Petra Ruemmele

Secondary sclerosing cholangitis

Secondary sclerosing cholangitis (SSC) is a chronic cholestatic biliary disease, characterized by inflammation, obliterative fibrosis of the bile ducts, stricture formation and progressive destruction of the biliary tree that leads to biliary cirrhosis. SSC is thought to develop as a consequence of known injuries or secondary to pathological processes of the biliary tree. The most frequently described causes of SSC are longstanding biliary obstruction, surgical trauma to the bile duct and ischemic injury to the biliary tree in liver allografts. SSC may also follow intra-arterial chemotherapy. Sclerosing cholangitis in critically ill patients is a largely unrecognized new form of SSC, and is associated with rapid progression to liver cirrhosis. The mechanisms leading to cholangiopathy in critically ill patients are widely unknown; however, the available clinical data indicate that ischemic injury to the intrahepatic biliary tree may be one of the earliest events in the development of this severe form of sclerosing cholangitis. Therapeutic options for most forms of SSC are limited, and patients with SSC who do not undergo transplantation have significantly reduced survival compared with patients with primary sclerosing cholangitis. Sclerosing cholangitis in critically ill patients, in particular, is associated with rapid disease progression and poor outcome.

Distinction of hereditary nonpolyposis colorectal cancer and sporadic microsatellite-unstable colorectal cancer through quantification of MLH1 methylation by real-time PCR.

Purpose: Promoter hypermethylation occurs frequently in tumors and leads to silencing of tumor-relevant genes like tumor suppressor genes. In a subset of sporadic colorectal cancers (CRC), inactivation of the mismatch repair gene MLH1 due to promoter methylation causes high level of microsatellite instability (MSI-H). MSI-H is also a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) in which mismatch repair inactivation results from germ-line mutations. For differentiation of sporadic and hereditary MSI-H tumor patients, MLH1 promoter methylation analysis is a promising tool but is not yet used in daily diagnostics because only qualitative techniques without standardization are available. The aim of this study is to establish a reliable and quantitative MLH1 methylation analysis technique and to define valid MLH1 methylation cutoff values for HNPCC diagnostics. Experimental Design: We developed a new real-time PCR–based technique to detect and quantify methylation of both proximal and distal hMLH1 promoter regions. We established and validated this technique in a cohort of 108 CRCs [94 MSI-H and 16 microsatellite stable (MSS) cases] comprising a reference (n = 58) and a tester tumor group (n = 50). Results: The reference tumor group contained 28 HNPCC with proven germ-line mutations or positive Amsterdam I criteria (median age, 37 years) and loss of MLH1 expression, 14 sporadic MSI-H CRC tumors with loss of MLH1 expression and BRAF V600E mutation (median age, 80.5 years), and 16 sporadic MSS CRC (median age, 76.5 years). No MLH1 promoter methylation could be found in any MSS tumors. HNPCC patients showed no or low level of MLH1 promoter methylation. A cutoff value of 18% methylation extent could be determined in this study to define MLH1 hypermethylation specific for sporadic MSI-H cases. Methylation could also be verified qualitatively by melting point analysis. BRAF V600E mutations were not detected in any HNPCC patients (n = 22 informative cases). Conclusion: According to the present data, quantitative MLH1 methylation analysis in MSI-H CRC is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC. The detection of a BRAF V600E mutation further supports the exclusion of HNPCC.

Gene expression profiling of colorectal cancer and metastases divides tumours according to their clinicopathological stage

Gene expression profiling of matched colorectal carcinomas and metastases could reveal key molecular events involved in tumour progression and metastasis. Expression profiles have been created from 25 colorectal carcinomas (CRCs, pT1–4), corresponding normal colonic mucosa, and 14 liver metastases using cDNA arrays containing 1176 cancer‐related genes (Clontech). Hierarchical clustering clearly distinguished carcinomas from non‐cancerous tissues, separated tumours into high‐stage (pT4 and extensive lymph node or distant metastases) and low‐stage (≤pT3) groups, and correlated with the histopathological classification in 87% (33/38 cases). Most primary tumours and matched liver metastases clustered on terminal branches of the dendrogram. Statistical analysis (Mann–Whitney U‐test) revealed 40 tumour‐specific genes (29 up‐regulated, 11 down‐regulated) which allowed identification of malignant tissue samples by cluster analysis. A specific expression signature in matching metastases was not found, but a set of 23 classifier genes with statistically significant expression patterns in high‐ and low‐stage tumours was identified. These genes may represent important targets in colorectal carcinogenesis and might provide useful clinicopathological tools in the management of colorectal cancer. Copyright

Histopathologic Features and Microsatellite Instability of Cancers of the Papilla of Vater and Their Precursor Lesions

The prevalence and development of microsatellite instability (MSI) and underlying mismatch repair (MMR) deficiency in the carcinogenesis of adenocarcinomas of the papilla of Vater and their precursor lesions are not well established. We analyzed 120 ampullary adenomas (31 pure adenomas and 89 carcinoma-associated adenomas) and 170 pure adenocarcinomas for MSI, immunohistochemical expression of MMR proteins and specific histopathologic features. The most common histologic subtype was intestinal (46.5%), followed by pancreatobiliary (23.5%), poorly differentiated adenocarcinomas (12.9%), intestinal-mucinous (8.2%), and invasive papillary carcinomas (5.3%). Eight of 89 adenomas (9%) and 15/144 carcinomas (10%) showed high microsatellite instability (MSI-H), 10/89 adenomas (11%) and 5/144 carcinomas (4%) showed low microsatellite instability (MSI-L), and 71/89 adenomas (80%) and 124/144 carcinomas (86%) were microsatellite stable (MSS). MSI analysis from carcinomas contiguous with an adenomatous component (n=54) exhibited concordant results in 6/8 (75%) MSI-H and 42/46 (91.3%) MSS tumors. Of 14 carcinomas with MSI-H, 7 showed loss of MLH1 and 5/6 (83%) MLH1 promoter methylation, and 2 carcinomas showed simultaneous loss of MSH2 and MSH6. Two carcinomas and 3 adenomas with MSI-H revealed exclusive loss of MSH6. MSI-H cancers were significantly associated with intestinal mucinous subtype (P<0.001), high tumor grade (P=0.003), expansive growth pattern (P=0.044), and marked lymphoid host response (P=0.004). Patients with MSI-H carcinoma had a significantly longer overall survival (P=0.0082) than those with MSI-L or MSS tumors. Our findings indicate that the MSI-phenotype is an early event, which develops at the stage of adenoma and is reliably detectable in the precursor lesion. The MMR deficient molecular pathway of carcinogenesis is associated with a histopathologic phenotype in ampullary cancer, similar to the one that has been well described in colon cancer.

Tumor development in murine ulcerative colitis depends on MyD88 signaling of colonic F4/80 + CD11b high Gr1 low macrophages

Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13-producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11b(high)Gr1(low) M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate-induced colitis, F4/80+CD11b(high)Gr1(intermediate) macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88-deficient (Myd88-/-) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88-/- mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11b(high)Gr1(intermediate) macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.

Bile duct damage after cold storage of deceased donor livers predicts biliary complications after liver transplantation

BACKGROUND & AIMS The aim of this study was to examine the development of biliary epithelial damage between organ retrieval and transplantation and its clinical relevance for patients. METHODS Common bile duct samples during donor hepatectomy, after cold storage, and after reperfusion were compared to healthy controls by hematoxylin and eosin (H&E) staining and immunofluorescence for tight junction protein 1 and Claudin-1. A bile duct damage score to quantify biliary epithelial injury was developed and correlated with recipient and donor data and patient outcome. RESULTS Control (N=16) and donor hepatectomy bile ducts (N=10) showed regular epithelial morphology and tight junction architecture. After cold storage (N=37; p=0.0119), and even more after reperfusion (N=62; p=0.0002), epithelial damage, as quantified by the bile duct damage score, was markedly increased, and both tight junction proteins were detected with inappropriate morphology. Patients with major bile duct damage after cold storage had a significantly increased risk of biliary complications (relative risk 18.75; p<0.0001) and graft loss (p=0.0004). CONCLUSIONS In many cases, the common bile duct epithelium shows considerable damage after cold ischemia with further damage occurring after reperfusion. The extent of epithelial damage can be quantified by our newly developed bile duct damage score and is a prognostic parameter for biliary complications and graft loss. Possibly, in an intraoperative histological examination, this bile duct damage score may influence decision-making in transplantation surgery.

Low-Dose Chemotherapy in Combination with COX-2 Inhibitors and PPAR-Gamma Agonists in Recurrent High-Grade Gliomas : A Phase II Study

Objectives: Combined treatment approaches targeting tumor as well as other cells contributing to tumor progression may control chemorefractory malignancies. Methods: A phase II trial was initiated to analyze the activity of continuously administered pioglitazone and rofecoxib combined with low-dose chemotherapy (capecitabine or temozolomide) in patients with high-grade gliomas (glioblastoma or anaplastic glioma). Results: Fourteen patients were evaluable for response and toxicity. Major side effects were palmoplantar erythema, edema and motor neuropathy grade 3. Disease stabilizations lasting longer than 3 months were noted in 4 of 14 patients (29%). Clinical responses did not correspond to immunohistochemical staining for cyclooxygenase 2, peroxisome proliferator-activated receptor-γ and CD31. Discussion: The study demonstrates that this novel regimen is moderately active and well tolerated in patients with high-grade gliomas. As a comparably small proportion of patients responded, the regimen might only be suitable for a subset of highly selected patients.

Silencing of MGMT expression by promoter hypermethylation in the metaplasia–dysplasia–carcinoma sequence of Barrett’s esophagus

To determine the relevance of MGMT in Barretts carcinogenesis, we analyzed promotor hypermethylation and expression of MGMT in Barretts adenocarcinomas and its paired precursor lesions from 133 patients using a methylation-specific PCR, real-time RT-PCR and immunohistochemistry. Hypermethylation was detected in 78.9% of esophageal adenocarcinomas, in 100% of Barretts intraepithelial neoplasia, in 88.9% of Barretts metaplasia, but only in 21.4% of normal esophageal mucosa samples (P<0.001) and correlated significantly with downregulation of MGMT transcripts (P=0.048) and protein expression (P=0.02). Decrease of protein expression was significantly correlated with progressed stage of disease, lymph node invasion and tumor size. We conclude, that aberrant promoter methylation of MGMT is a frequent and early event during tumorigenesis of Barretts esophagus. High prevalence of MGMT hypermethylation may represent a candidate marker for improved diagnosis and targeted therapy in Barretts adenocarcinoma.

Aberrant methylation of DAPK in long-standing ulcerative colitis and ulcerative colitis-associated carcinoma

Death-associated protein kinase (DAPK) has pro-apoptotic functions and participates in various apoptotic systems. DAPK acts as a tumor suppressor, and its inactivation by promoter hypermethylation has been frequently observed in various human cancers. As alterations of pro-apoptotic genes might cause instability in the balance of cell-turnover during chronic inflammatory processes, epigenetic silencing of DAPK might be involved in the carcinogenesis of ulcerative colitis-associated carcinoma (UCC). To evaluate the role of DAPK in the inflammation-driven carcinogenesis of ulcerative colitis (UC), we analyzed promoter hypermethylation and protein expression of DAPK using methylation-specific PCR and immunohistochemistry in 43 UCCs and paired UC-background mucosa, as well as in UC-background mucosa of 50 patients without UCC. The frequency of methylation of DAPK in UCCs was low (27.6%) compared to overall non-neoplastic UC-background mucosa (48.3%; p=0.02) and sporadic colorectal carcinoma (57.4%, p=0.019). The difference in the methylation frequency in UC-background mucosa in patients without UCC (54.2%), compared to those with UCC (40.0%), was not significant (p=0.141). Promoter methylation correlated significantly with decreased DAPK protein expression (p<0.001) and severity of inflammatory activity (p=0.024). In unmethylated UC-background mucosa, DAPK protein expression increased with activity of UC-associated inflammation, suggesting a protective role of the pro-apoptotic DAPK during the chronic inflammatory process of UC. Thus, inactivation of DAPK by promoter hypermethylation might be crucial for accumulation of DNA damage in inflamed mucosa of UC, and might therefore contribute to the initiation of the neoplastic process and development of UC-associated carcinoma.

Outcome of patients with ischemic-like cholangiopathy with secondary sclerosing cholangitis after liver transplantation

Abstract Background and aims. Sclerosing cholangitis in critically ill patients (SC-CIP) with sepsis and acute respiratory distress syndrome (ARDS) is a cholestatic liver disease with a rapid progression to liver cirrhosis and hepatic failure. Data on outcome of these patients after liver transplantation (LT) are sparse. Patients and methods. Eleven patients (46 ± 12 years; mean labMELD-score: 27 ± 7) with SC-CIP underwent LT. Six patients had severe polytrauma with multiple bone fractures, sepsis and ARDS. Five non-traumatic patients acquired SC-CIP during long-term intensive-care-unit stays due to sepsis and ARDS. Time to diagnosis, the microbiologic results and the survival rates after LT were evaluated. Results. SC-CIP was diagnosed by endoscopic retrograde cholangiopancreatography (ERCP) within 3 ± 1 months after manifestation of cholestasis and histologically confirmed in explanted livers. The predominant microorganisms isolated in bile were: Enterococcus and Candida albicans. Mean follow-up after LT was 28 ± 20 months. One female patient (non-traumatic) died due to sepsis 26 days after LT. All other patients left the hospital alive, but two (non-traumatic) patients died from sepsis, and one (traumatic) patient died in a hemorrhagic shock, thereafter. Seven of 11 patients (5 with polytrauma) are still alive and have a good quality of life. The survival of the SC-CIP patients after LT was comparable with that of patients transplanted due to alcoholic liver cirrhosis. Conclusion. SC-CIP develops rapidly within several months. Enterococcus and C. albicans were the main isolated microorganisms in the bile. Sepsis was the main cause of death after LT. Overall, SC-CIP is a good indication for LT in selected patients.