-precession and flow protocols. Percentiles were computed for age 8 to 20 years (99 subjects) because breath-holds were more consistent in this group. When indexed for body surface area or height, the centile curves of ventricular volumetric parameters showed allometric increase until adolescence, when a plateau was reached, with values comparable to published adult reference data. In contrast, ventricular mass centiles increased without plateau. There was a significant sex difference, with centiles reflecting larger values in boys than in girls ( P <0.05) when ventricular volumes were indexed to body surface area or height but not when indexed to weight (exception: mass). There was excellent agreement of axial and short-axis volumetry and of volumetric and flow-derived stroke volumes. Conclusions— Percentiles for ventricular volumes and mass in healthy children have been established to serve as reference values in pediatric heart disease. Significant sex differences were noted when indexing volumes to body surface area or height. Unisex centiles related to weight may be considered for chamber volumes albeit not for mass. Received February 19, 2009; accepted October 1, 2009. # CLINICAL PERSPECTIVE {#article-title-2}Background—Cardiac MRI is important in the treatment of children with congenital heart disease, but sufficient normative data are lacking. For ventricular volumes and mass, we sought to deliver reference centiles and to investigate sex effects. Methods and Results—We included 114 healthy children and adolescents, uniformly distributed spanning an age range of 4 to 20 years, as required by the Lambda-Mu-Sigma method to achieve a percentile distribution, thus avoiding arbitrary age categories. Subjects underwent axial volumetry (1.5-T scanner) using standardized 2D steady-state free-precession and flow protocols. Percentiles were computed for age 8 to 20 years (99 subjects) because breath-holds were more consistent in this group. When indexed for body surface area or height, the centile curves of ventricular volumetric parameters showed allometric increase until adolescence, when a plateau was reached, with values comparable to published adult reference data. In contrast, ventricular mass centiles increased without plateau. There was a significant sex difference, with centiles reflecting larger values in boys than in girls (P<0.05) when ventricular volumes were indexed to body surface area or height but not when indexed to weight (exception: mass). There was excellent agreement of axial and short-axis volumetry and of volumetric and flow-derived stroke volumes. Conclusions—Percentiles for ventricular volumes and mass in healthy children have been established to serve as reference values in pediatric heart disease. Significant sex differences were noted when indexing volumes to body surface area or height. Unisex centiles related to weight may be considered for chamber volumes albeit not for mass.

Thymoquinone Triggers Inactivation of the Stress Response Pathway Sensor CHEK1 and Contributes to Apoptosis in Colorectal Cancer Cells

There are few reports describing the role of p53-dependent gene repression in apoptotic cell death. To identify such apoptosis-associated p53 target genes, we used the pro-oxidant plant-derived drug thymoquinone and compared p53+/+ and p53-/- colon cancer cells HCT116. The p53 wild-type (wt) status correlated with more pronounced DNA damage and higher apoptosis after thymoquinone treatment. A significant up-regulation of the survival gene CHEK1 was observed in p53-/- cells in response to thymoquinone due to the lack of transcriptional repression of p53. In p53-/- cells, transfection with p53-wt vector and CHEK1 small interfering RNA treatment decreased CHEK1 mRNA and protein levels and restored apoptosis to the levels of the p53+/+ cells. p53-/- cells transplanted to nude mice treated with thymoquinone up-regulated CHEK1 expression and did not undergo apoptosis unlike p53+/+ cells. Immunofluorescence analysis revealed that the apoptosis resistance in p53-/- cells after thymoquinone treatment might be conveyed by shuttling of CHEK1 into the nucleus. We confirmed the in vivo existence of this CHEK1/p53 link in human colorectal cancer, showing that tumors lacking p53 had higher levels of CHEK1, which was accompanied by poorer apoptosis. CHEK1 overexpression was correlated with advanced tumor stages (P = 0.03), proximal tumor localization (P = 0.02), and worse prognosis (1.9-fold risk, univariate Cox regression; Kaplan-Meier, P = 0.04). We suggest that the inhibition of the stress response sensor CHEK1 might contribute to the antineoplastic activity of specific DNA-damaging drugs.

Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models

We have shown that thymoquinone (TQ) is a potent antitumor agent in human colorectal cancer cells. In this study, we evaluated TQs therapeutic potential in two different mice colon cancer models [ 1,2 ‐dimethyl hydrazine (DMH) and xenografts]. We also examined TQ effects on the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor invasion in vitro. Mice were treated with saline, TQ, DMH, or combinations once per week for 30 weeks and the multiplicity, size and distribution of aberrant crypt foci (ACF) and tumors were determined at weeks 10, 20 and 30. TQ injected intraperitoneally (i.p.) significantly reduced the numbers and sizes of ACF at week 10; ACF numbers were reduced by 86%. Tumor multiplicity was reduced at week 20 from 17.8 in the DMH group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long‐term; tumors did not re‐grow even when TQ injection was discontinued for 10 weeks. In a xenograft model of HCT116 colon cancer cells, TQ significantly (P < 0.05) delayed the growth of the tumor cells. Using a matrigel artificial basement membrane invasion assay, we demonstrated that sub‐cyto‐toxic doses of TQ (40μM) decreased C26 cell invasion by 50% and suppressed growth in three‐dimensional spheroids. Apoptotic signs seen morphologically were increased significantly in TQ‐treated spheroids. TUNEL staining of xenografts and immunostaining for caspase 3 cleavage in DMH tumors confirmed increased apoptosis in mouse tumors in response to TQ. These data should encourage further in vivo testing and support the potential use of TQ as a therapeutic agent in human colorectal cancer.

Cigarette smoking induces atrial fibrosis in humans via nicotine

Background: Cigarette smoking (CS) promotes endothelial dysfunction and atherosclerosis in the vascular bed. The impact of smoking on atrial myocardium is not defined in humans. Objective: To determine the effect of CS on the development of interstitial fibrosis in atrial myocardium. Design: Case–control study. Patients: 95 patients (46 smokers and 49 non-smokers) undergoing coronary artery bypass grafting (CABG). Main outcome measures: Amount of atrial fibrosis, collagen I, III and IV expression pattern, and quantitative reverse transcriptase-PCR. Occurrence of postoperative atrial fibrillation (AF). Results: In the study population, patient age correlated significantly with the amount of atrial fibrosis (r = 0.18; p<0.05). Nicotine misuse (pack years) was identified as the only factor related to atrial fibrosis in smokers (r = 0.311; p<0.05). The amount of fibrosis was higher in patients with postoperative AF (22.9% (6.2%) vs. 27.0% (8.2%); p<0.05). To show a causal relationship between CS and atrial fibrosis, atrial tissue slices from non-smokers (n = 8) were cultured in the presence of nicotine base (185 and 740 nmol/l). Nicotine base induced mRNA expression of collagen III (up to 10-fold) in a concentration-dependent manner resembling the immunohistological collagen expression pattern observed in CS. Conclusion: CS contributes to the development of atrial fibrosis via nicotine. Atrial fibrosis by itself has been shown to provide an arrhythmogenic substrate, which may increase the likelihood of the occurrence of atrial arrhythmias, including postoperative AF.

The Number of Lymph Node Metastases in Gastric Cancer Correlates with the Angiotensin I–Converting Enzyme Gene Insertion/Deletion Polymorphism

Purpose: In the present study, we aimed to substantiate the putative significance of angiotensin I–converting enzyme (ACE) on gastric cancer biology by investigating the influence of its gene polymorphism on gastric cancer progression. Experimental Design: Genomic DNA was purified from peripheral blood mononuclear cells or tissue specimens. Amplified ACE gene fragments were separated on agarose gels. D or I alleles were identified by the presence of 190- or 490-bp fragments, respectively. Local expression of ACE was investigated by immunohistochemistry. Results: Twenty-four of 113 (21%) gastric cancer patients had the II, 57 (51%) the ID, and 32 (28%) the DD genotype. The distribution of the ACE genotypes did not differ significantly from the control group of 189 patients without gastric cancer. However, the ACE genotypes correlated with the number of lymph node metastases and the Unio Internationale Contra Cancrum (UICC) tumor stage. Patients with the II genotype had a highly significantly smaller number of lymph node metastases (P < 0.001) and a significantly lower UICC tumor stage (P = 0.01) than patients with the DD genotype. No correlation was found between tumor type, tumor location, local tumor growth, distant metastases, and the ACE genotype. The expression of ACE in gastric cancer was investigated by immunohistochemistry in 100 of 113 patients. ACE was expressed by endothelial cells in all (100%) specimens and by tumor cells in 56 (56%) specimens. Conclusions: Our study shows that ACE is expressed locally in gastric cancer and that the gene polymorphism influences metastatic behavior.

Frequency and spectrum of congenital heart defects among live births in Germany

BackgroundCongenital heart defects (CHD) are the most common single organ malformations in humans. A comprehensive study was initiated within the Competence Network for Congenital Heart Defects to assess population-based nationwide prevalence data for Germany.MethodsStudy register of demographic and medical data of live births with CHD born between July 2006 and June 2007.ResultsSeven thousand two hundred forty-five live births and infants with CHD were registered in Germany by 260 participating institutions (prevalence 107.6 per 10,000 live births). The most common lesions were ventricular septal defect, atrial septal defect and valvular pulmonary stenosis with 52.7, 18.3 and 6.6 per 10,000 live births, respectively. A single ventricle, tetralogy of Fallot and the complete transposition of the great arteries were the most common severe cardiac lesions (3.0, 2.7 and 2.3 per 10,000 live births). Parents reported that prenatal echocardiography had been performed in 53.8% of severe CHD cases with a cardiac defect detected in 77.5% of them.ConclusionThe reported prevalences of severe CHD are within the range of regional and European comparative data. The prenatal detection rate of severe cardiovascular malformations is comparable to contemporary European registries. Postnatal diagnosis of the CHD has been made early in life.

Retention of the Arginine Allele in Codon 72 of the p53 Gene Correlates with Poor Apoptosis in Head and Neck Cancer

The allele constitution at codon 72 of the p53 gene (CGC-arginine or CCC-proline) plays a major role in inducing apoptosis in p53 mutant cells. To verify this, we determined GC-status, p53-mutations, and p53-loss of heterozygosity (LOH) in a group of 54 squamous cell carcinomas of the head and neck (SCCHN). A novel approach, using a one-step real-time PCR analysis with fluorescent hybridization probes, was applied to detect the GC status in tumors and corresponding blood samples. p53 mutations in exons 4 to 8 were detected by PCR-SSCP-sequencing analysis. Apoptosis was determined immunohistochemically using antibodies against Fas, FasL, p53, Bcl2, and terminal deoxy-transferase-mediated dUTP nick end labeling (TUNEL) staining. The overall frequency of p53-LOH in SCCHN was 45.2%. In cases of LOH, there was a preferential loss of the proline allele, which was associated with an up-regulation of Bcl2 and lack of co-expression of Fas/FasL and, thus, impaired apoptosis (P < 0.001). Apoptosis was not observed in tumors carrying the arginine allele. p53 mutations were detected in 29.6% of SCCHN and preferentially occurred at the arginine allele (P = 0.01). p53 alterations were more frequently observed in tumors of the oral cavity, oropharynx and hypopharynx, whereas they were rare in larynx carcinomas (P = 0.07). The p53-LOH status was not found to be significantly correlated with sex, age, TNM-status, or tumor grading. We conclude that apoptosis is correlated with the allelic status of codon 72 in SCCHN. Homozygous proline 72 appears to be an important regulator of apoptosis via the Fas/FasL pathway in SCCHN.

Morphine self-administration in µ-opioid receptor-deficient mice

Abstract. Morphine-induced place preference was demonstrated recently in wild-type mice, whereas this conditioned behaviour was not observed in µ-opioid receptor-deficient mice. In the present study, we investigated locomotor effects of subcutaneously (s.c.) injected morphine as well as intracerebroventricular (i.c.v.) morphine self-administration in µ-opioid receptor-knockout mice.After s.c. morphine injection, locomotor activity significantly increased in wild-type animals. As expected, in the self-administration test the rate of self-administration constantly increased in wild-type mice reflecting reward effects of morphine. This increase was independent of locomotor/motor activity. In contrast, self-administration rates and locomotor/motor activity significantly decreased in the receptor-deficient animals. It was shown that this aversive effect might partly be due to κ-opioid receptor interaction.