Molecular differences between arterial and venous grafts in the first year after coronary artery bypass grafting.

Despite commonly used for coronary artery bypass surgery, saphenous vein (SV) grafts have significantly lower patency rates in comparison to internal thoracic artery (ITA) grafts, which might be due to the structural characteristics of the vessel wall but also due to differences in oxidative stress adaptation and molecular signaling and regulation. This human post mortem study included a total of 150 human bypass grafts (75 SV grafts and 75 ITA grafts) obtained from 60 patients divided into five groups due to the time period of implantation: group 1: baseline group without grafting; group 2: 1 day; group 3: > 1 day-1 week; group 4: > 1 week-1 month; group 5: > 1 month-1 year. Pieces of 3 mm length were fixed with formaldehyde, dehydrated, wax embedded, cut into sections of 3 µm thickness, and histologically and immunohistochemically examined. Over the whole time period, we observed a lower neointima formation and a better preserved media in ITA grafts with a higher percentage of TNF-α, PDGFR-α, and VEGF-A in nearly all vessel wall layers, a higher amount of MMP-7, MMP-9, EGFR, and bFGF positive cells in SV grafts and a timely different peak not only between ITA and SV grafts but also within the various vessel wall layers of both graft types. Since most of the examined growth factors, growth factor receptors and cytokines are regulated by MAPKs, our results suggest an activation of different pathways in both vessel graft types immediately after bypass grafting.

Major Pathologic Response after Induction Therapy Has a Long-Term Impact on Survival and Tumor Recurrence in Stage IIIA/B Locally Advanced NSCLC.

BACKGROUND: Major pathologic response (MPR) determines favorable outcome in locally advanced non-small cell lung cancer after induction therapy (IT) followed by lung resection. The aim of this retrospective study was to identify the prognostic relevance of MPR in long-term interval. METHODS: In 55 patients, the survival rate according to MPR and non-MPR was estimated by Kaplan-Meier method and compared using log-rank, Breslow, and Tarone-Ware tests. RESULTS: The IT included chemoradiation with 50.4 Gy (range: 45-56.4 Gy) combined with platinum-based chemotherapy in 52 patients (94.5%) and platinum-based chemotherapy in 3 patients (5.5%). Perioperative morbidity and 30-day mortality were 36 and 3.6%, respectively. The estimated 5-year postoperative and progressive-free survivals were statistically significantly improved in MPR versus non-MPR with 53.5 versus 18% and 49.4 versus 18.5%, respectively. According to the log-rank, Breslow, and Tarone-Ware tests, the MPR demonstrates prognostic significance in early, long-term, and whole postoperative interval. CONCLUSION: MPR is associated with a robust correlation to long-term postoperative and recurrence-free survival improvement, and can potentially simplify the multidisciplinary debate and allow further stratification of adjuvant treatment in multimodality therapy.

Confocal Laser Endomicroscopy for Diagnosing Malignant Pleural Effusions.

BACKGROUND Confocal laser endomicroscopy (CLE) enables "in vivo" microscopic tissue diagnosis based on tissue reflectance or tissue fluorescence upon application of fluorescence agents. The aim of the present study was to evaluate CLE as a new diagnostic approach for differentiation between malignant versus non-malignant pleural effusions. MATERIAL AND METHODS In 100 patients with pleural effusions, thoracentesis was performed. Cresyl violet and acriflavine were used as contrast agents for probe-based CLE of effusions. CLE video sequences were assessed by 4 independent investigators (2 experienced in this technique, 2 with only basic knowledge). In addition, all CLE samples were evaluated by an expert pathologist (p). Results were compared with conventional cytology of effusions and histology of cell blocks. RESULTS CLE reliably permitted identification of malignant cells in pleural effusions. Sensitivity for detection of malignant effusions was 87% (p: 87%) and 81% (p: 72%) for acriflavine and cresyl violet, respectively. With regard to specificity, acriflavine and cresyl violet yielded a mean value of 99% (p: 100%) and 92% (p: 100%). CONCLUSIONS In this pilot study, CLE permitted simple and rapid detection of malignant pleural effusions. Larger prospective studies are warranted to corroborate our findings.

Pathologic complete response after induction therapy-the role of surgery in stage IIIA/B locally advanced non-small cell lung cancer.

BACKGROUND: Pathologic complete response (pCR) is dominant prognostic factor determining favorable outcome in locally advanced non-small cell lung cancer (NSCLC) after induction therapy (IT). There is no non-operative diagnostics that adequately estimates the pCR. Aim of this retrospective study was to assess the correlation between clinical and pathological factors in patients with pCR. METHODS: Twenty-five patients with pCR after curative lung resection following IT were assessed using univariate and multivariate Cox regression and descriptive analysis. The survival rate was estimated by Kaplan-Meier method. RESULTS: The IT included chemoradiation with median doses of 50.4 Gy (range, 45-59.4 Gy) combined with platinum-based chemotherapy in 23 patients (92%) and induction platinum-based chemotherapy in 2 patients (8%). Clinical tumor stage before IT was IIIA in 21, IIIB in 4 patients. Mean interval between IT and surgery was 8.1±3.0 weeks. Perioperative morbidity and 30-day mortality was 32% and 4%, respectively. There was no significant correlation of pCR and different clinical and pathological factors. The estimated 5-year long-term survival (LTS) and progressive-free survival (PFS) was 57% and 54%, respectively. The median LTS and PFS was not reached. CONCLUSIONS: pCR in patients with locally advanced NSCLC following IT is an independent prognostic factor, without correlation with pathological and clinical factors. Non-operative accurate assessment of pCR is currently impossible. Surgical resection enables secure identification of pCR and might improve the patient stratification for additive therapy.

Gene therapy with antisense oligonucleotides silencing c-myc reduces neointima formation and vessel wall thickness in a mouse model of vein graft disease.

Gene therapy for avoiding intimal hyperplasia of vein grafts after coronary artery bypass grafting is still discussed controversially. A promising application of gene therapy in vein grafts is the use of antisense oligonucleotides to block the expression of genes encoding cell cycle regulatory proteins in vascular smooth muscle cells. C-myc, either directly or by regulating the expression of other proteins, controls cell proliferation, apoptosis and cell survival, tissue remodeling, angiogenesis, cell metabolism, production of inflammatory and anti-inflammatory cytokines, and also participates in cell transformation. Forty C57BL/6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a previously described cuff technique. Twenty mice received periadventitial administration of antisense oligonucleotides directed against c-myc (treatment group), the other twenty mice received no treatment (control group). All vein grafts were harvested two weeks after surgery, dehydrated, wax embedded, cut into slides of 2 µm thickness, stained and histologically and immunohistochemically examined under light microscope. In our study, we could show the promising effects of antisense oligonucleotide treatment in a mouse model of vein graft disease including the significant reduction of neointimal, media and total vessel wall thickness with a significantly lower percentage of SMA positive cells, elastic fibres and acid mucopolysaccharides in the neointima and media, a decreased vascularization, and a lower expression of PDGFR ß, MMP-9 and VEGF-A positive cells throughout the whole vein graft wall.

Comparison of PD-L1 mRNA Expression Measured with the CheckPoint Typer® Assay with PD-L1 Protein Expression Assessed with Immunohistochemistry in Non-small Cell Lung Cancer.

BACKGROUND: Immunohistochemical (IHC) assessment of programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) has become important since the development of anti-PD-1/-PD-L1 directed drugs. Various PD-L1 antibodies and cut-offs have been used in different trials to predict response to these drugs, thus comparison of those studies is difficult. We compared PD-L1 mRNA expression measured by RT-qPCR with PD-L1 protein expression evaluated by IHC. Moreover, we investigated the impact of different tumour tissue acquisition methods on the reliability of PD-L1 measurement techniques. MATERIALS AND METHODS: NSCLC cases (N=22), including n=9 mediastinal lymph node biopsies acquired by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and n=5 metastases, were evaluated prospectively for PD-L1 protein on tumor cells (TC) and immune cells (IC) using E1L3N and 28-8 antibodies and PD-L1 mRNA using the CheckPoint TYPER® assay. RESULTS: In primary NSCLC tissues, agreement between PD-L1 mRNA and TC staining using the 28-8 antibody was excellent (ĸ=0.85, p=0.0002). Comparing both PD-L1 antibodies against each other showed a kappa value of 0.58 (p=0.0106). In EBUS-TBNA, PD-L1 mRNA correlated perfectly with the 28-8 antibody (ĸ=1.0, p=0.0023). PD-L1 mRNA levels significantly differed when comparing 28-8 TC staining of tumours >49% with 1-49% and 0% (p=0.0040; p=0.0081, respectively). In metastatic lesions, differences between PD-L1 mRNA and IHC became apparent (ĸ=0.2, p=0.2525). CONCLUSION: Testing of PD-L1 mRNA and 28-8 IHC showed an excellent agreement in NSCLC samples including mediastinal lymph node biopsies. Since PD-L1 expression in >50% TC detected by 28-8 IHC can be reliably detected by RT-qPCR, quantitative PD-L1 mRNA determination should be considered as an alternative to IHC as there is no interobserver variability in RNA results.

Stem cell therapy with skeletal myoblasts accelerates neointima formation in a mouse model of vein graft disease.

Although still a matter of controversial discussion, skeletal myoblasts are one of the options for stem cell transplantation improving cardiac function after myocardial infarction, exhibiting several advantages including the availability, the ability of self-renewal and differentiation, and the lack of ethical and immunological problems. The aim of this study was to investigate the impact of stem cell therapy with skeletal myoblasts on experimental venous bypass grafts in a mouse model of vein graft disease. Forty C57BL/6J mice underwent bypass grafting interposing a venous bypass graft of the donor mouse into the carotid artery of the recipient mouse. Twenty mice received periadventitially treatment with 1 million fluorescence labeled skeletal myoblasts suspended in culture medium (treatment group), the other twenty mice received only culture medium without myoblasts (control group). Two weeks after bypass surgery, the vein grafts of all 40 mice were harvested, stained and histologically investigated under light and immunofluorescence microscope. Against our expectations, skeletal myoblasts stayed in place and were still located in the adventitia after bypass grafting. Additionally, vein grafts of the myoblast group revealed a 2fold increased neoneointima formation, a decreased media thickness, a slightly increased neovascularization, a higher percentage of reendothelialization and also a slightly higher percentage of PDGFR ɑ, PDGFR ß, MMP-7 and MMP-9 positive cells, suggesting a paracrine mechanism responsible for accelerated neointima formation. In conclusion, the results of our study do not support the use of skeletal myoblast for the treatment of vein graft disease after coronary artery bypass surgery.

FAM96A is a novel pro-apoptotic tumor suppressor in gastrointestinal stromal tumors.

The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), "fibroblast-like cells" (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription-polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SCs stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.

The activating protein 1 transcription factor basic leucine zipper transcription factor, ATF-like (BATF), regulates lymphocyte- and mast cell-driven immune responses in the setting of allergic asthma.

BACKGROUND: Mice without the basic leucine zipper transcription factor, ATF-like (BATF) gene (Batf(-/-)) lack TH17 and follicular helper T cells, which demonstrates that Batf is a transcription factor important for T- and B-cell differentiation. OBJECTIVE: In this study we examined whether BATF expression would influence allergic asthma. METHODS: In a cohort of preschool control children and children with asthma, we analyzed BATF mRNA expression using real-time PCR in PBMCs. In a murine model of allergic asthma, we analyzed differences in this allergic disease between wild-type, Batf transgenic, and Batf(-/-) mice. RESULTS: In the absence of corticosteroid treatment, children with recurrent asthma have a significant increase in BATF mRNA expression in their PBMCs. Batf(-/-) mice display a significant reduction in the pathophysiologic responses seen in asthmatic wild-type littermates. Moreover, we discovered a decrease in IL-3 production and IL-3-dependent mast cell development in Batf(-/-) mice. By contrast, IFN-γ was induced in lung CD4(+) and CD8(+) T cells. Intranasal delivery of anti-IFN-γ antibodies induced airway hyperresponsiveness and inflammation in wild-type but not in Batf(-/-) mice. Transgenic overexpression of Batf under the control of the CD2 promoter/enhancer augmented lung inflammation and IgE levels in the setting of experimental asthma. CONCLUSION: BATF is increased in non-steroid-treated asthmatic children. Targeting BATF expression resulted in amelioration of the pathophysiologic responses seen in children with allergic asthma, and BATF has emerged as a novel target for antiasthma interventions.

A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes.

Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.

Prognostic and predictive value of estrogen receptor 1 expression in completely resected non-small cell lung cancer.

Adjuvant chemotherapy (ACT) leads to a modest improvement in survival among patients with completely resected non-small cell lung cancer (NSCLC) but molecular predictors are still rare. Publicly available gene microarray, clinical and follow-up data from two different studies on early-stage NSCLC were used to determine the expression of estrogen receptor 1 (ESR1). Expression values were calculated against clinical and survival data in a training set (n = 138) and a test set (subpopulation from the adjuvant JBR.10 study) allowing the determination of the prognostic effect of ESR1 in the observational arm as well as the predictive effect of ESR1 regarding ACT. Data were well balanced in terms of ESR1 expression. ESR1 high expression was of significant positive prognostic value in the training set and this could be confirmed in the test set cohort (hazard ratio for overall survival 0.248, 95% confidence interval: 0.088-0.701; p = 0.008). Additionally, ESR1 low tumors showed a benefit from ACT in terms of 5-year survival (33.3% observation arm and 77.8% ACT arm; p = 0.003), whereas patients with ESR1 high tumors did not have any benefit from ACT (test of interaction p = 0.024). ESR1 is an independent positive prognostic factor for survival in early-stage NSCLC patients. Patients with ESR1 high tumors did not benefit from ACT.

Phosphorothioate-modified CpG oligodeoxynucleotides mimic autoantigens and reveal a potential role for Toll-like receptor 9 in receptor revision.

Re-expression of recombinase activating genes (RAG) in mature B cells may support autoreactivity by enabling revision of the B-cell receptor (BCR). Recent reports suggest that administration of Toll-like receptor 9 (TLR9) -stimulating CpG oligodeoxynucleotides (ODN) could trigger the manifestation of autoimmune disease and that TLR are involved in the selection processes eliminating autoreactive BCR. The mechanisms involved remain to be elucidated. This prompted us to ask, whether TLR9 could be involved in receptor revision. We found that phosphorothioate-modified CpG ODN (CpG(PTO)) induced expression of Ku70 and re-expression of RAG-1 in human peripheral blood B lymphocytes and Igλ expression in sorted Igκ(+) B cells. Further results revealed unselective binding specificity of CpG(PTO) -induced immunoglobulin and suggested that CpG(PTO) engage and/or mimic IgM receptor signalling, an important prerequisite for the initialization of receptor editing or revision. Altogether, our data describe a potential role for TLR9 in receptor revision and suggest that CpG(PTO) could mimic chromatin-bearing autoantigens by simultaneously engaging the BCR and TLR9 on IgM(+) B cells.

Heat shock protein 90-sheltered overexpression of insulin-like growth factor 1 receptor contributes to malignancy of thymic epithelial tumors.

PURPOSE: The underlying molecular mechanisms of thymic epithelial malignancies (TEMs) are poorly understood. Consequently, there is a lack of efficacious targeted therapies and patient prognosis remains dismal, particularly for advanced TEMs. We sought to investigate protumorigenic mechanism relevant to this understudied cancer. EXPERIMENTAL DESIGN: Recently established cell lines derived from thymic epithelial tumors were used as a model system. The antitumor activity of specific heat shock protein 90 (Hsp90) inhibitors was investigated by an analysis of cell viability, cell cycle, and apoptosis using MTT-assays and flow cytometry. Western blotting was used to investigate the altered expression of Hsp90 clients. Pharmacological inhibitors against select Hsp90 clients, as well as RNAi, were employed to test the relevance of each client independently. Tissue microarray analysis was performed to match the in vitro findings with observations obtained from patient-derived samples. RESULTS: Hsp90 inhibition significantly reduces cell viability of thymic carcinoma cells, induces cell cycle arrest and apoptosis, and blocks invasiveness. Hsp90 inhibition triggers the degradation of multiple oncogenic clients, for example insulin-like growth factor 1 receptor (IGF-1R), CDK4, and the inactivation of PI3K/Akt and RAF/Erk signaling. Mechanistically, the IGF/IGF-1R-signaling axis contributes to the establishment of the antiapoptotic phenotype of thymic cancer cells. Finally, IGF-1R is overexpressed in advanced TEMs. CONCLUSIONS: We have unraveled a novel protumorigenic mechanism in TEMs, namely Hsp90-capacitated overexpression of IGF-1R, which confers apoptosis evasion in malignant thymic epithelial cells. Our data indicate that Hsp90 inhibition, which simultaneously blocks multiple cancer hallmarks, represents a therapeutic strategy in TEMs that may merit evaluation in clinical trials.

A typical thymic carcinoid tumour within a thymolipoma: report of a case and review of combined tumours of the thymus.

Thymolipomas are rare tumours located in the anterior mediastinum. Sometimes these tumours may be combined with thymomas or lymphomas. We present a unique case of a thymic carcinoid arising within a thymolipoma. A 68-year-old patient presented with chronic chest and neck pain, which was initially thought to be caused by coronary artery disease. A chest x-ray, exercise tolerance test and coronary angiography were unremarkable. The following CT scan of the neck and chest showed a small tumour in the anterior mediastinum. A robotic-assisted thymectomy was performed and histological examination revealed a neuroendocrine tumour of the thymus within a thymolipoma. The patient was discharged 3 days after surgery in good general condition.

CASTLE tumour of the neck: a rare location of a malignant tumour of the thymus.

We present the case of a 62-year-old woman who consulted her physician in December 2005, suffering from a mass at the left lower anterior neck with rapid enlargement. Intraoperative frozen section was highly suspicious of a CASTLE tumour (carcinomas showing thymus-like differentiation). Finally, immunohistochemical investigation revealing positivity for CK5/6, c-kit (CD117) and CD5 as well as negativity for thyroglobulin, calcitonin, vimentin and TTF-1 confirmed the diagnosis. Due to lymph node metastases, radiochemotherapy was performed. Fifteen months after the initial diagnosis disseminated pulmonary metastases were found and treated with cisplatin based chemotherapy, which led to a stabilisation of the disease. In June 2008, computed tomography showed progress of the pulmonary metastases, making further chemotherapeutical treatment necessary. Although treatment was changed in October 2008, the staging evaluation in January 2009 revealed further progress of the metastatic disease. Currently, the patient is still alive, but receives no medical treatment at the moment.

CTCF gene mutations in invasive ductal breast cancer.

The CTCF gene encodes for a transcriptional repressor of the c-myc oncogene and has previously been mapped to one of the smallest regions of overlapping interstitial deletions on chromosome 16q22.1 in invasive breast cancer. This chromosomal region is frequently deleted in both invasive lobular and ductal breast carcinomas. However, no target genes have been identified in invasive ductal breast cancer. We examined CTCF protein expression in 18 invasive ductal breast carcinomas using immunohistochemistry. Additionally, loss of heterozygosity (LOH) at chromosome 16q22.1 was determined and the complete cDNA sequence of CTCF was screened for mutations. Immunohistochemically, 17 tumours showed a moderate to strong nuclear staining for CTCF, one case was completely negative. Sequencing analysis revealed a tumour-specific truncating 14 bp insertion with silencing of the wild type allele in this case. In one further case we found a missense mutation that was shown not to be tumour-specific. Concordant with the antiproliferative effects of the CTCF protein in vivo, CTCF may be involved in tumour initiation or proliferation in individual cases of invasive ductal breast carcinoma.

The impact of comorbidity on the overall survival and the cause of death in patients after colorectal cancer resection.

BACKGROUND: Retrospective investigation to identify associations between certain patient characteristics and survival in 531 patients with resected colorectal cancer (CRC). Special reference is given to a standardized comorbidity. METHODS: To compare different levels of exposure we determined hazard ratios (HR) in Cox proportional hazards models for survival times and odds ratios (OR) in logistic regression models. RESULTS: Overall survival was associated with tumor stages (III+IV vs. I+II; HR 7.48), tumor differentiation (low vs. high; HR 1.84), blood transfusions (>2 vs. < or =2; HR 1.88), and comorbidity (Charlson Comorbidity Index >2 vs. < or =2; HR 1.77). Low tumor stage (I+II vs. III+IV; OR 11.1), elevated Charlson Comorbidity Index (>2 vs. < or =2; OR 3.83), and longer ICU stay (>2 days vs. < or =2 days; OR 3.40) more frequently lead to non-cancer-related death than to cancer-related death. CONCLUSION: Standardized comorbidity should be considered as a factor in survival studies of CRC.