Genes related to Hepatocellular (HCC) progression in Living donor (LD) and Deceased Donor (DD) Liver Transplant
National Institute of Diabetes & Digestive & Kidney Diseases
National Cancer Institute
Grant Number: 1RO1-DK069859
Welcome to the HCC molecular study. This proposal is nested in a network of leading liver transplantation centers established to accrue and follow a sufficient number of patients being considered for, and undergoing, LDLT in order to provide generalizable results from adequately powered studies. This network has established the Adult to Adult Living Donor Liver Transplantation Cohort Study (A2ALL) http://www.nih-a2all.org/ that will conduct both retrospective and prospective studies of LDLT. For comparison, A2ALL also recruits recipients of cadaveric livers. Six centers from the A2ALL group (Virginia Commonwealth University, Northwestern University, UCLA, UCSF, UNC, and University of Pennsylvania) plus the Data Collection Center (University of Michigan) will participate in the study. The Viral Epidemiology Branch Laboratory (National Cancer Institute) will support this study by providing a critical arm of the quality control program and data analysis.
Participant Institutions:
Mission and Statement: From the Principal Investigator
Funded by a grant from the National Institute of Diabetes & Digestive & Kidney Diseases
National Cancer Institute (RO1-DK069859), the GR2HHC Collaborative Group Project will focus on the application of developing technologies for gene expression profiling and complex trait genetics to advance our understanding of Hepatocellular carcinoma (HCC).
This proposal has the potential to recognize the genes implied in the origin and progression of HCV-HCC. In this application we propose the study of the molecular biology of HCC including the study of cirrhotic liver, pre-neoplatic lesions, and HCC at different stages. In addition, we will prospectively study the patients undergoing liver transplantation (since they are enlisted for LT) allowing the identification of key genes involved in recurrence or survival. Ultimately, the results from this proposal could provide a rationale for a molecular classification of the tumor able to predict outcomes and a guide for treatments. These studies will be the result of an outstanding multi-center collaborative effort, involving surgeons, hepatologists, molecular biologists, epidemiologists, and biostatisticians.
Thank you for your interest in our Project.
Robert A Fisher, M.D.
H.M. Lee Professor of Surgery and Pediatrics
Program Director Liver Transplant Program
Department of Surgery
Division of Transplantation Surgery
PROGRAM OVERVIEW
Principal Investigator: Dr Robert Fisher
Background and Significance: The incidence of liver cancer has been rising in US during the past 20-30 years, possibly due to the increased number of liver cirrhosis after hepatitis C infection. Because at the time of diagnosis with hepatocellular carcinoma (HCC) the malignancy already is at late stage and usually present with liver cirrhosis, surgical resection, the only curative treatment, is not an option for most of these patients. Liver transplantation represents the only potential curative treatment for HCC. However, the shortage of liver organ donation remains the major obstacle for liver transplantation. Making things worse, recurrence after liver transplantation remains one of the major obstacles to further prolonging survival of HCC patients. Investigators propose to identify and determine the changes in expression of genes that may be potentially involved in the development of HCC; in the progression from the liver cirrhosis to HCC or from transplantable to non-transplantable disease status; and in the prediction of post-transplantation survival. Findings of the proposed study will help distinguish HCC patients who would be best served by various treatment strategies. It is especially important to identify individual patients who receive the liver transplantation will have the best outcome given the limited number of organ donors available.
SPECIFIC AIMS
AIM 1: To investigate the genes involved in viral carcinogenesis and tumor initiation in patients with HCV with and without HCC, we will examine hepatic gene expression and LOH analysis in explanted livers from HCV-infected liver transplant recipients. We will compare gene expression and LOH among different patients, as well as from different sections of the same patient’s liver. Tissue specimens will range from early HCV cirrhosis to HCV cirrhosis and HCC. Normal liver tissue (from donors deceased donors) will be used for comparison analysis.
AIM 2: To examine the genes implicated in tumor progression in patients with HCV-HCC while waiting for transplantation, we will identify gene patterns that discriminate tumor progression from non-progression. To accomplish this, gene expression microarrays will be performed using RNA from liver biopsies at diagnosis time and/or prior to ablative procedure in HCV-HCC patients listed for transplantation.
AIM 3: To determine the potential role of these genetic markers in predicting post-transplantation outcomes in HCV-infected patients with HCC, we will perform survival analysis. Outcomes measures will be overall survival and cancer-free survival. In these analyses we will examine potential associations with genetic markers, clinical-pathological characteristics, and waiting time differences with LDLT compared to Deceased Donor Liver Transplant (DDLT).
Study organization: The total study duration will be five years consisting of 2 years of patient accrual and 3 years of patient follow-up. Patients will be recruited at every participating center, where clinical evaluation, collection of demographic information, requested tests, ablative procedures, and liver transplantation would be performed. All study data will be entered into the BioDBx electronic data entry system by study coordinators at each study site. This data will be encrypted and transferred to the DCC and stored on a secure server at the University of Michigan. All the requested specimens will be sent to Virginia Commonwealth University (VCU) monthly under the required shipping conditions and with the appropriated forms. The molecular studies will be performed at the Molecular Laboratory at VCU with the cooperation of the Viral Epidemiology Branch Laboratory (National Cancer Institute-NCI) evaluating 5% of the total samples (inter-laboratory quality control). Data analysis will be performed by VCU and NCI. The Tables and graphics below show the study aims with the minimum sample size required for statistical analysis, the schematic for patient flow through the study, the availability of samples from the A2ALL study, and the time-dependent analysis.
Gene Expression Analysis
Sub-Project leader: Valeria Mas, Ph.D.
The major risk factors for HCC development are now well defined and some of the multiple steps involved in hepatocarcinogenesis have been elucidated in recent years (3). However, no clear picture of how and in what sequence these factors interact at the molecular level has emerged yet. Malignant transformation of hepatocytes may occur as a consequence of various etiologies, such as chronic viral hepatitis, alcohol, and metabolic disorders, in the context of increased cellular turnover induced by chronic liver injury, regeneration and cirrhosis. Overwhelming lines of epidemiologic evidence have indicated that chronic infection with HCV poses a major risk towards the development of HCC (25,27,28). However, it remains controversial whether HCV plays a direct role in the pathogenesis of HCV-associated HCC or whether it merely serves an indirect role. Chronic inflammation and cirrhosis, accompanied by regenerative process, function as a tumor promoter, providing a pathway from chronic HCV infection to HCC. HCC usually arises after 2–4 decades of infection, typically in the context of underlying cirrhosis. The occurrence of HCC without cirrhosis is uncommon. By understanding the transition from benign through dysplastic to malignant nodules, we propose the study of the gene expression patterns at different stages, including: 1-Early cirrhosis, 2- Advanced cirrhosis, 3- Hyperplastic nodules, 4- HCC (T1-4) (Aim 1).
Recent advances in diagnostic modalities, such as imaging techniques and the measurement of serum tumor markers, have improved the rate of early detection of HCC, and therapeutic approaches to HCC have also progressed (1-6). However, the long-term survival of HCC patients continues to be poor because of the high incidence of recurrence within the liver after initial treatment (52-56). Systematic analysis of gene expression patterns provides an insight into the biology and pathogenesis of HCC. A molecular understanding of HCC progression before liver transplantation is an important step toward the identification of predictive markers and more specific targets for HCC recurrence. Two tumors in the same diagnostic category may disseminate at drastically different rates or show completely different responses to therapy. These extreme differences in the outcomes indicate that the biology and behavior of the tumors are different. In this step of the study we will assess two important aspects of the disease: HCC staging and prognosis. We propose that if a molecular method predicting early recurrence could be achieved with high accuracy, it would be possible to make better decisions about the use of the different treatment options (Aim 2).
For monitoring the relative mRNA abundance of human genes GeneChip Human Genome U133A 2.0 will be used (Affymetrix). The GeneChip Human Genome U133A 2.0 is a single array representing 14,500 well-characterized human genes that can be used to explore human biology and disease progression. This microarray analyzes the expression level of 18,400 transcripts and variants. Sequences used in the design of the array were selected from GenBank, dbEST and RefSeq. Sequences were further analyzed for correct orientation, false priming, false clustering, alternative splicing and alternative polyadenilation (Affymetrix, Santa Clara, CA). Our selection of the Affymetrix GeneChipplatform assures that our molecular-based models will be accessible to other researchers and amenable to use in other sites.
Bioinformatics and Statistics Core
Sub-Project Leader: Kellie J Archer, Ph.D.
The Bioinformatics and Statistics Core will provide the bioinformatic and statistical support for this project. All data will flow to the Core for both statistical analysis and archiving. This will include developing and monitoring experimental designs for statistical integrity, performing data analyses for GR2HCC Project Aims using appropriate statistical, machine learning, and bioinformatic methods, and organizing the complex data sets generated by the Project to promote accessibilty to individual Investigators. Of particular importance is the integration of genomic data with clinical data entered into the web site at Scripps. Additionally, the Core will supervise the selection and statistical validation of the gene candidate set using the gene expression data. We plan to use a combination of traditional statistical methods as well as various supervised (class prediction) and unsupervised (clustering) machine learning methods
Quality Control (QC) Program
Laboratory QC (VCU: Valeria Mas, PhD-Kellie Archer, PhD)
1) Quality control protocol.
Study initiation: IRB approval required for study initiation.
Sub- specific Aims:
A- Quality of the samples to be analyzed:
- To evaluate tissue collection and storage at every Participating Center.
- To evaluate shipment conditions at every Participating Center.
- To study differences in RNA quality for explanted liver vs. needle biopsies.
- To evaluate pathological evaluation results at the different Institutions.
Inter-laboratory QC (VCU: Valeria Mas, PhD-Kellie Archer, PhD; NCI: Thomas O’Brien, MD, MPH, Ruth Pfeiffer, PhD)
The molecular studies will be performed at the Molecular Laboratory at VCU with the cooperation of the Viral Epidemiology Branch Laboratory (National Cancer Institute-NCI) evaluating 5% of the total samples (inter-laboratory quality control). Data analysis will be performed by VCU and NCI.
QC Protocol: Study Initiation
B- Interlaboratory Quality Control:
- To perform for a set of samples, RNA isolations using the same protocol at two different centers (VCU; Dr Mas and NCI: Dr O’Brien) Tissue samples will be provided by VCU.
- To run cDNA microarrays for those samples at the two different labs (cDNA, IVT, hybridization reactions using U133A 2.0 GeneChip (Affymetrix, Santa Clara, CA))
QC protocol design
C- Interpretation of the QC protocol initiation results, discussion with all the centers.
Link To:
Samples collected
Quality Control results from the RNA isolated from samples submitted from participant centers
Samples analyzed
Cell files
Public data:
Papers
Genes Associated With Progression and Recurrence of Hepatocellular Carcinoma in Hepatitis C Patients Waiting and Undergoing Liver Transplantation: Preliminary Results
Presentations
Session Minutes:
9/28/2006 Session Minutes
11/2/2006 Session Minutes
2/22/2007 Session Minutes