C. Desai

LC-MS/MS-based serum proteomics for identification of candidate biomarkers for hepatocellular carcinoma

Associating changes in protein levels with the onset of cancer has been widely investigated to identify clinically relevant diagnostic biomarkers. In the present study, we analyzed sera from 205 patients recruited in the United States and Egypt for biomarker discovery using label‐free proteomic analysis by LC‐MS/MS. We performed untargeted proteomic analysis of sera to identify candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. We further evaluated the significance of 101 proteins in sera from the same 205 patients through targeted quantitation by MRM on a triple quadrupole mass spectrometer. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the United States and Egyptian cohorts. Among the 21 candidates, ten were previously reported as HCC‐associated proteins (eight exhibiting consistent trends with our observation), whereas 11 are new candidates discovered by this study. Pathway analysis based on the significant proteins reveals upregulation of the complement and coagulation cascades pathway and downregulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. The results of this study demonstrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to evaluate changes in protein levels and discover novel diagnostic biomarkers. All MS data have been deposited in the ProteomeXchange with identifier PXD001171 (http://proteomecentral.proteomexchange.org/dataset/PXD001171).

LC-MS profiling of N-Glycans derived from human serum samples for biomarker discovery in hepatocellular carcinoma.

Defining clinically relevant biomarkers for early stage hepatocellular carcinoma (HCC) in a high-risk population of cirrhotic patients has potentially far-reaching implications for disease management and patient health. Changes in glycan levels have been associated with the onset of numerous diseases including cancer. In the present study, we used liquid chromatography coupled with electrospray ionization mass spectrometry (LC–ESI-MS) to analyze N-glycans in sera from 183 participants recruited in Egypt and the U.S. and identified candidate biomarkers that distinguish HCC cases from cirrhotic controls. N-Glycans were released from serum proteins and permethylated prior to the LC–ESI-MS analysis. Through two complementary LC–ESI-MS quantitation approaches, global profiling and targeted quantitation, we identified 11 N-glycans with statistically significant differences between HCC cases and cirrhotic controls. These glycans can further be categorized into four structurally related clusters, matching closely with the implications of important glycosyltransferases in cancer progression and metastasis. The results of this study illustrate the power of the integrative approach combining complementary LC–ESI-MS based quantitation approaches to investigate changes in N-glycan levels between HCC cases and patients with liver cirrhosis.

Survival of cystic fibrosis patients undergoing liver and liver-lung transplantations.

BACKGROUND We evaluated the outcome of combined liver-lung transplantation (L-LTx) in cystic fibrosis (CF) patients with liver transplantation (LTx) for CF liver disease. METHODS The United Network for Organ Sharing (UNOS) data were analyzed from October 1987 to August 2009. RESULTS Of 294 patients (210 children), 265 (90.1%) received an LTx and 29, an L-LTx. Patient survival was: adult LTx, 80%, 74%, and 67% at 1, 3, and 5 years, and L-LTx, 72%, 61.4%, and 61.4% (P = .7); pediatric LTx, 85%, 82%, and 74% at 1, 3, and 5 years, and L-LTx, 83%, 83%, and 83% (P = .4). Pediatric patients had a slight survival advantage over adults for LTx (P = .08). Graft survival, not affected by immunosuppression regimens, was similar to patient survival. CONCLUSIONS The outcome of L-LTx appears similar to LTx in CF providing support for the prospect of a combined transplant.

Developing Trends in the Intestinal Transplant Waitlist

The United Network for Organ Sharing database was examined for trends in the intestinal transplant (ITx) waitlist from 1993 to 2012, dividing into listings for isolated ITx versus liver‐intestine transplant (L‐ITx). Registrants added to the waitlist increased from 59/year in 1993 to 317/year in 2006, then declined to 124/year in 2012; Spline modeling showed a significant change in the trend in 2006, p < 0.001. The largest group of registrants, <1 year of age, determined the trend for the entire population; other pediatric age groups remained stable, adult registrants increased until 2012. The largest proportion of new registrants were for L‐ITx, compared to isolated ITx; the change in the trend in 2006 for L‐ITx was highly significant, p < 0.001, but not isolated ITx, p = 0.270. New registrants for L‐ITx, <1 year of age, had the greatest increase and decrease. New registrants for isolated ITx remained constant in all pediatric age groups. Waitlist mortality increased to a peak around 2002, highest for L‐ITx, in patients <1 year of age and adults. Deaths among all pediatric age groups awaiting L‐ITx have decreased; adult L‐ITx deaths have dropped less dramatically. Improved care of infants with intestinal failure has led to reduced referrals for L‐ITx.

Outcome of Intestinal Transplants for Patients With Crohn's Disease

BACKGROUND The pathophysiology of Crohns disease (CD) is related to immune dysregulation making it unique among indications for intestinal transplants (ITx). We examined whether outcomes of ITx for CD are any worse than the overall ITx population. METHODS United Network for Organ Sharing Standard Transplant Analysis and Research files were analyzed. Adult ITx recipients from 1987 to 2009 were included. RESULTS Of 86 primary ITx for CD, 61 (70%) had isolated ITx and 25 (30%) had liver-ITx (L-ITx). The 1-, 3-, and 5-year patient survival for isolated ITx was 85%, 67%, and 54%; for L-ITx, 63%, 47%, and 41% (P = .04). The graft survival at 1, 3, and 5 years was 85%, 55%, and 45% for isolated ITx recipients and 63%, 47%, and 41% for L-ITx recipients (Wilcoxons test, P = .04). Patient and graft survival was better in era 2 (January 2001 through August 2009) than in era 1 (October 1987 through December 2000). In the regression analysis of long-term outcome of adults undergoing ITx, recipient age > 40 years and hospitalization prior to transplantation were negative predictors of outcome. CONCLUSION Patient and graft survival for CD patients is not inferior to other indications for ITx.

Pseudolymphoma (reactive lymphoid hyperplasia) of the liver: A clinical challenge.

Reactive lymphoid hyperplasia (RLH), also known as pseudolymphoma or nodular lymphoid lesion of the liver is an extremely rare condition, and only 51 hepatic RLH cases have been described in the literature since the first case was described in 1981. The majority of these cases were asymptomatic and incidentally found through radiological imaging. The precise etiology of hepatic RLH is still unknown, but relative high prevalence of autoimmune disorder in these cases suggests an immune-based liver disorder. Imaging features of hepatic RLH often suggest malignant lesions such as hepatocellular carcinoma and cholangiocarcinoma. In this report, we discuss two cases of hepatic RLH in patients with autoimmune hepatitis. We also present pathologic and magnetic resonance imaging findings, including one case utilizing a hepatocellular contrast agent, Eovist. Definitive diagnosis of hepatic RLH often requires surgical excision.

Paired Kidney Donor Exchanges and Antibody Reduction Therapy: Novel Methods to Ameliorate Disparate Access to Living Donor Kidney Transplantation in Ethnic Minorities

BACKGROUND Currently ethnic minority patients comprise 60% of patients listed for kidney transplantation in the US; however, they receive only 55% of deceased donor renal transplants and 25% of living donor renal transplants. Ethnic disparities in access to kidney transplantation result in increased morbidity and mortality for minority patients with end-stage renal disease. Because these patients remain dialysis dependent for longer durations, they are more prone to the development of HLA antibodies that further delay the possibility of receiving a successful kidney transplant. STUDY DESIGN Two to 4 pretransplant and post-transplant plasma exchanges and i.v. immunoglobulin were used to lower donor-specific antibody levels to less than 1:16 dilution; cell lytic therapy was used additionally in some cases. Match pairing by virtual cross-matching was performed to identify the maximal exchange benefit. Sixty candidates for renal transplantation were placed into 4 paired kidney exchanges and/or underwent antibody reduction therapy. RESULTS Sixty living donor renal transplants were performed by paired exchange pools and/or antibody reduction therapy in recipients whose original intended donors had ABO or HLA incompatibilities or both (24 desensitization and 36 paired kidney exchanges). Successful transplants were performed in 38 ethnic minorities, of which 33 were African American. Twenty-two recipients were white. Graft and patient survival was 100% at 6 months; graft function (mean serum creatinine 1.4 g/dL) and acute rejection rates (20%) have been comparable to traditional live donor kidney transplantation. CONCLUSIONS Paired kidney donor exchange pools with antibody reduction therapy can allow successful transplant in difficult to match recipients. This approach can address kidney transplant disparities.

Reuse of liver allografts from brain-dead liver transplant recipients

Sirs, A unique method of expanding the donor pool is to reuse allograft from recipients. Although there are case reports documenting retransplantation of livers, the concept has not been routinely adopted [1–7]. From June 2007, we implemented a policy of reusing liver allografts. We present three cases and extrapolate from our experience the potential for liver reprocurement. The first of our recipients was a 17-year-old girl with acute liver failure (ALF) after acetaminophen overdose. The patient progressed to grade 4 encephalopathy, severe coagulopathy, and renal failure. The intracranial pressure (ICP) ranged from 15 to 20 mmHg and the cerebral perfusion pressure ranged from 50 to 60 mmHg. A liver graft (Table 1) was procured in a standard fashion and the piggyback technique with a lateral cavoplasty was used to perform the recipient operation. The anastomosis involved the donor proper hepatic artery to the recipient common hepatic artery; 5 units of packed red blood cells (PRBC) and 8 units of fresh frozen plasma (FFP) were necessary. Postoperatively, the recipient progressed to brain-death. The family consented to organ donation. The second recipient of this liver was a 61-year-old white man with hepatitis C virus (HCV) and hepatocellular carcinoma (HCC – within Milan criteria) [United Network of Organ Sharing (UNOS) Model for End-Stage Liver Disease (MELD) score – 25 and the laboratory MELD score – 16]. After liver procurement, the inferior vena cava at the site of lateral cavoplasty was over-sewn. The recipient operation was done with the standard caval replacement technique. The hepatic artery was anastomosed without a conduit and the bile duct anastomosis was performed with a T-tube. No blood products were necessary. The patient was discharged from hospital on postoperative day 8. The first recipient in our second case was a 17-yearold boy (Table 1), with ALF from acetaminophen toxicity. He developed grade 4 encephalopathy; however, severe coagulopathy precluded ICP monitoring. A CT scan revealed mild cerebral edema and brainstem reflexes were intact. A liver allograft (Table 1) was transplanted with vena caval replacement. The hepatic artery was anastomosed without a conduit. The biliary anastomosis was end-to-end without a T-tube; 14 units of PRBC, 23 units of FFP, 10 units of cryoprecipitate, and factor VII were transfused. The patient, however, progressed to brain-death. The family consented for donation. The second recipient of this liver was a 57-year-old AfricanAmerican man with HCV cirrhosis and HCC within Milan criteria [UNOS MELD score – 25 and laboratory MELD score – 9]. The transplant was done with a standard caval replacement technique. The hepatic artery was anastomosed without a conduit and the biliary anastomosis was performed end-to-end without a T-tube. Two units of PRBC and FFP were transfused. Liver enzymes normalized on day 20 (Table 1). The patient was discharged 10 days after surgery. He had one episode of acute cellular rejection after 14 months, treated with steroids. The first recipient in our third case was a 16-year-old African-American boy with ALF after an overdose of an unknown drug, presenting with severe acidosis and coagulopathy. The donor liver (Table 1) had completely replaced right and left hepatic arteries, which were reconstructed on the back table. The transplant involved the piggyback technique; the suprahepatic vena cava was anastomosed to the ostia of the hepatic veins. The patient made an uneventful recovery. Fifty-two months following transplantation, the patient was declared brain-dead from a gunshot to the head. The family consented to donation. The second recipient of this liver was a 34-year-old African-American woman with cirrhosis from autoimmune hepatitis [MELD score of 40]. She had type 1 diabetes mellitus and renal failure requiring dialysis. The liver was procured with careful hilar dissection to avoid disturbance to the blood supply of the bile duct. The liver was transplanted using a standard technique with anastomoses of supra and infrahepatic vena cava. The donor’s common hepatic artery (without disturbing the reconstructed replaced vessels) was anastomosed to the recipient’s common hepatic artery and the bile duct was anastomosed in an end-to-end fashion. The right kid-

Complex Arterial Reconstruction in Multivisceral Transplantation

We report the case of a successful multivisceral transplant in which both donor and recipient presented aberrant anatomy of the celiac‐mesenteric axis requiring five separate arterial anastomoses to reconstruct the blood inflow to the graft.

Metabolomic Characterization of Hepatocellular Carcinoma in Patients with Liver Cirrhosis for Biomarker Discovery

Background: Metabolomics plays an important role in providing insight into the etiology and mechanisms of hepatocellular carcinoma (HCC). This is accomplished by a comprehensive analysis of patterns involved in metabolic alterations in human specimens. This study compares the levels of plasma metabolites in HCC cases versus cirrhotic patients and evaluates the ability of candidate metabolites in distinguishing the two groups. Also, it investigates the combined use of metabolites and clinical covariates for detection of HCC in patients with liver cirrhosis. Methods: Untargeted analysis of metabolites in plasma from 128 subjects (63 HCC cases and 65 cirrhotic controls) was conducted using gas chromatography coupled to mass spectrometry (GC-MS). This was followed by targeted evaluation of selected metabolites. LASSO regression was used to select a set of metabolites and clinical covariates that are associated with HCC. The performance of candidate biomarkers in distinguishing HCC from cirrhosis was evaluated through a leave-one-out cross-validation based on area under the receiver operating characteristics (ROC) curve. Results: We identified 11 metabolites and three clinical covariates that differentiated HCC cases from cirrhotic controls. Combining these features in a panel for disease classification using support vector machines (SVM) yielded better area under the ROC curve compared with alpha-fetoprotein (AFP). Conclusions: This study demonstrates the combination of metabolites and clinical covariates as an effective approach for early detection of HCC in patients with liver cirrhosis. Impact: Further investigation of these findings may improve understanding of HCC pathophysiology and possible implication of the metabolites in HCC prevention and diagnosis. Cancer Epidemiol Biomarkers Prev; 26(5); 675–83. ©2016 AACR.