Joerg Haier

Extent of surgery and recurrence rate of hidradenitis suppurativa

Abstract Hidradenitis suppurativa (HS) is a chronic fistula- and abscess-forming disease of the cutis and subcutis of unknown etiology. Disease recurrence is frequent and may cause severe complications. We analyzed patients with HS who underwent surgery between 1976 and 1997. The operative procedures were divided into drainage procedures (n=6), limited regional (n=14), and radical wide excisions (n=11). The extent of surgery was examined in terms of the clinical course and late postoperative sequelae of HS. At a mean follow-up of 72 months, we found developed locoregional recurrent HS in 45% of patients. There was 100% recurrence after drainage, 42.8% after limited, and 27% after radical excision (P<0.05). HS recurred after a median interval of 3 months for drainage, 11 months for limited excision, and 20 months for radical excision (P<0.05). The disease-free interval continued up to 35 months. Long-term sequelae included penile amputation and a case of fatal squamous cell carcinoma. Although radical wide excision of the HS-affected cutis is associated with the lowest recurrence rate, it is still considerable and warrants long-term follow-up.

EP300--a miRNA-regulated metastasis suppressor gene in ductal adenocarcinomas of the pancreas.

Genetic and epigenetic alterations during development of pancreatic ductal adenocarcinomas (PDACs) are well known. This study investigates genetic and epigenetic data together with tumor biology to find specific alterations responsible for metastasis formation. Using 16 human PDAC cell lines in a murine orthotopic PDAC model, local infiltration and metastatic spread were assessed by standardized dissemination scores. The cell lines were further classified into 3 hierarchical groups according to their metastatic potential. Their mRNA and microRNA (miRNA) expression was profiled via mRNA‐microarray as well as Taqman Low Density Array, and validated by single quantitative RT‐PCR and Western blotting. In the highly metastatic group, a significant induction of EP300 targeting miRNAs miR‐194 (fold change: 26.88), miR‐200b (fold change: 61.65), miR‐200c (fold change: 19.44) and miR‐429 (fold change: 21.67) (p < 0.05) was detected. Corresponding to this, decreased expression of EP300 mRNA (p < 0.0001) and protein (p < 0.05) were detected in the highly metastatic PDAC cell lines with liver metastases compared to the nonmetastatic or marginally metastatic cell lines, while no correlation with local tumor growth was found. In conclusion, epigenetic alterations with upregulated EP300 targeting miRNAs miR‐194, miR‐200b, miR‐200c and miR‐429 are related to reduced EP300 mRNA and protein in PDAC. These results demonstrate that miRNAs might be able to modulate the expression of metastasis‐specific suppressor genes and metastatic behavior in PDAC, suggesting diagnostic and therapeutic opportunities for EP300 and its targeting miRNAs in PDAC.

Expression of tight and adherens junction proteins in ulcerative colitis associated colorectal carcinoma: upregulation of claudin-1, claudin-3, claudin-4, and β-catenin

BackgroundTight junction (TJ) proteins play a critical role in cellular adhesion, glandular differentiation, and cellular proliferation. The function of these proteins is compromised in a number of intestinal diseases, including ulcerative colitis that has an increased incidence for colorectal carcinoma (CAC). The aim of this study was to determine the expression of TJ proteins, claudin-1–4, occludin, ZO-1, and the adherens junction (AJ) protein β-catenin in CAC.MethodsSixteen colectomy specimens with CAC, adjoining intraepithelial neoplasia, and normal mucosa were studied by immunofluorescence. A semiquantitative evaluation of all investigated proteins was performed by scoring the staining intensity, and the TJ and AJ protein expression in neoplastic cells was compared to normal and intraepithelial neoplastic colonic mucosa.ResultsUsing an intensity scoring system, mucosa of crypts and surfaces of CAC exhibited significantly elevated expression levels of claudin-1, claudin-3, claudin-4, and β-catenin compared to intraepithelial neoplasia and normal mucosa (p < 0.05). These data were confirmed by a comparative score. The expression of claudin-2, occludin, and ZO-1 showed no differences between the groups.ConclusionTJ proteins claudin-1, claudin-3, claudin-4, and the AJ protein β-catenin are overexpressed in CAC. This suggests that these proteins may become potential markers and targets in CAC.

Epigenetic markers for chemosensitivity and chemoresistance in pancreatic cancer--a review.

Adjuvant first‐line gemcitabine monochemotherapy presents a standard treatment for patients with advanced pancreatic adenocarcinoma and improves overall survival in chemosensitive patients. Nonetheless, 6‐month progression‐free survival remains below 15%, despite interdisciplinary approaches. The success of gemcitabine treatment is disappointing and—in the absence of reliable tumor markers—challenging to quantify. Epigenetic alterations have been recently identified to take on important roles in cancer development and possibly cancer treatment. In this context, microRNAs are becoming increasingly acknowledged as useful biomarkers for classifying cancers and providing information on their chemo‐ and radiosensitivity. This review illustrates the potential of genetic and epigenetic markers in the prediction of chemosensitivity in pancreatic cancer patients and in the monitoring of their response rates to adjuvant therapy.

Role of Tumor Microenvironment on Gene Expression in Pancreatic Cancer Tumor Models

OBJECTIVES The microenvironment is known to be a relevant factor of influence on tumor growth and metastasis in pancreatic ductal adenocarcinoma (PDAC). To determine the influence of the microenvironment on changes in gene expression, we analyzed gene expression in different PDAC tissues. METHODS Four human PDAC cell lines were introduced into a murine PDAC model with two insertion techniques: injection and implantation. Gene expression profiles of the cell lines growing in vitro and in vivo (ectopically and orthotopically) were established by microarray and validated by RT-PCR. RESULTS Significant differences were found in the gene expression profiles of the in vitro versus in vivo tissues (P < 0.05), while no differences were found between the in vivo tissues. Analyzing the orthotopic tumors derived from the injection and implantation methods, similar gene expression patterns with 0%-18% significantly differentially expressed genes between tumors of the two different methods were observed (analysis of variance [ANOVA]; P < 0.0001). CONCLUSIONS Gene expression from cell lines growing in vitro differed from the expression patterns of the same cells growing in vivo, while the localization of the growing tumor cells did not significantly alter gene expression. These data demonstrate that the implantation and injection techniques used in this study yield similar results and may be compared with each other.

Epigenetic regulation and role of metastasis suppressor genes in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is distinguished by rapid dissemination. Thus, genetic and/or epigenetic deregulation of metastasis suppressor genes (MSG) is a likely event during early pancreatic carcinogenesis and a potential diagnostic marker for the disease. We investigated 9 known MSGs for their role in the dissemination of PDAC and examined their promoters for methylation and its use in PDAC detection.MethodsMRNA expression of 9 MSGs was determined in 18 PDAC cell lines by quantitative RT-PCR and promoter methylation was analyzed by Methylation Specific PCR and validated by Bisulfite Sequencing PCR. These data were compared to the cell lines’ in vivo metastatic and invasive potential that had been previously established. Statistical analysis was performed with SPSS 20 using 2-tailed Spearman’s correlation with P < 0.05 being considered significant.ResultsComplete downregulation of MSG-mRNA expression in PDAC cell lines vs. normal pancreatic RNA occurred in only 1 of 9 investigated genes. 3 MSGs (CDH1, TIMP3 and KiSS-1) were significantly methylated. Methylation only correlated to loss of mRNA expression in CDH1 (P < 0.05). Bisulfite Sequencing PCR showed distinct methylation patterns, termed constant and variable methylation, which could distinguish methylation-regulated from non methylation-regulated genes. Higher MSG mRNA-expression did not correlate to less aggressive PDAC-phenotypes (P > 0.14).ConclusionsGenes with metastasis suppressing functions in other tumor entities did not show evidence of assuming the same role in PDAC. Inactivation of MSGs by promoter methylation was an infrequent event and unsuitable as a diagnostic marker of PDAC. A distinct methylation pattern was identified, that resulted in reduced mRNA expression in all cases. Thus, constant methylation patterns could predict regulatory significance of a promoter’s methylation prior to expression analysis and hence present an additional tool during target gene selection.

SERPINB5 Promoter Hypomethylation Differentiates Pancreatic Ductal Adenocarcinoma From Pancreatitis.

ObjectivesThe diagnosis of pancreatic ductal adenocarcinoma (PDAC) is challenging in the setting of pancreatitis. We investigated SERPINB5 for its impact on PDAC tumor biology and its use as a diagnostic marker for PDAC in the setting of pancreatitis. MethodsPatient samples from PDAC primary tumors, PDAC lymph node metastases, and pancreatitis were investigated for SERPINB5 promoter methylation by methylation-specific polymerase chain reaction (PCR). Six PDAC cell lines were investigated in vitro and in vivo using an orthotopic mouse model to generate primary tumors and metastases. SERPINB5 mRNA expression, protein expression, and promoter methylation were determined by quantitative reverse transcriptase–PCR, methylation-specific PCR, and Western Blot. ResultsIn patient samples, detection of an unmethylated SERPINB5 promoter differentiated pancreatitis from PDAC with a sensitivity of 57% and a specificity of 95% (P < 0.001). SERPINB5 was not deregulated in primary tumors versus metastases, but primary tumors without SERPINB5 protein expression had significantly reduced viability (P = 0.02). ConclusionsSERPINB5 seems to assume an oncogenic role in PDAC. In clinical samples, detection of unmethylated SERPINB5 was a specific marker for PDAC even in the context of pancreatitis and may provide the basis for a liquid biopsy option to detect PDAC.

Serum microRNA signatures differentiate pancreatic ductal adenocarcinoma from pancreatitis and healthy pancreas

Introduction Pancreatic ductal adenocarcinoma (PDAC) is recognized for its lethality which is largely due to late clinical manifestation combined with rapid dissemination. In order to move forward the time of diagnosis, specific, sensitive and easily accessible markers are essential. MicroRNAs are an emerging class of molecules involved in tumorigenesis and metastasis. Additionally, they are disease specific and highly stable in blood, predestined to become a novel class of biomarkers. Methods 96 patients were enrolled from 2009 to 2010: PDAC: n=53, pancreatitis: n=30, healthy control: n=13. RNA was isolated from serum samples of each patient using a modified TRIzol method and artificial miRNAs were spiked in at 5nM. MicroRNA-microarray was carried out by febit GmbH (Heidelberg, Germany) for 19 PDAC, 15 pancreatitis and 8 control samples. Significantly differentially expressed miRNAs were validated by quantitative reverse transcriptase PCR in all samples. Results The following miRNAs were validated to be upregulated in PDAC vs. both pancreatitis and healthy controls: miR-190, −1246, −26a, −3152, −1265, −924, while miR-509-3-5p, −519c-5p, −501-3p, −548s-195* were downregulated. Using the validated miRNAs, PDAC was differentiated from pancreatitis with a sensitivity of 0.84 and specificity of 0.93. Conclusions This is the first investigation of circulating miRNAs as biomarkers for PDAC that has made use of microarrays to investigate global expression rather than employing qRT-PCR to focus on specific miRNAs. Thus, novel biomarker candidates have been identified. Further functional analysis of these miRNAs will uncover their significance and role in the progression of PDAC. (Supported by a grant from Foerderverein Peter Geiger e.V.).

T1916 Differentiation, Origin and Mesothelial Adhesive Potential Affect Growth and Metastasis of Ductal Adenocarcinoma of the Pancreas

Background: Adenocarcinomas of the exocrine pancreas belong to the most aggressive malignancies with an overall 5-year survival rate of still less than 5%. The early development of distant metastases, such as peritoneal carcinomatosis, is a specific characteristic of this particular tumor entity, but their mechanisms are poorly understood. In a series of newly established orthotopic models for pancreatic carcinomas tumor growth, metastasis and their potential for mesothelial adhesion as one of the first steps of metastasis formation were investigated. Methods: 16 cell lines of wellto undifferentiated pancreatic cancers from different tumor sites (primary or metastatic lesions) were used to implant subcutaneous donor tumors and subsequent orthotopic transplantation in nude mice. After 12 weeks primary tumor volume, local infiltration, and patterns of systemic metastases were assessed using a standardized dissemination score. This In Vivo behaviour was compared with In Vitro tumor cell adhesion to mesothelial cells. The data was tested for significant differences using Scheffeand t-test (SPSS 13.0). Results: In Vivo experiments resulted in a tumor take rate of 100%. Differences regarding tumor size, infiltration and metastatic spread were found depending on differentiation and origin of the cell lines. Less differentiated cells of primary tumors and metastasis caused higher dissemination scores than better-differentiated cells (p<0.05). A significant increase (p<0.05) of tumor growth, infiltration and metastasis was also seen for cells originating from metastases compared to those from primary tumors. Adhesion assays revealed an adhesive potential at mesothelium of all cell lines (21-95%). Primary tumors with well-moderate differentiation presented significantly increased adhesion rates (mean 74-95%) compared to poorly-undifferentiated primary tumors (mean 31-55%) and well-moderately differentiated (mean 21-44%) or poorly-undifferentiated (mean 2545%) metastases [p<0.05]. The maximum adhesion rates were found in average 76 min after addition of pancreatic cells to mesothelial monolayers (observation period 90min). Conclusion: These experimental systems are an interesting tool for the investigation of mechanisms or pancreatic cancer progression and demonstrate that grade of differentiation and origins are relevant factors for tumor growth and metastatic spread. The correlation of pancreatic cells adhesion at the mesothelium with their origin and differentiation suggests that this adhesive potential is a required, but not rate-limiting step for the formation of peritoneal carcinomatosis in this tumor entity.