The management of papillary microcarcinoma (PMC) of the thyroid is controversial, especially after partial thyroid resection for benign thyroid disease. In order to detect prognostic factors for PMC, we analyzed 116 patients with PMC for encapsulation status and lymph node metastases. Between 10/1992 and 12/2010, 116 patients with PMC have been operated in our department (87 females, 29 males, median age 49 years). Eighty per cent of PMCs were diagnosed postoperatively. Seventy-six patients (66%) received a more extended resection with either thyroidectomy, near total thyroidectomy, or Dunhill operation either primarily or after completion operation, whereas 40 patients (34%) had only partial resection. Fifty patients (43%) received radioiodine (RIA) ablation. Lymph node metastases were found in 21 patients (18%). Univariate analysis showed four risk factors to be significantly associated with the risk of lymph node metastasis (p<0.05): male gender, younger age, age group<50 years and nonencapsulation of the tumor. Multivariate analysis demonstrated statistical significance for gender and tumor capsulation status. The tumor capsulation status also correlated with tumor multifocality. Our data show that the risk of lymph node metastases is significantly higher in partially or nonencapsulated PMC than in encapsulated specimens. We therefore suggest that the WHO classification should be extended to a compulsory notification of the encapsulation status in PMC.

Disseminierte Tumorzellen solider Tumoren in Knochenmark und Lymphknoten wie auch der Nachweis zirkulierender Tumorzellen im Blut der Patienten dienen zunehmend als Surrogatmarker für systemische Therapieansätze. Zur Detektion epithelialer Zellen in den genannten Gewebekompartimenten wird insbesondere das Zelloberflächenprotein EpCAM (CD326) verwendet (u.a. beim FDA-anerkannten CellSearch-System) und mit Edrecolomab und Catumaxomab stehen zudem zwei zugelassene therapeutische Antikörper zur Verfügung. Da bislang keine systematischen Daten zur Prävalenz von disseminierten EpCAM-positiven Tumorzellen vorliegen, haben wir EpCAM-positive Zellen aus dem Knochenmark von Patienten mit unterschiedlichen Karzinomen des Magen-Darm-Traktes isoliert.

Mature cystic teratomas are often found in gonadal sites, but are very rarely located extragonadally, for example, in retroperitoneum, mediastinum, central nervous system, lung, or liver. In the literature, only 10 cases of cystic teratoma originating from the diaphragm have been reported. Here, we report for the first time a metachronous occurrence of a benign mature cystic teratoma in the left diaphragm together with a serotonin-producing neuroendocrine tumor of the ileum. The 51-year-old, female patient received a partial resection of the ileum due to a neuroendocrine tumor (pT3N1M0) 4 years ago. Furthermore, she was operated for a benign cystadenoma of the right ovary 3 years ago. In her past medical history, she had an appendectomy in her childhood and a subtotal thyroidectomy 10 years ago. To our knowledge, this is the first report describing the metachronous occurrence of benign mature cystic teratoma in the diaphragm and a highly differentiated neuroendocrine tumor of the ileum. The possible coincidence of both diseases is discussed.

Patients with ESCC (squamous cell carcinoma of the esophagus) are most commonly diagnosed with locally advanced tumor stages. Early metastatic disease and late diagnosis are common reasons responsible for this tumor's poor clinical outcome. The prognosis of esophageal cancer is very poor because patients usually do not have symptoms in early disease stages. Squamous cell carcinoma of the esophagus frequently complicates patients with multiple co-morbidities and these patients often require interdisciplinary diagnosis and treatment procedures. At present time, neoadjuvant radiation therapy and chemotherapy followed by surgery are regarded as the international standard of care. Meta-analyses have confirmed that this approach provides the patient with better local tumor control and an increased overall survival rate. It is recommended that patients with positive tumor response to neoadjuvant therapy and who are poor surgical candidates should consider definitive radiochemotherapy without surgery as a treatment option. In future, EGFR antibodies may also be administered to patients during therapy to improve the current treatment effectiveness. Positron-emission tomography proves to be an early response-imaging tool used to evaluate the effect of the neoadjuvant therapy and could be used as a predictive factor for the survival rate in ESCC. The percentage proportions of residual tumor cells in the histopathological analyses represent a gold standard for evaluating the response rate to radiochemotherapy. In the future, early response evaluation and molecular biological tests could be important diagnostic tools in influencing the treatment decisions of ESCC patients.

Prior in vivo studies supported the concept that Mallory bodies (MBs) are aggresomes of cytokeratins 8 and 18. However, to test this hypothesis an in vitro model is needed to study the dynamics of MB formation. Such a study is difficult because MBs have never been induced in tissue culture. Therefore, MBs were first induced in vivo in drug-primed mice and then primary cultures of hepatocytes from these mice were studied. Two approaches were utilized: 1. Primary cultures were transfected with plasmids containing the sequence for cytokeratin 18 (CK 18) tagged with green fluorescent protein (GFP). 2. Immunofluorescent staining was used to localize the ubiquitin-proteasome pathway components involved in MB-aggresome complex formation in primary hepatocyte cultures. The cells were double stained with a ubiquitin antibody and one of the following antibodies: CK 8, CK 18, tubulin, mutant ubiquitin (UBB+1), transglutaminase, phosphothreonine, and the 20S and 26S proteasome subunits P25 and Tbp7, respectively. In the first approach, fluorescence was observed in keratin filaments and MBs 48 h after the cells were transfected with the CK 18 GFP plasmid. Nascent cytokeratin 18 was preferentially concentrated in MBs. Less fluorescence was observed in the normal keratin filaments. This indicated that MBs continued to form in vitro. The immunofluorescent staining of the hepatocytes showed that CK 8 and 18, ubiquitin, mutant ubiquitin (UBB+1), P25, Tbp7, phosphothreonine, tubulin, and transglutaminase were all located at the border or the interior of the MB. These results support the concept that MBs are aggresomes of CK 8 and CK18 and are a result of inhibition of the ubiquitin-proteasome pathway of protein degradation possibly caused by UBB+1.

Previous studies on the immunoreactivity of β-catenin, MUC1 and c-met in gastric carcinomas regarding survival and clinico-pathological features led to contradictory results. Therefore, a series of 94 diffuse-type and mixed-type subcardial gastric carcinomas according to the Laurén classification were investigated to elucidate possible correlations with clinico-pathological and prognostic data. An immunohistochemical study was performed to detect the expression of β-catenin, MUC1 and c-met. Loss of membranous/cytoplasmic β-catenin expression in the tumour centre correlated with pT, loss at the invasion front with pTNM stage. MUC1 expression in the tumour centre correlated with lymph node metastasis and pTNM stage. c-Met did not show such associations. In multivariate survival analysis, loss of membranous/ cytoplasmic β-catenin expression as well as a strong MUC1 expression at the tumour invasion front represent independent predictors of a worse prognosis. On the other hand, c-met expression did not exhibit any prognostic value in this study.

Background: The Epithelial-Mesenchymal-Transition (EMT) is a critical event in the metastatic cascade [1]. Loss of E-Cadherin seems to be an important step in initiating this cell program [2]. The aim of the present work is to elucidate the expression profiles of E-Cadherin and Snail in colorectal cancer (CRC), as well as illustrating the influence of Snail, Twist1 and Twist2 in colorectal adenomas, as prestage of CRC. Methods: Tissue-Micro-Arrays (TMA) of 250 patients were prepared from formalinfixed and paraffin embedded (FFPE) samples and an immunohistochemical staining using the ABCMethod was performed against Snail and E-Cadherin. RNA was extracted from FFPE adenomas (n = 41) and normal colon mucosa (n = 10). Serial sections of the adenomas were stained for Snail and E-Cadherin, as well. A qRT-PCR was used to evaluate the expression of E-Cadherin, Snail, Twist1 and Twist2 in the adenomas and normal tissue. Results: While Snail was detected in 72 % of the CRC and in 96 % E-Cadherin was down-regulated, we could not detect a significant correlation between these findings. There was no prognostic correlation between Snail and overall survival. We could, however, detect a significant correlation between Snail (78 % positive, p < 0.05), Twist1 (38 % positive, p = 0.3) and Twist2 (61 % positive, p < 0.05) and loss of E-Cadherin in adenomas, as well as a significant correlation between Snail staining and loss of E-Cadherin (67 % Snail positive, p = 0.02). When Snail and Twist2 or Snail and Twist1 were co-expressed, we found a highly significant (p = 0.001, p = 0.003, respectively) correlation to loss of E-Cadherin. Conclusion: We could detect Snail in a high percentage of CRCs, but without a significant impact on E-Cadherin expression. A possible explanation is the activation of other important EMT-pathways (e. g. WNT) [3, 4]. We found a highly significant correlation between Snail, Twist1 and Twist2 expression and the loss of E-Cadherin on transcriptional level and between Snail and E-Cadherin on protein level in colorectal adenomas, thus showing a possible influence on the progression towards malignancy in these precursors of CRC.

Introduction: Up-regulation of heme oxygenase (HO)-1 has been extensively described to decrease ischemia/reperfusion (I/R) injury in the liver. Most agents which have been used for liver preconditioning are not suitable for clinical application. Aim of this study was to evaluate the potential of Hemoglobin-Glutamer 200 (HbG200) preconditioning to decrease extended cold I/R injury. Material & Methods: The time course of HO-1 expression in the rat liver after intravenous application of a calculated HbG200 volume was evaluated during 24 hrs by repeated Western blot analysis, to determine maximum HO-1 expression. The calculated immediate post infusion concentration was 3,9 g/dl polymerized hemoglobin. Wistar rat livers were harvested and stored at 4 °C in UW-solution for 8 hrs (group A, n = 7) or 44 hrs (group B-D, n = 7 each) and reperfused at 37 °C in the isolated perfused rat liver (IPRL) with a sanguineous perfusate for 180 min. In group C and D rats were preconditioned by intravenous application of HbG200, 12 hrs before organ harvest, at maximum HO-1 expression. In group D, HO activity was inhibited one hour before organ harvest by intraperitoneal administration of SnPP. Results: HbG200 infusion increased serum hemoglobin by 3.2 g/dl in rats, resulting in maximum HO-1 expression in the liver after 12 hrs. HbG200 preconditioning of the liver significantly (p < 0.05) decreased ALT and AST values in group C (121 ± 14 u/l and 236 ± 21 u/l) compared to group B (424 ± 109 u/l and 492 ± 146 u/l) after 180min of reperfusion. SnPP abolished the preconditioning effect of HbG200, which resulted in highest ALT and AST values in group D (603 ± 51 u/l and 683 ± 38 u/l). Portal flow was significantly (p < 0.05) improved by HbG200 preconditioning (group C) compared to control group B. Bile production after 180min of reperfusion was also significantly increased (p < 0.01) in group C (0,91 ± 0,16 ml) compared to group B (0.54 ± 0.2 ml). Conclusion: HbG200 is a ready to use, cell free hemoglobin solution and easy to administer. Preconditioning of the liver by intravenous HbG200 application proved to be very effective to reduce reperfusion injury after extended cold liver preservation.

Cytochrome P450 epoxygenases (CYP450) have been recently shown to promote malignant progression. Here we investigated the mRNA and protein expression and potential clinical relevance of CYP2C9 in esophageal cancer. Highest expression was detected in esophageal adenocarcinoma (EAC; n = 78) and adjacent esophageal mucosa (NEM; n = 79). Levels of CYP2C9 in EAC and NEM were significantly higher compared to esophageal squamous cell carcinoma (ESCC; n = 105). Early tumor stages and well-differentiated tumors showed a significantly higher CYP2C9 expression compared to progressed tumors. Moreover, CYP2C9 expression was correlated to high Ki-67 labeling indices in EAC and Ki-67 positive tumor cells in EAC and ESCC. Selective inhibition of CYP2C9 decreased tumor cell proliferation (KYSE30, PT1590 and OE19) in vitro, which was abolished by 11,12-epoxyeicosatrienoic acid (11,12-EET). Cell-cycle analysis using FACS revealed that inhibition of CYP2C9 leads to a G0/G1 phase cell-cycle arrest. CYP2C9 seems to be relevant for early esophageal cancer development by promoting tumor cell proliferation. Pharmacological inhibition of CYP2C9 might contribute to a more efficient therapy in CYP2C9 highly expressing esophageal cancers.

The oxygen-regulated transcription factor subunit hypoxia inducible factor-1alpha (HIF-1alpha) is involved in angiogenesis, energy metabolism, cell survival, and inflammation. We examined the protein expression of HIF-1alpha within the progression of Barrett's sequence as well as the type and degree of the environmental inflammatory reaction. Squamous epithelium (SE), metaplastic, low- and high-grade dysplastic lesions, and tumor tissue of 57 resection specimens from patients with Barrett's adenocarcinoma were immunohistochemically analyzed. Active and chronic inflammatory reactions were classified according to the Updated Sydney System. HIF-1alpha protein expression increased significantly from SE to Barrett's metaplasia (BM) (P < 0.0001). From metaplasia through low- and high-grade dysplasia to cancer, no further increase could be detected. Active and chronic inflammation were also significantly different between SE and BM (P < 0.0001) but not during further progression in the sequence. HIF-1alpha protein expression did not correlate with histopathologic parameters or survival. HIF-1alpha protein expression pattern resembles the active and chronic environmental inflammatory reaction. All were significantly increased in metaplasia compared to SE without further change in tumor development. HIF-1alpha protein expression appears to be associated with inflammatory processes in the development of BM.

Epithelial-mesenchymal transition (EMT) seems to be a critical event for invasion and metastasis of carcinoma cells [1]. E-cadherin down-regulation is a critical step in this mechanism [2]. The aim of the present work was to study the expression profiles of E- cadherin transcription regulators SIP1, Snail and Twist in human colorectal adenomas as a pre-stage of colorectal cancer and to correlate the expression patterns with E-cadherin expression.

EpCAM represents a relevant therapeutic target in antibody-based tumor therapy. In our study, we investigated the potential of EpCAM as a therapeutic molecular target in the treatment of esophageal squamous cell carcinoma (ESCC) by assessing the expression of EpCAM using a tissue microarray (TMA) comprising the primary tumor tissue of 118 patients. The EpCAM protein expression in the tumors was analyzed by immunohistochemistry and verified by quantitative real-time PCR in a subgroup of cases. Whereas no expression was detected in normal esophageal squamous epithelium, 64% of the investigate tumors expressed EpCAM on a low 1+(12%), moderate 2+(19%), and strong 3+ (32%) level, respectively. Expression was predominantly heterogeneous, while subgroup exhibited an enhanced EpCAM expression at the tumor invasion front. Strong (3+) expression of EpCAM in the primary tumor tissue correlated significantly with a limited overall survival of the patients (logrank test, p=0.0479). Aside from invasion depth (pT) and nodal involvement (pN), strong (3+) expression of EpCAM in the primary tumor was established as an independent progrnostic marker in the multivariate analysis. Consistently, strong (3+) EpCAM protein expression in the tumor tissue correlated with an increased detection of the transcript in the corresponding subset of cases as determined by qPCR. Thus, the prognostic relevance of its overexpression strongly suggests EpCAM as a promising molecular target for antibody-based adjuvant therapy in ESCC.

Background: The evidence of lymph node metastasis is decisive for the prognosis of patients with rectal carcinoma. The aim of this study is to evaluate the prevalence and the clinical importance of disseminated tumour cells in perirectal lymph nodes from patients with rectal carcinoma UICC stage I, considered free of disease by conventional histopathology. Patients and Methods: We used the epithelium-specific monoclonal anti-EpCAM antibody Ber-EP4 in immunohistochemistry to discover disseminated tumour cells in 756 regional lymph nodes (∼20 lymph nodes per patient) judged as tumour-free by conventional histopathology in 38 patients with rectal adenocarcinoma staged as pT1-2, pN0, M0, G1-3, R0. MOPC 21 served as isotyp control. We correlated the results with clinical follow-up data (median follow-up 67 months, range 11–123 months). Results: EpCAM-positive cells were detected in the lymph nodes from 9 of 38 patients (23.7%). Originally 1 of 8 cases (12.5%) was juged as pT1, pN0 and 8 of 30 cases (26.7%) were juged as pT2, pN0 by conventional histopathology. Patients without any EpCAM-positive lymph node cells showed over a follow-up of 10 years postoperative no evidence of cancer recurrence. 4 of 9 patients with EpCAM-positive lymph nodes showed a tumour recurrence including 1 local and 3 distant metastases. 5 years postoperative already 40% of these patients showed a cancer relapse. There were significant differences in recurrence rates (4 of 9 versus 0 of 29; p=0.0064) and in 10-year relapse-free survival rates (p=0.0015) between patients with and without EpCAM-positive lymph node cells. Conclusion: EpCAM-positive cells were detected in perirectal lymph nodes of a significant number of patients with rectal carcinoma UICC stage I. Our results suggest that immunohistochemical investigations with the anti-EpCAM antibody Ber-EP4 in lymph nodes may more accurately identify patients who may be at high risk for rectal cancer recurrence than does routine histopathology and who could therefore benefit from adjuvant therapy.

Recent studies have shown an association between the GNAS1 T393C polymorphism and clinical outcome for various solid tumors. In this study, we genotyped 51 patients from an observational trial on cisplatin/5-FU-based neoadjuvant radiochemotherapy of locally advanced esophageal cancer (cT2-4, Nx, M0) and genotyping was correlated with histomorphological tumor regression. The C-allele frequency in esophageal cancer patients was 0.49. Pearson's χ 2 -test showed a significant ( P <0.05) association between tumor regression grades and T393C genotypes. Overall, 63% of the patients in the T-allele group (TT+CT) were minor responders with more than 10% residual vital tumor cells in resection specimens, whereas T(−) genotypes (CC) showed a major histopathological response with less than 10% residual vital tumor cells in 80%. The results support the role of the T393C polymorphism as a predictive molecular marker for tumor response to cisplatin/5-FU-based radiochemotherapy in esophageal cancer.

Microsatellite analysis is an important tool in clinical research and molecular diagnostics because microsatellite instability (MSI) occurs frequently in various types of cancer. Approximately 10-15% of colorectal, gastric and endometrial carcinomas are associated with MSI, and this has an impact on clinical prognosis. The microsatellite loci Bat25, Bat26, D2S123, D5S346 and D17S250, recommended by the Bethesda guidelines, were analysed by microfluidic-based on-chip electrophoresis in 40 cases of colon carcinoma with known MSI status. In all cases, microfluidic separation of the PCR amplicons resulted in highly resolved, distinct patterns of each of the five microsatellite loci. Detection of MSI could be demonstrated by microsatellite-loci-associated, well-defined deviations in the electropherogram profiles of tumour and non-tumour material, and confirmed the classification of MSI cases performed by conventional technology. In conclusion, microfluidic chip technology is a simple and reliable approach for MSI detection that allows label-free and very fast analysis of microsatellite amplicons.

The Wnt-pathway, with nuclear beta-catenin as a main effector protein, can induce Epithelial-Mesenchymal Transition (EMT) and indirectly chromosomal instability (CIN). Here, we investigated for disseminated tumor cells (DTC) expressing nuclear beta-catenin and if this phenotype is associated with specific genomic alterations. Therefore, we established a double-immunfluorescence labelling protocol to determine the staining pattern of beta-catenin in single disseminated tumor cells. Single CK-positive cells were isolated by micromanipulation, their DNA was globally amplified by an adapter-linker PCR method and their genomes were subsequently screened for chromosomal gains and losses using comparative genomic hybridization (CGH). We detected 34 CK-positive cells in 20 bone marrow samples of 68 CRC patients (29%). CGH analysis revealed a higher mean chromosomal aberration number (11,4) for cells expressing beta-catenin in the nucleus compared to those with cytoplasmatic or absent beta-catenin staining (4,1). Our results demonstrated a variable expression of beta-catenin on early DTCs detected in bone marrow of CRC patients. However, a significant number showed nuclear beta-catenin staining, indicating activated Wnt signaling. Interestingly, cells with nuclear beta-catenin displayed the highest level of chromosomal instability. Ongoing research will clarify, whether these cells confer a higher risk for metastatic relapse.

Predicting the malignant potential of gastrointestinal stromal tumors (GISTs) remains difficult. We assessed the value of serosal penetration, an established prognostic factor in solid tumors, to determine the clinical outcome in patients with GISTs. From 1996-2002, 25 consecutive patients with GIST underwent surgical resection at our Department. The histopathological presence of serosal penetration was assessed to predict clinical outcome. In addition, the established histopathological classification system by Franquemont (modified by using the Ki-67 proliferation index), was applied to each study patient. A Ki-67 index > or =5% (p<0.001) and a mitotic rate > or =5/50 high-power fields (p<0.047) significantly correlated with a shorter survival, whereas a tumor size >5 cm (p=0.07) tended towards a worse prognosis. The survival of patient groups defined by Franquemont (p=0.03) were of prognostic relevance. The presence of serosal penetration significantly correlated (p<0.01) with a shorter survival. Our data suggest that the presence of serosal penetration is a negative prognostic factor for GISTs. Serosal penetration may become a useful additional parameter for the classification of the malignant potential of GISTs. Since our data are merely hypothesis-generating, serosal penetration should be evaluated in large prospective databases.