Proteomic analysis of HCV cirrhosis and HCV-induced HCC: identifying biomarkers for monitoring HCV-cirrhotic patients awaiting liver transplantation.

BACKGROUND: Progression from chronic hepatitis C virus (HCV) infection to cirrhosis and hepatocellular carcinoma (HCC) results in protein changes in the peripheral blood. We evaluated global protein expression in plasma samples of HCV-cirrhotic and HCV-cirrhotic-HCC patients. PATIENTS AND METHODS: Plasma samples from 25 HCV-cirrhotic-HCC and 10 HCV-cirrhotic patients were quantitatively evaluated for protein expression. Tryptic peptides were analyzed using Thermo linear ion-trap mass spectrometer (LTQ) coupled with a Surveyor HPLC system (Thermo). SEQUEST and X!Tandem database search algorithms were used for peptide sequence identification. Protein relative quantification was performed using the area under the curve from the select ion chromatogram. A significant fold change between groups was based on controlling the false discovery rate (FDR) at less than 5%. RESULTS: We identified and quantified 2320 proteins from the analysis of the different protein pattern between HCV-cirrhosis and HCV-HCC samples. Gene ontology terms classified the more important biologic process related to these proteins as signal transduction, regulation of transcription DNA-dependent, protein amino acid phosphorylation, cell adhesion, transport, and immune response. Seven proteins showed significant expression changes with a FDR less than 5% between cirrhosis and tumor groups. Moreover, 18 proteins showed significant expression changes (FDR <5%) when plasma samples from HCV-cirrhosis were compared with early HCV-HCC. CONCLUSIONS: Differential protein expression was observed between samples from HCV patients with cirrhosis with and without HCC. Also, differences were observed between early and advanced HCV-HCC samples. This study provides important information for discovery of potential biomarkers for early HCC diagnosis in HCV cirrhotic patients.

Genes involved in viral carcinogenesis and tumor initiation in hepatitis C virus-induced hepatocellular carcinoma.

The role of chronic hepatitis C virus (HCV) in the pathogenesis of HCV-associated hepatocellular carcinoma (HCC) remains controversial. To understand the transition from benign to malignant, we studied the gene expression patterns in liver tissues at different stages, including normal, cirrhosis, and different HCC stages. We studied 108 liver tissue samples obtained from 88 distinct patients (41 HCV-cirrhotic tissues, 17 HCV-cirrhotic tissues from patients with HCC, and 47 HCV-HCC tissues). Differentially expressed genes (DEG) were studied by use of high-density oligonucleotide arrays. Among probe sets identified as differentially expressed via the F test, all pairwise comparisons were performed. Cirrhotic tissues with and without concomitant HCC were further evaluated, and a classifier was used to predict whether the tissue type was associated with HCC. Differential expression profiles were analyzed using Interaction Networks and Functional Analysis. Characteristic gene signatures were identified when normal tissue was compared with cirrhosis, cirrhosis with early HCC, and normal with HCC. Pathway analysis classified the cellular and biological functions of the DEG as related to cellular growth and proliferation, cell death and inflammatory disease in cirrhosis; cell death, cell cycle, DNA replication, and immune response in early HCCs; and cell death, cell growth and proliferation, cell cycle, and DNA repair in advanced HCCs. Characteristic gene signatures were identified at different stages of HCV-HCC progression. A set of genes were identified to predict whether the cirrhotic tissue was associated with HCC.

Gene expression patterns in deceased donor kidneys developing delayed graft function after kidney transplantation.

BACKGROUND: Delayed graft function (DGF) after kidney transplantation (KTx) ranges between 2% and 50%. The mechanisms leading to DGF deserve special interest because DGF exerts negative influences on long-term outcomes. We studied gene expression profiles in deceased donor kidney (DDK) biopsies with and without DGF. METHODS: Gene expression profiling was performed on donor kidney tissues from 33 DDK with the use of microarrays. DDK were classified as grafts with immediate function (non-DGF; n=21) and grafts with DGF (n=12). DGF was defined as a dialysis requirement in the first week after transplantation. Demographic donor and recipient information was collected. The robust-multiarray average method was used to estimate probe set expression summaries. Logistic regression was used to identify genes significantly associated with DGF development. RESULTS: Patients were followed for 3 months after KTx. Thirty-eight probe sets (n=36 genes) were univariably differentially expressed in DDK with DGF when compared with DDK with non-DGF (alpha=0.001). Sixty-nine probe sets (n=65 genes) were differentially expressed in DDK with DGF when compared with DDK with non-DGF after adjusting for cold ischemia time (alpha=0.001). Gene ontology terms classified the overexpressed genes in DDK with DGF as principally related to cell cycle/growth (e.g., IGFBP5, CSNK2A2), signal transduction (e.g., RASGRP3), immune response (e.g., CD83, BCL3, MX1), and metabolism (e.g., ENPP4, GBA3). TNFRSF1B was overexpressed in DDK with DGF. CONCLUSIONS: Cold ischemia time was a predictor of DGF independently of the preservation method. We identified a set of 36 genes candidates of DGF in DDK, with genes involved in the inflammatory response being the more important.

Molecular pathways involved in loss of kidney graft function with tubular atrophy and interstitial fibrosis.

Loss of kidney graft function with tubular atrophy (TA) and interstitial fibrosis (IF) causes most kidney allograft losses. We aimed to identify the molecular pathways involved in IF/TA progression. Kidney biopsies from normal kidneys (n = 24), normal allografts (n = 6), and allografts with IF/TA (n = 17) were analyzed using high-density oligonucleotide microarray. Probe set level tests of hypotheses tests were conducted to identify genes with a significant trend in gene expression across the three groups using Jonckheere-Terpstra test for trend. Interaction networks and functional analysis were used. An unsupervised hierarchical clustering analysis showed that all the IF/TA samples were associated with high correlation. Gene ontology classified the differentially expressed genes as related to immune response, inflammation, and matrix deposition. Chemokines (CX), CX receptor (for example, CCL5 and CXCR4), interleukin, and interleukin receptor (for example, IL-8 and IL10RA) genes were overexpressed in IF/TA samples compared with normal allografts and normal kidneys. Genes involved in apoptosis (for example, CASP4 and CASP5) were importantly overexpressed in IF/TA. Genes related to angiogenesis (for example, ANGPTL3, ANGPT2, and VEGF) were downregulated in IF/TA. Genes related to matrix production-deposition were upregulated in IF/TA. A distinctive gene expression pattern was observed in IF/TA samples compared with normal allografts and normal kidneys. We were able to establish a trend in gene expression for genes involved in different pathways among the studied groups. The top-scored networks were related to immune response, inflammation, and cell-to-cell interaction, showing the importance of chronic inflammation in progressive graft deterioration.

Angiogenesis soluble factors as hepatocellular carcinoma noninvasive markers for monitoring hepatitis C virus cirrhotic patients awaiting liver transplantation.

BACKGROUND: Physiological angiogenesis occurs during liver regeneration, leading to the formation of new functional sinusoids. Pathological angiogenesis occurs in hepatocellular carcinoma (HCC). We aimed to evaluate the expression of angiogenic factors in hepatitis C virus (HCV)-HCC tissues and the utility of angiogenesis soluble factors as noninvasive markers of HCC and tumor growth. METHODS: Thirty-eight HCV-HCC tumors with 10 corresponding nontumor cirrhotic tissues, as well as 42 independent HCV cirrhotic and 6 normal liver tissues were studied using high-density oligonucleotide arrays. Human angiogenesis microarray was used for the protein detection of EGF, TIMP-1, TIMP-2, HGF, angiopn-1, angiopn-2, VEGF-A, IP-10, PDGF, KGF, angiogenin, VEGF-D, ICAM-1, and FGF in plasma samples from 40 patients (30 HCCs and 10 HCV cirrhosis). RESULTS: From the gene expression analysis of the HCV-HCC tumors compared to normal livers, we found an important number of genes related to angiogenesis differentially expressed (alpha=0.01), including VEGF, PDGF, AGPTL2, ANG, EGFL6, EGFR, angiopn-1, angiopn-2, ICAM2, TIMP-2, among others. Moreover, angiogenic genes were also differentially expressed when HCV-HCC samples were compared to HCV cirrhotic tissues (alpha=0.01; VEGF, EGFL3, EGFR, VEGFB, among others). Ten out of 14 angiogenic proteins analyzed were statistically differentially expressed between HCV cirrhosis and HCV-HCC groups (TIMP-1, TIMP-2, HGF, angiopn-1, angiopn-2, VEGF-A, IP-10, PDGF, KGF, and FGF; P<0.05). In addition, we observed that angiopn-2 was the most significant predictor (area under the curve: 0.83). CONCLUSION: Differentially expressed angiogenesis genes were observed between HCV patients with and without HCC. Soluble angiogenic factors might be useful for monitoring high-risk HCV patients.

Evaluation of gene panel mRNAs in urine samples of kidney transplant recipients as a non-invasive tool of graft function.

Non-invasive monitoring may be useful after kidney transplantation (KT), particularly for predicting acute rejection (AR). It is less clear whether chronic allograft nephropathy (CAN) is also associated with changes in urine cells. To identify non-invasive markers of allograft function in kidney transplant patients (KTP), mRNA levels of AGT, TGF-beta1, EGFR, IFN-gamma, TSP-1, and IL-10 in urine (Ur) samples were studied using QRT-PCR. Ninety-five KTP and 111 Ur samples were evaluated. Patients (Pts) were divided as, within six months (N = 31), and with more than six months post-KT (N = 64). KTP with more than six months post-KT were classified as KTP with stable kidney function (SKF) (N = 32), KTP with SKF (creatinine < 2 mg/dL) and proteinuria > 500 mg/24 h (N = 18), and KTP with biopsy proven CAN (N = 14). F-test was used to test for equality of variances between groups. IL-10 mRNA was decreased in Ur samples from KTP with less than six months post-KT (P = 0.005). For KTR groups with more than six months post-KT, AGT and EGFR mRNA were statistically different among KTP with SKF, KTP with SKF and proteinuria, and CAN Pts (P = 0.003, and P = 0.01), with KTP with SKF having higher mean expression. TSP-1 mRNA levels also were significantly different among these three groups (P = 0.04), with higher expression observed in CAN Pts. Using the random forest algorithm, AGT, EGFR, and TGF-beta1 were identified as predictors of CAN, SKF, SKF with proteinuria. A characteristic pattern of mRNA levels in the different KTP groups was observed indicating that the mRNA levels in Ur cells might reflect allograft function.

Genes associated with progression and recurrence of hepatocellular carcinoma in hepatitis C patients waiting and undergoing liver transplantation: preliminary results.

BACKGROUND: Liver transplantation (LT) represents a curative treatment for small hepatocellular carcinoma (HCC). Potentially curable higher-stage HCC patients are denied LT due to the lack of cancer markers that predict progression and recurrence. METHODS: Thirty-eight candidates for LT with hepatitis C virus (HCV) cirrhosis and HCC were studied. Gene expression (Gexp) analysis of tumor samples was performed using microarrays. RESULTS: Twenty patients underwent transplantation, 13 progressed while waiting for transplantation, 4 are alive awaiting transplantation, and 1 died without progression while waiting for LT. Differences in GExp among patients who underwent LT or did not progress (n=25) versus those whose disease progressed while waiting for LT (n=13) were assessed. Thus, 54 probe sets (Pset) were significantly differentially expressed. Among LT patients, 10 Psets were differentially expressed between LT patients with the same explanted stage that recurred (n=5) versus LT patients who did not recur (n=5). Ninety-eight Psets were significantly associated with survival at the alpha=0.005 level. CONCLUSIONS: Here, we have identified genes associated with HCC progression in HCV-HCC patients awaiting LT transplantation. A limited number of genes were related to overall survival and cancer-free survival after LT. Incorporation of these molecular markers could help to improve organ allocation for HCV-HCC patients.

Establishing the molecular pathways involved in chronic allograft nephropathy for testing new noninvasive diagnostic markers.

BACKGROUND: Chronic allograft nephropathy (CAN) is a cause of graft loss. The multistage processes that result in CAN are poorly understood. Noninvasive assays for detecting allograft dysfunction and predicting long-term outcomes are a priority in transplantation (Tx). METHODS: Renal tissue from kidney transplant patients (KTP) with CAN (n=11) and normal kidneys (NK; n=7) were studied using microarrays. Markers resulting from the microarray analysis (transforming growth factor [TGF]-beta, epidermal growth factor receptor [EGFR], angiotensinogen [AGT]) were tested in urine (Ur) and peripheral blood (PB) samples from the CAN patients (collected at the biopsy time) using reverse-transcriptase real-time polymerase chain reaction. Ur and PB samples from long-term KTP with stable renal function (SRF; n=20) were used as control. RESULTS: Assuming unequal variances between CAN and NK, using a false discovery rate of 0.005, and running 1,000 of all possible permutations, 728 probe sets were differentially expressed. Genes related to fibrosis and extracellular matrix deposition (i.e., TGF-beta, laminin, gamma 2, metalloproteinases-9, and collagen type IX alpha 3) were up-regulated. Genes related to immunoglobulins, B cells, T-cell receptor, nuclear factor of activated T cells, and cytokine and chemokines receptors were also upregulated. EGFR and growth factor receptor activity (FGFR)2 were downregulated in CAN samples. AGT, EGFR, and TGF-beta levels were statistical different in urine but not in blood samples of CAN patients when compared to KTP with SRF (P<0.001, P=0.04, and P<0.001, respectively). CONCLUSIONS: Genes related to fibrosis, extracellular matrix deposition, and immune response were found up-regulated in CAN. Markers resulting from the microarray analysis were differentially expressed in Ur samples of the CAN patients and in concordance with the microarray profiles.

Differentially expressed genes between early and advanced hepatocellular carcinoma (HCC) as a potential tool for selecting liver transplant recipients.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Liver transplantation (LT) represents a curative treatment for "small" HCC. Preoperative staging is critical in selecting optimal candidates for LT to optimize the use of this scarce resource. From December 1997 to February 2004, 148 patients diagnosed with cirrhosis and HCC were evaluated at our center. After staging, the patients were listed for LT according to United Network for Organ Sharing (UNOS) criteria. When pretransplant liver MRIs were compared with the findings of the explanted livers, 8 of 35 patients (22.8%) were understaged. Three of the 8 patients (37.5%) had recurrence post-LT. A retrospective gene expression profiling study was done using microarray technology for tumor samples in the pretransplant hepatitis C virus (HCV)-HCC understaged patients and in a contemporaneous group of HCV-HCC patients that were accurately staged. Two sample t tests comparing the early versus advanced HCV-HCCs with respect to gene expression showed an important set of genes differentially expressed among the samples. Hierarchical clustering analysis of the gene expression profiling classified 93.8% of the total tumor samples and 85.7% of the understaged samples in concordance with the explanted pathological staging. We found a distinctive pattern of gene expression between early and advanced HCV-HCCs. These results suggest that gene expression profiling could improve the pre-LT HCC staging to more closely mimic the explant pathology. Whether gene expression profiling of HCC will be refined to the point of predicting potential metastatic biologic behavior to predict post-LT recurrence will require longitudinal prospective study of this gene array technology.

Molecular techniques for identifying HCC origin and biology after orthotopic liver transplantation.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Liver transplantation represents the potentially curative treatment for small HCC. Recurrence after surgical resection and liver transplantation remains one of the major obstacles in further prolonging survival of patients with HCC. In the new liver, HCC might be of recipient or donor origin. One approach for investigating this question is by performing human identification and/or engraftment analysis. Distinction between recurrent and de novo HCC after orthotopic liver transplantation could allow for the development of important clinical and therapeutic strategies. Polymerase chain reaction amplification of highly polymorphic short tandem repeat DNA sequences, gene expression profiling, and fluorescence in situ hybridization were applied in a patient who developed a second HCC after orthotopic liver transplantation from an opposite gender donor. These techniques provided consistent evidence that the second HCC was a recurrence of the primary tumor.

Study of mRNA growth factors in urinary cells of kidney transplant recipients as predictors of chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN) is responsible for a significant proportion of graft loss. Current diagnostic methods for CAN are inadequate, and noninvasive assays for detecting allograft dysfunction/rejection and predicting long-term outcomes are a priority in transplantation. METHODS: Urine samples were collected from 48 kidney transplant recipients (KTR): 18 recipients with stable graft function (creatinine levels<2.0 mg/dL) and proteinuria of less than 500 mg/24 hr (Group 1); 18 recipients with stable graft function and proteinuria of greater than 500 mg/24 hr (Group 2); and 12 recipients with biopsy confirmed CAN. Urinary cell levels of transforming growth factor-beta1 (TGF-beta1) mRNA or epidermal growth factor (EGF) mRNA were measured using real-time quantitative PCR assay, and levels were correlated with renal allograft status. The integrity of RNA isolated from urine sediments was also assessed using the Agilent 2100 Bioanalyzer. RESULTS: Urinary cell TGF-beta1 mRNA levels were higher in the CAN group compared to Group 1 (P<0.0001) or Group 2 (P<0.0001). Urinary cell EGF mRNA levels were higher in Group 1 compared to Group 2 (P<0.0001) or the CAN group (P<0.0001). There were no significant differences in the urinary cell levels of EGF mRNA between patients with greater than 500 mg/24 hr proteinuria (Group 2) and the CAN group (P=0.75). CONCLUSION: These results demonstrate that urinary cell TGF-beta1 mRNA levels distinguish CAN patients from long-term transplant patients with stable renal function and varied levels of proteinuria. Urinary cells may be a good resource for the noninvasive diagnosis of CAN.

Apolipoprotein E genotypes as predictors of high-risk groups for developing hyperlipidemia in kidney transplant recipients undergoing sirolimus treatment.

BACKGROUND: Hypercholesterolemia (HCHL) and hypertriglyceridemia (HTRG) have emerged as the most significant metabolic consequences of therapy with sirolimus. Lipid status can be exacerbated by a variety of factors in the posttransplant setting, including genetic factors. Apoliprotein E (Apo E) polymorphism is an established genetic risk factor for hyperlipidemia. We studied the association between Apo E gene polymorphisms and lipids after kidney transplantation in patients undergoing sirolimus treatment. METHODS: We studied 98 kidney transplant patients (KTP) with stable renal allograft undergoing sirolimus treatment: 39 with HCHL and HTRG within 90 days postsirolimus treatment (PST) and 59 without hyperlipidemia PST. Apo E genotyping was performed using INNO-LiPA-ApoE. RESULTS: The cholesterol and the triglyceride values between the groups were 323.3+/-71.6 vs. 180.9+/-31.2 mg/dL (P<0.001) and 318.9+/-97.2 vs. 159.7+/-38.7 mg/dL (P<0.001). There was a significant difference in the genotype distribution of the hyperlipidemia and normal groups (P=0.009) with the percentages in each group as follows: E2/2 and E3/2: 12.8 vs. 5.1%; E3/3: 69.2% vs. 86.4%; and E4/3 and E4/4: 18.0% vs. 8.5%. We observed a higher number of patients with the genotype E3/3 in the group without hyperlipidemia PST (P=0.039). E3/2 and E4/4 genotype frequencies were higher in patients with hyperlipidemia PST. LDL levels in the hyperlipidemia PST group was statistical significant higher (P<0.001) and we observed an association between Apo E allelic distribution and LDL (P=0.005). CONCLUSIONS: Genetic factors, as Apo E genotypes, could allow the early identification of patients who are at a high risk for developing hyperlipidemia PST.