Daniel G. Maluf

MicroRNAs as Biomarkers in Solid Organ Transplantation

Important progress has been made in improving short‐term outcomes in solid organ transplantation. However, long‐term outcomes have not improved during the last decades. There is a critical need for biomarkers of donor quality, early diagnosis of graft injury and treatment response. MicroRNAs (miRNAs) are a class of small single‐stranded noncoding RNAs that function through translational repression of specific target mRNAs. MiRNA expression has been associated with different diseases and physiological conditions. Moreover, miRNAs have been detected in different biological fluids and these circulating miRNAs can distinguish diseased individuals from healthy controls. The noninvasive nature of circulating miRNA detection, their disease specificity and the availability of accurate techniques for detecting and monitoring these molecules has encouraged a pursuit of miRNA biomarker research and the evaluation of specific applications in the transplant field. miRNA expression might develop as excellent biomarkers of allograft injury and function. In this minireview, we summarize the main accomplishments of recently published reports focused on the identification of miRNAs as biomarkers in organ quality, ischemia‐reperfusion injury, acute rejection, tolerance and chronic allograft dysfunction emphasizing their mechanistic and clinical potential applications and describing their methodological limitations.

Effects of interferon treatment on liver histology and allograft rejection in patients with recurrent hepatitis C following liver transplantation

Recurrent hepatitis C after liver transplantation remains a significant cause of graft loss and retransplantation. Although treatment of recurrent hepatitis C with interferon‐based regimens has become widely accepted as safe and can lead to sustained virologic clearance of hepatitis C virus (HCV) RNA, long‐term histologic improvement and the risk of precipitating graft rejection remain controversial. The present study is a retrospective evaluation of the clinical and histological consequences of treating recurrent hepatitis C with interferon‐based therapy in a selected group of liver transplant recipients. Twenty‐three liver transplant recipients with recurrent hepatitis C and histologic evidence of progressive fibrosis completed at least 6 months of interferon, 83% of whom received pegylated‐interferon α‐2b; only 4 tolerated ribavirin. Overall, 11 patients (48%) had undetectable HCV RNA at the end of 6 months of treatment. Of these patients, 3 remained HCV RNA–negative on maintenance interferon monotherapy for 33 months, and the other 8 (35%) completed treatment and remained HCV RNA–undetectable 24 weeks after discontinuation of interferon. Overall necroinflammatory activity in liver biopsies obtained 2 years after HCV RNA became undetectable decreased significantly (7.73 ± 2.37 vs. 5.64 ± 2.94 units before and after treatment, respectively; P = .016). However, 5 of these 11 patients had no histologic improvement in follow‐up liver histology. Liver biopsies in the 12 nonresponders demonstrated disease progression. Of the 23 patients treated with interferon, 8 (35%) had evidence of acute or chronic rejection on posttreatment liver biopsy, most of whom had no previous history of rejection (P < .01 for comparison of pretreatment and posttreatment prevalence of histologic rejection), and 2 experienced graft loss from chronic rejection, requiring retransplantation. In conclusion, interferon treatment of recurrent hepatitis C does not consistently improve histologic disease after virologic response, and it may increase the risk of allograft rejection. (Liver Transpl 2004;10:850–858.)

Histologic recurrence of chronic hepatitis C virus in patients after living donor and deceased donor liver transplantation

Hepatitis C virus (HCV) recurs in nearly all patients after liver transplantation. This recurrence is associated with progressive fibrosis and graft loss. It remains unclear whether the natural course of HCV recurrence is altered in patients who undergo living donor liver transplantation (LDLT). We conducted a prospective, controlled trial using protocol liver biopsies to evaluate the histologic outcome of recurrent HCV in 23 patients who underwent LDLT and 53 patients who underwent transplantation with a deceased donor liver (DDLT) during the same period of time. Patients who did not survive at least 6 months after transplantation or who had hepatocellular carcinoma or any other coexistent liver disease were excluded from analysis. All patients underwent protocol liver biopsy at 6 months and at 12 months and at yearly intervals thereafter. The mean age, sex, racial distribution, and serum HCV RNA and the percentage of patients with genotype 1 were similar in the 2 groups of patients. The model for end‐stage liver disease score at the time of transplantation was slightly lower in patients who underwent LDLT, but this difference was not significant. The distribution of immunosuppression agents used, the mean doses of calcineurin agents, the use of mycophenolate mofetil, and the dose and tapering schedule for prednisone were similar in both groups of patients. The mean duration of follow‐up was 40 months. No significant difference in either graft or patient survival or the percentage of patients who developed acute rejection was noted in the 2 groups of patients. At 48 months, graft and patient survival were 82% and 82% and 75% and 79% for patients who underwent DDLT and LDLT, respectively. The degree of hepatic inflammation increased stepwise over 3 years but was not significantly different in the 2 patient groups. In contrast, the mean fibrosis score and the percentage of patients with fibrosis increased stepwise after DDLT but appeared to plateau 12 months after LDLT. At 36 months, fibrosis was present in 78% of DDLT patients, and mean fibrosis score was 1.9, compared with 59% with fibrosis and a mean score of .9 after LDLT. In conclusion, these data strongly suggest that fibrosis progression from recurrent HCV is not more severe in patients after LDLT. (Liver Transpl 2004;10:1248–1255.)

A prospective evaluation of fibrosis progression in patients with recurrent hepatitis C virus following liver transplantation

Recurrence of hepatitis C virus (HCV) following liver transplantation (LT) is universal. A subset of these patients develop advanced fibrosis and cirrhosis and it is believed that this leads to increased posttransplantation mortality. The specific aims of this study were to determine the incidence of advanced fibrosis and those factors associated with this process, and to evaluate causes for mortality in patients with recurrent HCV. A total of 227 patients who underwent LT with chronic HCV were monitored prospectively. The mean age of this group at LT was 49.5 yr; 76% were male and 85% were Caucasian. Fibrosis progression was monitored by protocol liver biopsy, initially performed 6 months after LT and then at 6‐ to 24‐month intervals. Advanced fibrosis, defined as the bridging fibrosis or cirrhosis, developed in 1%, 11%, 25%, and 41% of patients after 1, 3, 5, and 6‐10 yr, respectively. Acute cellular rejection hepatic steatosis, a persistent elevation in serum alanine aminotransferase and donor‐race were associated with the development of advanced fibrosis. In contrast, the development of advanced fibrosis was not affected by the use of interferon prior to undergoing LT, cytomegalovirus disease, or donor age. A total of 60 patients (26%) died over 15 yr of follow‐up. Although graft failure accounted for 45% of deaths in patients with advanced fibrosis, this represented only 8% of all deaths in patients with recurrent HCV. Sepsis was the most common cause of death and this was observed with similar frequency in patients who developed advanced fibrosis (45%) and in those with less advanced fibrosis (47%). In conclusion, approximately 41% of patients with recurrent HCV developed advanced fibrosis 6‐10 yr after LT. However, complications associated with sepsis, not recurrent cirrhosis, was the most common cause of death in patients with recurrent HCV and this was similar in patients with or without advanced fibrosis. Liver Transpl 13:975–983, 2007.

MicroRNA profiles in allograft tissues and paired urines associate with chronic allograft dysfunction with IF/TA

Despite the advances in immunosuppression, renal allograft attrition over time remains unabated due to chronic allograft dysfunction (CAD) with interstitial fibrosis (IF) and tubular atrophy (TA). We aimed to evaluate microRNA (miRNA) signatures in CAD with IF/TA and appraise correlation with paired urine samples and potential utility in prospective evaluation of graft function. MiRNA signatures were established between CAD with IF/TA versus normal allografts by microarray. Validation of the microarray results and prospective evaluation of urine samples was performed using real‐time quantitative‐PCR (RT‐qPCR). Fifty‐six miRNAs were identified in samples with CAD‐IF/TA. Five miRNAs were selected for further validation based on array fold change, p‐value and in silico predicted mRNA targets. We confirmed the differential expression of these five miRNAs by RT‐qPCR using an independent set of samples. Differential expression was detected for miR‐142‐3p, miR‐204, miR‐107 and miR‐211 (p < 0.001) and miR‐32 (p < 0.05). Furthermore, differential expression of miR‐142‐3p (p < 0.01), miR‐204 (p < 0.01) and miR‐211 (p < 0.05) was also observed between patient groups in urine samples. A characteristic miRNA signature for IF/TA that correlates with paired urine samples was identified. These results support the potential use of miRNAs as noninvasive markers of IF/TA and for monitoring graft function.

Surveillance for Hepatocellular Carcinoma in Patients with Cirrhosis Improves Outcome

OBJECTIVE Liver transplantation has become an effective treatment for cirrhotic patients with early-stage hepatocellular carcinoma. We hypothesized that the quality of surveillance for hepatocellular carcinoma influences prognosis by affecting access to liver transplantation. METHODS A total of 269 patients with cirrhosis and hepatocellular carcinoma were retrospectively categorized into 3 groups according to quality of surveillance: standard-of-care (n=172) (group 1); substandard surveillance (n=48) (group 2); and absence of surveillance in patients not recognized to be cirrhotic (n=59) (group 3). RESULTS Three-year survival in the 60 patients who underwent liver transplantation was 81% versus 12% for patients who did not undergo transplantation (P<.001). The percentages of patients who underwent transplantation according to tumor stage at diagnosis (T1, T2, T3, and T4) were 58%, 35%, 10%, and 1%, respectively. Hepatocellular carcinoma was diagnosed at stages 1 and 2 in 70% of patients in group 1, 37% of patients in group 2, and only 18% of patients in group 3 (P <.001). Liver transplantation was performed in 32% of patients in group 1, 13% of patients in group 2, and 7% of patients in group 3 (P<.001). Three-year survival from cancer diagnosis in patients in group 3 (12%) was significantly worse than in patients in group 1 (39%) or group 2 (27%) (each P<.05). Eighty percent of patients in group 3 had subtle abnormalities of cirrhosis on routine laboratory tests. CONCLUSION The quality of surveillance has a direct impact on hepatocellular carcinoma stage at diagnosis, access to liver transplantation, and survival.

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.

Adult Living Donor Versus Deceased Donor Liver Transplantation: A 6‐Year Single Center Experience

No long‐term (>3 years) prospective comparison of adult‐to‐adult living donor liver transplantation (A2ALLTx) to adult deceased donor liver transplantation (ADDLTx) has been reported.

Utilization of extended donor criteria liver allograft : Is the elevated risk of failure independent of the model for end-stage liver disease score of the recipient?

Background. The goal of this analysis was to determine if outcomes from the use of extended criteria donor (ECD) livers were dependent upon the Model for End-Stage Liver Disease (MELD) score of the recipient. Methods. The Organ Procurement and Transplantation Network (OPTN) database as of March 4, 2006 was used for the analysis. Data from 12,056 adult liver transplant (LTx) recipients between June 1, 2002 and June 30, 2005 was analyzed. The donor risk index (DRI) was calculated as previously reported. A DRI of ≥1.7 was classified as ECD. Relative risk (RR) estimates were derived from Cox regression models adjusted for DRI, recipient MELD, age, sex, ethnicity, diagnosis, and year of transplant. Results. Data from 2,873 grafts falling in the ECD category (23.8%) and their recipients were analyzed. Recipients with low MELD scores (<15) received the highest proportion of ECD livers (33%). ECD livers were associated with a significant increase in the RR of graft failure within each MELD category. However, this effect held within each of the three MELD categories. Conclusion. The use of ECD grafts expands the organ pool at expense of increased RR of liver failure. Our analysis showed no significant interaction between DRI and MELD score of the recipient. The fact that there is no additional impact of ECD livers in recipients with high MELD scores suggests that this group of patients may benefit from this pool of grafts.

Non-resective ablation therapy for hepatocellular carcinoma: effectiveness measured by intention-to-treat and dropout from liver transplant waiting list

Abstract:  Background:  Orthotopic liver transplantation (OLT) for patients with small hepatocellular carcinoma (HCC) is widely accepted, and the usefulness of local ablation techniques as a bridge for liver transplantation is still under investigation.