Tim O. Lankisch

Implementation of proteomic biomarkers: Making it work

Eur J Clin Invest 2012; 42 (9): 1027–1036

Bile proteomic profiles differentiate cholangiocarcinoma from primary sclerosing cholangitis and choledocholithiasis

Early detection of malignant biliary tract diseases, especially cholangiocarcinoma (CC) in patients with primary sclerosing cholangitis (PSC), is very difficult and often comes too late to give the patient a therapeutic benefit. We hypothesize that bile proteomic analysis distinguishes CC from nonmalignant lesions. We used capillary electrophoresis mass spectrometry (CE‐MS) to identify disease‐specific peptide patterns in patients with choledocholithiasis (n = 16), PSC (n = 18), and CC (n = 16) in a training set. A model for differentiation of choledocholithiasis from PSC and CC (PSC/CC model) and another model distinguishing CC from PSC (CC model) were subsequently validated in independent cohorts (choledocholithiasis [n = 14], PSC [n = 18] and CC [n = 25]). Peptides were characterized by sequencing. Application of the PSC/CC model in the independent test cohort resulted in correct exclusion of 12/14 bile samples from patients with choledocholithiasis and identification of 40/43 patients with PSC or CC (86% specificity, 93% sensitivity). The corresponding receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.93 (95% confidence interval [CI]: 0.82‐0.98, P = 0.0001). The CC model succeeded in an accurate detection of 14/18 bile samples from patients with PSC and 21/25 samples with CC (78% specificity, 84% sensitivity) in the independent cohort, resulting in an AUC value of 0.87 (95% CI: 0.73‐0.95, P = 0.0001) in ROC analysis. Eight out of 10 samples of patients with CC complicating PSC were identified. Conclusion: Bile proteomic analysis discriminates benign conditions from CC accurately. This method may become a diagnostic tool in future as it offers a new possibility to diagnose malignant bile duct disease and thus enables efficient therapy particularly in patients with PSC. (HEPATOLOGY 2010;)

Gilbert's disease and atazanavir: from phenotype to UDP-glucuronosyltransferase haplotype.

Gilberts disease leads to intermittent non‐hemolytic hyperbilirubinemia by a reduction of hepatic bilirubin glucuronidation associated with the presence of the UDP‐glucuronosyltransferase (UGT) 1A1*28 polymorphism. It is considered benign because it does not result in hepatocellular damage. However, pharmacogenetic analyses have linked UGT1A1*28 to drug toxicity and cancer predisposition. The protease inhibitor atazanavir (ATV) is an inhibitor of hepatic UGT activity leading to hyperbilirubinemia in individual patients. Whether this is linked specifically to UGT1A1*28 or to more complex variants influencing glucuronidation is unclear. One hundred and six ATV‐treated patients were characterized and genotyped for UGT1A1*28, the UGT1A3 (‐66C) and UGT1A7 (‐57G) promoter variants, and UGT1A7129K/131K. ATV treatment increased median bilirubin levels from 10 to 41 μmol/L (P = .001) with hyperbilirubinemia exceeding 43 μmol/L in 37%. Hyperbilirubinemia over 43 μmol/L was significantly associated not only with UGT1A1*28 but also with UGT1A3‐66C, UGT1A7‐57G, and UGT1A7129K/131K, although these variants do not naturally occur in linkage dysequilibrium in blood donors. Homozygous combinations of UGT1A1*28 with the other variants increased from 7.4% (normal bilirubin to 42 μmol/L) to 41% to 46.1% (43 to >85 μmol/L), and 100% (>85 μmol/L). All six patients with hyperbilirubinemia greater than 85 μmol/L were homozygous for all four variants identifying a haplotype inherited on a single allele. In conclusion, the genetic variant associated with Gilberts disease is identified as part of a haplotype of four UGT1A variants spanning three genes at the UGT1A gene locus. This haplotype predisposes to hyperbilirubinemia in ATV treatment and may have an additional role as a pharmacogenomic risk factor for drug therapy. (HEPATOLOGY 2006;44:1324–1332.)

Urine proteomic analysis differentiates cholangiocarcinoma from primary sclerosing cholangitis and other benign biliary disorders

Background Diagnosis and curative treatment of cholangiocarcinoma (CC) often comes too late due to the lack of reliable tumour markers especially in patients with primary sclerosing cholangitis (PSC). The authors recently introduced bile proteomic analysis for CC diagnosis. Nevertheless, bile collection depends on invasive endoscopic retrograde cholangiography. The authors therefore evaluated urine proteomic analysis for non-invasive CC diagnosis. Methods Using capillary electrophoresis mass spectrometry the authors established a CC-specific peptide marker model based on the distribution of 42 peptides in 14 CC, 13 PSC and 14 benign biliary disorder (BBD) patients. Results In cross-sectional validation of 123 patients, the urine peptide marker model correctly classified 35 of 42 CC patients and 64 of 81 PSC and BBD patients with an area under the curve value of 0.87 (95% CI 0.80 to 0.92, p=0.0001, 83% sensitivity, 79% specificity). Evaluation of 101 normal controls resulted in 86% specificity. All 10 patients with CC on top of PSC were correctly classified. The majority of sequence-identified peptides are fragments of interstitial collagens with some of them also detected in blood indicating their extra-renal origin. Immunostaining of liver sections for matrix metallopeptidase 1 indicated increased activity of the interstitial collagenase in liver epithelial cells of CC patients. Conclusion The urine test differentiates CC from PSC and other BBD and may provide a new diagnostic non-invasive tool for PSC surveillance and CC detection.

Cutting Edge: Identification and Characterization of Human Intrahepatic CD49a+ NK Cells

Although NK cells are considered innate, recent studies in mice revealed the existence of a unique lineage of hepatic CD49a+DX5− NK cells with adaptive-like features. Development of this NK cell lineage is, in contrast to conventional NK cells, dependent on T-bet but not Eomes. In this study, we describe the identification of a T-bet+Eomes−CD49a+ NK cell subset readily detectable in the human liver, but not in afferent or efferent hepatic venous or peripheral blood. Human intrahepatic CD49a+ NK cells express killer cell Ig-like receptor and NKG2C, indicative of having undergone clonal-like expansion, are CD56bright, and express low levels of CD16, CD57, and perforin. After stimulation, CD49a+ NK cells express high levels of inflammatory cytokines but degranulate poorly. CD49a+ NK cells retain their phenotype after expansion in long-term in vitro cultures. These results demonstrate the presence of a likely human counterpart of mouse intrahepatic NK cells with adaptive-like features.

Endoscopic closure of esophageal intrathoracic leaks: stent versus endoscopic vacuum-assisted closure, a retrospective analysis

BACKGROUND AND STUDY AIM Placement of covered self-expanding metal or plastic stents (SEMS or SEPS) is an established method for managing intrathoracic leaks. Recently, endoscopic vacuum-assisted closure (EVAC) has been described as a new effective treatment option. Our aim was to compare stent placement with EVAC for nonsurgical closure of intrathoracic anastomotic leaks. PATIENTS AND METHODS In a retrospective analysis we were able to identify 39 patients who were treated with SEMS or SEPS and 32 patients who were treated with EVAC for intrathoracic leakage. In addition to successful fistula closure, we analyzed hospital mortality, number of endoscopic interventions, incidence of stenoses, and duration of hospitalization. RESULTS In a multivariate analysis, successful wound closure was independently associated with EVAC therapy (hazard ratio 2.997, 95 % confidence interval [95 %CI] 1.568 - 5.729; P = 0.001). The overall closure rate was significantly higher in the EVAC group (84.4 %) compared with the SEMS/SEPS group (53.8 %). No difference was found for hospitalization and hospital mortality. We found significantly more strictures in the stent group (28.2 % vs. 9.4 % with EVAC, P < 0,05). CONCLUSIONS EVAC is an effective endoscopic treatment option for intrathoracic leaks and showed higher effectiveness than stent placement in our cohort.

Gilbert's Syndrome and Irinotecan Toxicity: Combination with UDP-Glucuronosyltransferase 1A7 Variants Increases Risk

Background: Gilberts syndrome is characterized by a functional promoter single nucleotide polymorphism (SNP) of the UDP-glucuronosyltransferase (UGT) 1A1 gene and represents a pharmacogenetic risk factor for irinotecan toxicity, but study data remain controversial. The active CPT-11 metabolite 7-ethyl-10-hydroxycamptothecin is detoxified by several UGT1A proteins, which include UGT1A7 with a high specific activity that may contribute to the risk of irinotecan toxicity in Gilberts syndrome patients. Methods: Genotyping of the UGT1A1*28, UGT1A7 N129K/R131K, and UGT1A7-57T/G variants was done in 105 irinotecan-treated patients with metastatic colorectal cancer; adverse events were documented during all 297 treatment cycles and analyzed by Cochran-Mantel-Haenszel, Mann-Whitney, and χ2 tests. Results: The presence of UGT1A7 but not UGT1A1 variants was associated with at least one adverse event. In patients combining all three variants, thrombocytopenia and leukopenia were significantly more frequent. The overall incidence of adverse events was significantly higher (P = 0.0035) in carriers of the UGT1A risk alleles, who also had significantly higher rate of dose reductions. Conclusions: Irinotecan toxicity is more likely in patients with Gilberts syndrome carrying the UGT1A1*28 allele combined with reduced function UGT1A7 N129K/R131K and UGT1A7-57T/G SNP. Based on the ability of UGT1A7 to metabolize and eliminate the active irinotecan metabolite 7-ethyl-10-hydroxycamptothecin, the UGT1A1/UGT1A7 SNP combination haplotype appears to be a superior risk predictor than Gilberts syndrome alone. (Cancer Epidemiol Biomarkers Prev 2008;17(3):695–701)

Routine bile collection for microbiological analysis during cholangiography and its impact on the management of cholangitis.

BACKGROUND Antibiotic treatment of cholangitis is often insufficient because of inappropriate antibiotic use or bacterial resistance. OBJECTIVE To evaluate the role of routine bile collection during endoscopic retrograde cholangiography or percutaneous transhepatic cholangiography for microbiological analysis in the antibiotic management of cholangitis and to identify risk factors of bacteriobilia. DESIGN Prospective, observational, diagnostic study. SETTING Hannover Medical School, Hannover, Germany. PATIENTS AND INTERVENTION This study involved 243 consecutive patients undergoing endoscopic retrograde cholangiography/percutaneous transhepatic cholangiography for biliary complications after orthotopic liver transplantation (27%), malignancy (27%), primary sclerosing cholangitis (15%), benign strictures (11%), and choledocholithiasis (8%). MAIN OUTCOME MEASUREMENTS Microbiological examination of bile samples. RESULTS Patients with biliary stents or who were receiving repeated interventions after orthotopic liver transplantation were at increased risk of bacteriobilia (P < .05). The rate of gram-positive monomicrobial infection was higher in patients with primary sclerosing cholangitis (P < .01). In 40 examinations, patients presented with preprocedural cholangitis although they were receiving antibiotics. According to bile culture results, the antibiotic treatment was modified to a more specific therapy in 72.5% of patients. In patients who developed cholangitis after endoscopic retrograde cholangiography (27 examinations), specific antibiotic treatment was started or refined in 67% of cases, based on bile culture results. LIMITATIONS Contamination of samples during intervention cannot be totally excluded. CONCLUSION Orthotopic liver transplantation, biliary stenting, and repeated interventions are risk factors of bacteriobilia. In our patients with primary sclerosing cholangitis, gram-positive monomicrobial infections were more common. A bile sample collected during cholangiography for microbiological analysis is a simple, potentially valuable, diagnostic tool in patients with cholangitis. Each center should recognize its own patterns of infection to ensure ideal targeted therapy.

Genetic variability of aryl hydrocarbon receptor (AhR)-mediated regulation of the human UDP glucuronosyltransferase (UGT) 1A4 gene

UDP glucuronosyltransferases (UGTs) play an important role for drug detoxification and toxicity. UGT function is genetically modulated by single nucleotide polymorphisms (SNPs) which lead to the expression of functionally altered protein, or altered expression levels. UGT1A4 activity includes anticonvulsants, antidepressants and environmental mutagens. In this study the induction of the human UGT1A4 gene and a potential influence of genetic variation in its promoter region were analyzed. SNPs at bp -219 and -163 occurred in 9% among 109 blood donors reducing UGT1A4 transcription by 40%. UGT1A4 transcription was dioxin inducible. Reporter gene experiments identified 2 xenobiotic response elements (XRE), which were functionally confirmed by mutagenesis analyses, and binding was demonstrated by electromobility shift assays. Constitutive human UGT1A4 gene expression and induction was aryl hydrocarbon receptor (AhR)-dependent, and reduced in the presence of SNPs at bp -219 and -163. AhR-mediated regulation of the human UGT1A4 gene by two XRE and a modulation by naturally occurring genetic variability by SNPs is demonstrated, which indicates gene-environment interaction with potential relevance for drug metabolism.

Family 1 uridine-5′-diphosphate glucuronosyltransferases (UGT1A): from Gilbert’s syndrome to genetic organization and variability

The human UDP-glucuronosyltransferase 1A gene locus is organized to generate enzymes, which share a carboxyterminal portion and are unique at their aminoterminal variable region. Expression is tissue-specific and overlapping substrate specificities include a broad spectrum of endogenous and xenobiotic compounds as well as many therapeutic drugs targeted for detoxification and elimination by glucuronidation. The absence of glucuronidation leads to fatal hyperbilirubinemia. A remarkable interindividual variability of UDP-glucuronosyltransferases is evidenced by over 100 identified genetic variants leading to alterations of catalytic activites or transcription levels. Variant alleles with lower carcinogen detoxification activity have been associated with cancer risk such as colorectal cancer and hepatocellular carcinoma. Genetic variants and haplotypes have been identified as risk factors for unwanted drug effects of the anticancer drug irinotecan and the antiviral proteinase inhibitor atazanavir. Glucuronidation and its variability are likely to represent an important factor for individualized drug therapy and risk prediction impacting the drug development and licensing processes.