Dirk Uhlmann

Surgical and palliative management and outcome in 184 patients with hilar cholangiocarcinoma: palliative photodynamic therapy plus stenting is comparable to r1/r2 resection.

Objective:First, to analyze the strategy for 184 patients with hilar cholangiocarcinoma seen and treated at a single interdisciplinary hepatobiliary center during a 10-year period. Second, to compare long-term outcome in patients undergoing surgical or palliative treatment, and third to evaluate the role of photodynamic therapy in this concept. Summary Background Data:Tumor resection is attainable in a minority of patients (<30%). When resection is not possible, radiotherapy and/or chemotherapy have been found to be an ineffective palliative option. Recently, photodynamic therapy (PDT) has been evaluated as a palliative and neoadjuvant modality. Methods:Treatment and outcome data of 184 patients with hilar cholangiocarcinoma were analyzed prospectively between 1994 and 2004. Sixty patients underwent resection (8 after neoadjuvant PDT); 68 had PDT in addition to stenting and 56 had stenting alone. Results:The 30-day death rate after resection was 8.3%. Major complications occurred in 52%. The overall 1-, 3-, and 5-year survival rates were 69%, 30%, and 22%, respectively. R0, R1, and R2 resection resulted in 5-year survival rates of 27%, 10%, and 0%, respectively. Multivariate analysis identified R0 resection (P < 0.01), grading (P < 0.05), and on the limit to significance venous invasion (P = 0.06) as independent prognostic factors for survival. PDT and stenting resulted in longer median survival (12 vs. 6.4 months, P < 0.01), lower serum bilirubin levels (P < 0.05), and higher Karnofsky performance status (P < 0.01) as compared with stenting alone. Median survival after PDT and stenting, but not after stenting alone, did not differ from that after both R1 and R2 resection. Conclusion:Only complete tumor resection, including hepatic resection, enables long-term survival for patients with hilar cholangiocarcinoma. Palliative PDT and subsequent stenting resulted in longer survival than stenting alone and has a similar survival time compared with incomplete R1 and R2 resection. However, these improvements in palliative treatment by PDT will not change the concept of an aggressive resectional approach.

Identification of novel proteins associated with hepatocellular carcinomas using protein microarrays.

Characterization of the protein profiles expressed by hepatocellular carcinomas (HCCs) may identify the genes involved in hepatocellular carcinogenesis and offers the possibility of elucidating clinical biomarkers. In an effort to discover such proteins and pathways that are deregulated in hepatocellular carcinogenesis, cellular proteomes of matched normal liver cells and carcinoma were analysed by tissue microdissection and protein microarrays. Using protein microarrays made up of 83 different antibodies, it was possible to monitor alterations of the protein levels in HCC and non‐neoplastic liver tissue. Further analysis of altered proteins was performed using western blot analysis and tissue microarrays (TMAs) containing 210 HCC specimens and corresponding liver tissue. The protein microarray approach revealed differential expression between HCC and normal liver of 32 of the 83 proteins examined: 21 of these were up‐regulated and 11 down‐regulated. IGF (insulin growth factor) II, ADAM (a disintegrin and metalloproteases) 9, STAT (signal transducers and activators of transcription) 3, SOCS (suppressors of cytokine signalling) 3, and cyclin D1 were significantly up‐regulated and collagen I, SMAD 4, FHIT (fragile histidine triad), and SOCS1 were down‐regulated. The differential expression of these proteins was confirmed using western blot analysis and TMAs. Correlation of differentially regulated proteins with clinico‐pathological data showed that cyclin D1 and SOCS1 were associated with tumour prognosis in univariate analysis, but not multivariate analysis. These data indicate that the development of an array‐based approach for the determination of protein profiles in HCC may facilitate the identification of new proteins associated with carcinogenesis or prognosis. Copyright

Decreased expression of p27 protein is associated with advanced tumor stage in hepatocellular carcinoma

Reduced expression of the cyclin‐dependent kinase inhibitor p27 has previously been correlated with fatal clinical outcome in some tumors, including gastric, breast, and prostate cancers. For hepatocellular carcinoma, the findings are equivocal. In situ hybridization and immunohistochemistry were performed on a series of 203 curatively (R0) resected hepatocellular carcinomas and in corresponding non‐cancerous liver tissue to detect p27. Patients receiving liver transplantation were excluded. The results were correlated with histopathological stage according to the UICC system, Edmondson grade, several other histopathological factors of possible prognostic significance, and finally patient survival. Whereas p27 mRNA was expressed homogeneously in all carcinomas examined, the p27 protein was found in various amounts. The labeling index of p27 protein was significantly lower in advanced stages of the disease (P < 0.001, χ2 = 28.1). We observed decreased p27 protein in higher pT categories (P < 0.001, χ2 = 24.7) and in multiple tumor nodules (P < 0.001, χ2 = 9.3). Multivariate Cox survival analysis identified age, co‐existing cirrhosis, and Edmondson grade as independent prognostic factors. We conclude that evaluation of p27 in hepatocellular carcinoma is useful to predict stage of disease and may have clinical significance, e.g., in predicting optimal therapeutic regimes. Int. J. Cancer 89:350–355, 2000.

Genetic and epigenetic alterations of the INK4a-ARF pathway in cholangiocarcinoma

The INK4a–ARF locus, located on chromosome 9p21, encodes two cell‐cycle regulatory proteins, p16INK4a and p14ARF, acting through the Rb–CDK4 and p53 pathways. To study the contribution of each pathway in the tumourigenesis of cholangiocarcinoma, the alterations of p14ARF, p16INK4a, p53, and pRb were analysed. After microdissection, DNAs from 51 cholangiocarcinomas were analysed by methylation‐specific PCR (MSP), restriction‐enzyme related polymerase chain reaction (RE‐PCR), microsatellite analysis, mRNA expression, and DNA sequencing. Immunohistochemistry of p14ARF, p16INK4a, p53, and pRb was also performed. Promoter methylation of p14ARF was found in 13/51 cases (25%) and p16INK4a showed aberrant promoter methylation in 39/51 cases (76%) which correlated with loss of mRNA transcription. Two tumours (4%) had homozygous deletion of the INK4a–ARF locus. Specific mutations of both exons were not detected. p14ARF inactivation appeared in the context of an unmethylated p16INK4a promoter in eight of 13 cases (61%) of the carcinomas methylated at p14ARF. Mutations of p53 were found in 19 of 51 tumours (37%), and four of them (21%) harboured p14ARF inactivation. The pRb protein was detected in 30/51 (59%) tumours examined. The absence of pRB protein did not correlate with any of the examined parameters. Alterations of the INK4a–ARF locus, pRB or p53 status could not be established as independent prognostic factors in these tumours. These findings indicate that the INK4a–ARF locus is frequently inactivated in cholangiocarcinoma of the liver and occurs independently of the status of p53 or pRb. Copyright

Allele loss and epigenetic inactivation of 3p21.3 in malignant liver tumors

Previously, the RASSF1A, BLU and SEMAPHORIN 3B (SEMA3B) candidate tumor suppressor genes on chromosome 3p21.3 were found to be inactivated and downregulated by genetic and epigenetic changes in lung cancer. We analyzed the methylation status of RASSF1A, BLU and SEMA3B in 35 hepatocellular carcinomas (HCCs) and 15 cholangiocarcinomas (CCs) by methylation‐specific PCR and loss of heterozygosity (LOH) at 3p21.3 after microdissection. The presence of mRNA transcripts was confirmed by semiquantitative PCR. SEMA3B hypermethylation was found in 29/35 HCCs (83%) and in all (15/15) patients with CC. BLU promoter hypermethylation was detected in 7/35 (20%) HCCs and 3/15 (20%) CCs. In 2 corresponding specimens of hepatitis B virus‐related liver cirrhosis, BLU methylation was also observed, but not in uninvolved normal liver tissue. RASSF1A was methylated in 21/35 HCCs (60%) and in 10/15 CCs (67%). LOH at 3p21.3 occurred in 8/35 (23%) HCCs and 3/15 (20%) CCs. The presence of hypermethylation was statistically associated with LOH of SEMA3B and correlated with downregulation of mRNA transcripts. SEMA3B transcripts increased upon treatment of HCC cell lines with the demethylation compound 5‐aza‐2‐deoxycytidine. In conclusion, our data indicate that 2‐hit gene silencing of SEMA3B through epigenetic changes and allele loss is a common and important event in the carcinogenesis of malignant liver tumors.

Quality of life in chronic pancreatitis: a prospective trial comparing classical whipple procedure and duodenum-preserving pancreatic head resection.

Few data are available with respect to quality of life after pancreatic head resection in patients with chronic pancreatitis. The aim of this study was to compare the classical Whipple pancreatoduodenectomy (PD) with the Beger duodenum-preserving pancreatic head resection (DPPHR), in terms of quality of life, using standardized, valid, and reliable questionnaires. Sixty-five consecutive patients were included in this study. The PD procedure was chosen when pancreatic cancer could not be ruled out (n = 30); otherwise DPPHR was performed (n = 35). Quality of life was measured prospectively three times with the European Organization for Research and Treatment of Cancer (EORTC) Quality-of-Life Questionnaire (QLQ-C30) and the Gastrointestinal Quality-of-Life Index (GIQLI). Both procedures led to a significant improvement in quality of life, especially with regard to pain status. However, at the second follow-up examination (18 to 24 months postoperatively), all functional scales and the most important symptom scales of the EORTC QLQ-C30 revealed a better quality of life in the DPPHR group compared to the PD group. After classical PD, more patients seem to develop diabetes mellitus. The EORTC QLQ-C30 was found to be a better tool for quality-of-life assessment than the GIQLI in patients with chronic pancreatitis.

Evaluation of hepatic microcirculation by in vivo microscopy.

In vivo microscopy is an excellent technique for investigating the microcirculation and until recently the only one that allowed direct visualization. Rat liver has been widely studied, because microcirculatory disorders play a pivotal role in the pathogenesis of organ failure during hepatic ischemia, transplantation, hemorrhagic shock, endotoxemia, and sepsis. The state of the microcirculation is an important prognostic factor for the reestablishment of organ function after these injuries. This article introduces the most common procedures for in vivo microscopy of the rat liver, summarizes the available fluorescent dyes, and gives an overview of criteria for the expression and evaluation of microscopic findings. Particular emphasis is given to a description of the different parameters assessed by direct observation of hepatic microcirculation, such as perfusion rate, leukocyte-endothelium interactions, leukocyte velocities, and phagocytic activity. Examples of normal range values are given. This overview is intended to help those wanting to introduce this method into their research and who are embarking on intravital microscopy for the first time, and to enable them to decide which techniques are appropriate for answering special questions.

Involvement of endothelin/ nitric oxide balance in hepatic ischemia/ reperfusion injury

Introduction: Endothelin (ET) and nitric oxide (NO) act as opponents in the regulation of the hepatic microcirculation. During ischemia/reperfusion (I/R) ET levels are increased, whereas no rise in NO levels is observed. This imbalance may be responsible for microcirculatory disturbances. The aim of this study was to restore the delicate ET/NO balance to maintain the integrity of the hepatic microcirculation and to reduce I/R injury. Methods: Ischemia was induced by crossclamping of the hepatoduodenal ligament for 30 min with portal decompression using a splenocaval shunt (56 Wistar rats, 200–250 g). Sham operation, ischemia and treatment groups with the endothelin receptor antagonist (ERA) bosentan (1 mg/kg body weight i.v.) and the NO donor l-arginine (400 mg/kg body weight i.v.) were performed. For assessment of the microcirculation, sinusoidal perfusion rate, diameters of sinusoids and postsinusoidal venules, leukocyte endothelium interactions and velocity of free-flowing leukocytes were investigated by means of in vivo microscopy 30–90 min after reperfusion. Local hepatic tissue pO2 was measured prior to ischemia, 30 min and 60 min after reperfusion and aspartate aminotransferase (AST)/alanine aminotransferase (ALT) levels were investigated 2 h and 6 h after reperfusion. Results: After ischemia, sinusoidal and venular diameters were reduced to 76% and 85%, respectively, of sham operation group values (P<0.05), but were maintained at baseline in ERA (98/102%) and NO (102/105%) groups (P<0.05). Increased postischemic leukocyte sticking in sinusoids (144%) and venules (435%) was reduced by therapy to 110/253% (ERA) and 111/ 324% (NO), respectively (P<0.05). Perfusion rate was increased to 93% and 94% compared with 82% in the ischemia group (P<0.05). Concomitant with the improved microcirculation in therapy groups, local hepatic tissue pO2 was improved 30 min after reperfusion in the ERA (11.0 mmHg) and the l-arginine group (11.5 mmHg) relative to the ischemic group (6.9 mmHg) (P<0.05). In addition, postischemic AST/ALT increase was reduced by therapy. Conclusion: Our results indicate that maintenance of ET/NO balance by blocking ET receptors, as well as providing a NO donor, protects the liver microcirculation and reduces hepatic I/R injury.

EndothelinA Receptor Blockade Reduces Ischemia/Reperfusion Injury in Pig Pancreas Transplantation

Objective The effect of prophylactic administration of a selective endothelinA receptor antagonist (ETA-RA) on ischemia/reperfusion injury in an experimental model of graft pancreatitis after pancreas transplantation was evaluated. Summary Background Data It is well established that endothelin-1 (ET-1), a powerful vasoconstrictor, plays an important role in the development of pancreatitis. Recent studies have shown a beneficial effect of endothelin receptor antagonists in the therapy for experimental pancreatitis. Methods Relevant ischemia/reperfusion injury was induced in pig pancreas transplants after 6 hours hypothermic preservation in University of Wisconsin solution. The recipients were randomized into 2 groups: control pigs received isotonic saline and the treated group received the selective ETA-RA BSF 208075 at the beginning of reperfusion. On postoperative days 2 and 5, animals were relaparotomized to obtain tissue specimens. Blood monitoring included lipase, amylase, C-reactive protein, trypsinogen-activation peptide, thiobarbituric acid-reacting substances, and ET-1. Partial oxygen tension (ptiO2) was measured by a Clarke-type electrode and blood flow by laser doppler. A semiquantitative score index was used for assessment of histologic injury and for immunohistochemical analysis of ET-1 and ETA receptor expression. Tissue mRNA levels of prepro ET-1, ETA receptor, pro-interleukin (IL)-6, and pro-IL-1&bgr; were quantified using TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). Results Prophylactic treatment with ETA-RA significantly reduced the severity of graft pancreatitis evidenced by C-reactive protein. The finding of transient capillary perfusion at the beginning of reperfusion supports the application of the ETA-RA during this period. The dramatic increase of plasma ET-1 in the therapy group is a clear evidence of effective receptor blockade. Mean trypsinogen-activation peptide levels from the portal venous effluent, but not mean systemic plasma TAP values were significantly lower in the treated group. Analysis of ptiO2 and blood flow revealed a significant improvement of capillary perfusion and blood flow in the treated group and was associated with relevant reduction of tissue injury. Intrapancreatic ET-1 and IL-6 mRNA expression and ET-1 protein levels were significantly lower in the therapy group as compared with the control group. In contrast, ETA mRNA showed a marked up-regulation by ETA receptor blockade. Conclusion Application of a ETA-RA reduces ischemia/reperfusion induced graft pancreatitis in a pig transplantation model by improving microcirculation and reducing tissue injury.

Five-year follow-up of a prospective non-randomised study comparing duodenum-preserving pancreatic head resection with classic Whipple procedure in the treatment of chronic pancreatitis

BackgroundThree prospective randomised studies were conducted to compare pancreatoduodenectomy (PD) with duodenum-preserving pancreatic head resection (DPPHR) in patients suffering from chronic pancreatitis (cP). In these three series, the superiority of the duodenum-preserving technique with regard to quality of life (QOL) and pain relief has been demonstrated. Long-term follow-up investigations have not been published so far. The present paper reports on a 5-year follow-up study of a prospective, non-randomised trial comparing classic Whipple procedure (PD) with Beger DPPHR.Materials and methodsSeventy patients were initially enrolled in this study. Fifty-one patients were left for the present long-term outcome analysis (PD, n = 24; DPPHR, n = 27). The follow-up included the following parameters: QOL, pain intensity, endocrine and exocrine function, and body mass index (BMI).ResultsThe median follow-up was 63.5 (range 56–67) months. Two patients in the DPPHR group and none in the PD group underwent a re-operation. The QOL scores of the relevant symptom scales (nausea, pain, diarrhoea) and functional parameters (physical status, working ability, global QOL) were significantly better in the DPPHR group than in the PD group. Pain intensity as self-assessed by the patients was less pronounced in the DPPHR group (P < 0.001), whereas the frequency of acute episodes and analgesic medication did not differ between the two groups. No difference was observed between the two groups with regard to endocrine and exocrine function. The values of the median body mass index (BMI) in the PD group [23.4 (range 18.5–25.0) kg/m2] and in the DPPHR group [24.2 (range 17.9–27.8) kg/m2] were comparable. The 5-year outcome remained stable compared to the early post-operative data published elsewhere.ConclusionThis 5-year long-term outcome analysis documents the superiority of the Beger duodenum-preserving technique over the classic Whipple procedure in terms of QOL and pain intensity as self-assessed by the patients.