Jeremias Wohlschlaeger

Clinical, molecular, and genomic changes in response to a left ventricular assist device

The use of left ventricular assist devices in treating patients with end-stage heart failure has increased significantly in recent years, both as a bridge to transplantation and as destination therapy in those who are ineligible for cardiac transplantation. This increase is based largely on the results of several recently completed clinical trials with the new second-generation continuous-flow devices that showed significant improvements in survival, functional capacity, and quality of life. Additional information on the use of the first- and second-generation left ventricular assist devices has come from a recently released report spanning the years 2006 to 2009, from the Interagency Registry for Mechanically Assisted Circulatory Support, a National Heart, Lung, and Blood Institute-sponsored collaboration between the U.S. Food and Drug Administration, the Centers for Medicare and Medicaid Services, and the scientific community. The authors review the latest clinical trials and data from the registry, with tight integration of the landmark molecular, cellular, and genomic research that accompanies the reverse remodeling of the human heart in response to a left ventricular assist device and functional recovery that has been reported in a subset of these patients.

Survivin Determines Cardiac Function by Controlling Total Cardiomyocyte Number

Background— Survivin inhibits apoptosis and regulates cell division in many organs, but its function in the heart is unknown. Methods and Results— We show that cardiac-specific deletion of survivin resulted in premature cardiac death. The underlying cause was a dramatic reduction in total cardiomyocyte numbers as determined by a stereological method for quantification of cells per organ. The resulting increased hemodynamic load per cell led to progressive heart failure as assessed by echocardiography, magnetic resonance imaging, positron emission tomography, and invasive catheterization. The reduction in total cardiomyocyte number in α-myosin heavy chain (MHC)–survivin−/− mice was due to an ≈50% lower mitotic rate without increased apoptosis. This occurred at the expense of DNA accumulation because survivin-deficient cardiomyocytes displayed marked DNA polyploidy indicative of consecutive rounds of DNA replication without cell division. Survivin small interfering RNA knockdown in neonatal rat cardiomyocytes also led to polyploidization and cell cycle arrest without apoptosis. Adenoviral overexpression of survivin in cardiomyocytes inhibited doxorubicin-induced apoptosis, induced DNA synthesis, and promoted cell cycle progression. The phenotype of the αMHC-survivin−/− mice also allowed us to determine the minimum cardiomyocyte number sufficient for normal cardiac function. In human cardiomyopathy, survivin was potently induced in the failing heart and downregulated again after hemodynamic support by a left ventricular assist device. Its expression positively correlated with the mean cardiomyocyte DNA content. Conclusions— We suggest that the ontogenetically determined cardiomyocyte number may be an independent factor in the susceptibility to cardiac diseases. Through its profound impact on both cardiomyocyte replication and apoptosis, survivin may emerge as a promising new target for myocardial regeneration.

High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome

IntroductionFocal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy.MethodsWe analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis.ResultsElevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01).ConclusionsImmunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.

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.

Hemodynamic Support by Left Ventricular Assist Devices Reduces Cardiomyocyte DNA Content in the Failing Human Heart

Background— Whether adult cardiomyocytes have the capacity to regenerate in response to injury and, if so, to what extent are still issues of intense debate. In human heart failure, cardiomyocytes harbor a polyploid genome. A unique opportunity to study the mechanism of polyploidization is provided through the setting of hemodynamic support by left ventricular assist devices. Hence, the cardiomyocyte DNA content, nuclear morphology, and number of nuclei per cell were assessed before and after left ventricular assist device support. Methods and Results— In 23 paired myocardial samples, cardiomyocyte ploidy was investigated by DNA image cytometry, flow cytometry, and in situ hybridization. Nuclear cross-sectional area and perimeters were measured morphometrically, and the binucleated cardiomyocytes were counted. The median of the cardiomyocyte DNA content and the number of polyploid cardiomyocytes both declined significantly from 6.79 c to 4.7 c and 40.2% to 23%, whereas a significant increase in diploid cardiomyocytes from 33.4% to 50.3% and in binucleated cardiomyocytes from 4.5% to 10% after unloading was observed. Conclusions— The decrease in polyploidy and increase in diploidy after left ventricular assist device suggest a numeric increase in diploid cardiomyocytes (eg, through cell cycle progression with completion of mitosis or by increased stem cells). The cardiac regeneration that follows may serve as a morphological correlate of the recovery observed in some patients after unloading.

Significance of Folate Receptor Alpha and Thymidylate Synthase Protein Expression in Patients with Non–Small-Cell Lung Cancer Treated with Pemetrexed

Introduction: Folate receptor alpha (FRA) regulates cellular uptake of folates and antifolates. Information about FRA protein expression in metastatic non–small-cell lung cancer (NSCLC) is limited. We investigated FRA as a biomarker for pemetrexed-based chemotherapy and compared it with thymidylate synthase (TS), the main target of pemetrexed. Methods: Pretreatment tumor specimens from 207 patients with advanced NSCLC were assessed for FRA and TS protein expression by immunohistochemistry using the H-score (range, 0–300) and correlated to patients’ clinicopathological data, radiographic response, progression-free survival (PFS), and overall survival (OS). Results: Low total (cytoplasmic and nuclear) TS protein expression (H-score < 210) was associated with improved PFS (median: 5.6 versus 3.5 months; hazard ratio [HR] = 0.6379, p = 0.0131) and prolonged OS (median: 22.5 versus 11.5 months; HR = 0.5680,p = 0.0107). An association between lower TS levels and response to pemetrexed-based therapy was found—mean H-score 187 ± 5, median 180 for responders versus mean H-score 201 ± 4, median 210, for non-responders, p = 0.0244. High intracellular FRA expression (H-score ≥110) was associated with prolonged OS (28.9 versus 11.7 months, HR = 0.5316, p = 0.0040) and a trend for association with PFS (5.6 versus 4.1 months, HR = 0.7395, p = 0.0801) was noted. Membranous FRA expression was seen in 83% of patients, moreover, high membranous expression (H-score ≥20) was associated with improved PFS (5.6 versus 3.7 months, HR = 0.6445, p = 0.0306) and OS (22.1 versus 11.5 months, HR = 0.5378, p = 0.0131). Conclusions: A large number of NSCLC patients have high expression of FRA and/or a low level of TS expression. Expression levels of FRA and TS were associated with clinical benefit from pemetrexed therapy.

Activation of extracellular regulated kinases (ERK1/2) but not AKT predicts poor prognosis in colorectal carcinoma and is associated with k-ras mutations

Signal transduction and modulation represent central mechanisms in cellular processes such as cell-cycle regulation, oncogenesis, and apoptosis. The aim of this study was to determine the prognostic relevance of two kinases important in the regulation of cell proliferation and apoptosis in 135 colorectal cancer cases: AKT and extracellular regulated kinases (ERK1/2). We investigated the relationship of phospho-ERK1/2 (pERK1/2) and phospho-AKT (pAKT) with associated parameters (EGFR, COX-2, cyclin-D1), proliferative activity (Ki-67), and apoptosis (TUNEL) using immunohistochemistry. Additionally, the k-ras gene was screened for mutations to determine its putative association with ERK1/2 activation. Activation of ERK1/2 but not AKT correlated statistically with the presence of k-ras mutations (P = 0.015). Survival analysis of phospho-ERK1/2 immunoexpression showed a significant correlation with decreased overall survival (OS). The multivariate Cox regression analysis identified pERK1/2 as an independent prognostic parameter (P = 0.005). Activation of ERK1/2 in colorectal cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor. Furthermore, our data suggest that mutations of the k-ras oncogene may induce activation of ERK1/2. We propose immunohistochemical determination of pERK1/2 status as a promising candidate for the identification of high-risk patients who would benefit from new anticancer drugs targeting the ERK pathway.

Hypothermic reconditioning of porcine kidney grafts by short-term preimplantation machine perfusion.

Background. Clinical trial data suggest that continuous hypothermic machine perfusion (HMP) during the entire preservation period reduces the incidence of delayed graft function and improves graft survival. This study evaluates whether short-term MP after cold storage (CS) is also effective. Methods. Kidney function after cold preservation (4°C, 21 hr) and transplantation was studied in an autotransplant model using Landrace pigs (25–30 kg; n=5 per group) with 1 week follow-up. Preservation was performed by conventional CS or HMP with a modified Lifeport Kidney Transporter either continuously during the entire preservation period or only for 2 hr of hypothermic reconditioning (HR) subsequent to conventional CS. Results. HMP and HR similarly improved cortical microcirculation and significantly reduced maximal serum creatinine levels and recovery of creatinine clearance to normal values compared with CS. Fractional excretion of Na+ was unaltered after HMP and HR but significantly increased until postoperative day 5 on CS. On a molecular level, HR reduced innate immunoreactivity (toll-like receptor 4 expression and high mobility group protein B1 [HMGB-1] release) and normalized antiinflammatory tissue expression of von Kruppel-like Factor-2. Conclusion. Short-term reconditioning after CS proves to be as effective as continuous MP during the whole storage time. Because of its logistical convenience, the concept of an a posteriori treatment recommends itself to be evaluated in clinical trials.

Phosphorylation of p70S6 kinase predicts overall survival in patients with clear margin‐resected hepatocellular carcinoma

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitors play a key role in regulating signal transduction by blocking the mTOR pathway and combining anticancer and immunosuppressive properties. This study was undertaken to determine the prevalence and clinicopathological relevance of phospho‐p70S6 (p‐p70S6) kinase in hepatocellular carcinoma (HCC) and to investigate the effects of rapamycin on HCC in vitro.

Different micro-RNA expression profiles distinguish subtypes of neuroendocrine tumors of the lung: results of a profiling study

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides), non-coding, highly conserved single-stranded RNAs with posttranscriptional regulatory features, including the regulation of cell proliferation, differentiation, survival, and apoptosis. They are deregulated in a broad variety of tumors showing characteristic expression patterns and can, thus, be used as a diagnostic tool. In contrast to non-small cell carcinoma of the lung neuroendocrine lung tumors, encompassing typical and atypical carcinoids, small cell lung cancer and large cell neuroendocrine lung cancer, no data about deregulation of tumor entity-specific miRNAs are available to date. miRNA expression differences might give useful information about the biological characteristics of these tumors, as well as serve as helpful markers.In 12 pulmonary neuroendocrine tumors classified as either typical carcinoid, atypical, large cell neuroendocrine or small cell lung cancer, screening for 763 miRNAs known to be involved in pulmonary cancerogenesis was conducted by performing 384-well TaqMan low-density array real-time qPCR. In the entire cohort, 44 miRNAs were identified, which showed a significantly different miRNA expression. For 12 miRNAs, the difference was highly significant (P<0.01). Eight miRNAs showed a negative (miR-22, miR-29a, miR-29b, miR-29c, miR-367*; miR-504, miR-513C, miR-1200) and four miRNAs a positive (miR-18a, miR-15b*, miR-335*, miR-1201) correlation to the grade of tumor biology. The miRNAs let-7d; miR-19; miR-576-5p; miR-340*; miR-1286 are significantly associated with survival. Members of the miR-29 family seem to be extremely important in this group of tumors. We found a number of miRNAs, which showed a highly significant deregulation in pulmonary neuroendocrine tumors. Moreover, some of these deregulated miRNAs seem to allow discrimination of the various subtypes of pulmonary neuroendocrine tumors. Thus, the analysis of specific sets of miRNAs can be proposed as diagnostic and/or predictive markers in this group of neoplasias.