PAAR Abstract, 2000

Pharmacogenetics of Anticancer Agents Research Group

The co-existence of genetic polymorphisms in drug metabolizing enzymes, targets, receptors, and transporters, in the context of drug and non-drug influences, result in high frequencies of unusual drug reaction phenotypes. It is the goal of the PAAR Group to elucidate the contribution of such genetic polymorphisms to anticancer drug pharmacodynamics.

Projects

Liver Tissue Core

Investigator: Mary V. Relling

The Human Liver Tissue Core stores tissue from human liver donors and imposes standardized procedures for making subcellular fractions such as microsomes, cell membranes, RNA, DNA, and cytosolic fractions. The information is stored in the centralized access database, again created with the help of Dr. Nadkarni, at Yale. The liver resource is overseen by Drs. Mary Relling at St. Jude Children's Research Hospital and Eileen Dolan at the University of Chicago, and both work closely with Dr. Stephen Strom at the University of Pittsburgh. There are standardized procedures for accessing the resource and further information can be obtained by e-mailing mary.relling@stjude.org.

Lymphoblastoid Cell Line Core

Investigators: Amittha Wickrema

The Lymphoblastoid Cell Line Core has established 168 lymphoblastoid cell lines as 84 sets of two siblings from various ethnic backgrounds. Also maintained are cell lines from the Coriell Tissue Resource for PAAR phenotyping and genotyping studies.

All cell lines are maintained in cryogenic storage until a request is made to grow and provide cells, DNA or RNA for investigators. A database has been created and pharmGKB numbers assigned with assistance of Dr. Prakash from Yale and further information on the availability of cell lines can be obtained by e-mailing awickrem@medicine.bsd.uchicago.edu.

Molecular Genetics Core

Investigator: Edwin Cook and Soma Das

This Core is responsible for polymorphism identification and genotyping for the majority of the PAAR projects. A number of techniques are used for genotyping, and are individualized for each project. Genotyping methods are under continuing development, but currently include SNP genotyping by FP-TDI, SNaPshot, single-plex or duplex SBE-dHPLC, TaqMan, PCR-RFLP, and multiplexed microsatellite genotyping ranging from single markers to genome scans. Polymorphism identification is by PCR-direct sequencing with PolyPhred analysis linked to PAARManager. PAARManager, written by and under ongoing development by Xiaolin Wu facilitates online review of sequencing-based SNP identification include trace-viewing capability, integration of polymorphism data for multiple projects, a platform for integration of Molecular Genetics Core data with data from other cores, an interface for uploading data to the Genotyping Database (see below), and a graphical interface to track sequencing coverage.

Carboxylesterase Project

Investigator: M. Eileen Dolan

Dr. Dolan's CE project focuses on identifying polymorphisms (SNPs) in human carboxylesterase I and/or II responsible for phenotypic variability in CPT-11 conversion to SN-38. Using the human liver bank tissue resource, SNPs within exons and introns are identified. Possible splice variants of different transcript lengths are also identified and used to pinpoint intronic polymorphisms that may be important in inter-individual variability in these genetic targets.

UGT Project

Investigator: Mark J. Ratain

The UDP-glucuronosyltransferase transferases, UTG1A1, UGT1A9, and UTG2B7 are being assessed for their importance in the metabolism of SN-38, flavopiridol, morphine, and epirubicin. A study UGT1A haplotype structure is underway in Dr. Di Rienzo's laboratory.

CYP3A4/PXR Discovery Project

Investigator: Erin Schuetz

Dr. Schuetz has taken an approach of using outliers in terms of phenotypes to discover variability in CYP3A4, CYP3A5, its regulator PXR, as well as P-glycoprotein. Using the human liver tissue resource, transcript lengths are identified and used to pinpoint intronic polymorphisms that may be important in inter-individual variability in these genetic targets. Follow up genomic DNA sequencing is used to assess the frequency of genotypes in outlying individuals as well as in specific ethnic and racial subgroups sometimes using the Coriell tissue resources.

Pharmacodynamic Discovery Project

Investigator: Eileen Dolan

Dr. Dolan is determining whether there are genetic determinants of cellular susceptibility to chemotherapy (i.e. cisplatin) using EBV immortalized lymphoblastoid cell lines from CEPH families. Phenotypic variability in susceptibility to cisplatin is assessed by cell survival after exposure to drug. Data is analyzed to determine the degree of heritability. Microarray gene expression profiling experiments are employed to characterize the genes that influence the susceptibility to cisplatin. Together with genome wide linkage analysis, we are determining the most likely gene candidates responsible for sensitivity to drug.

Genotyping Database

Investigator: Wenjian Yang

We have established a secure, web based database with Oracle as backend for storing genotype at specific, well characterized loci in individual patients or subjects. The database stores detailed information on relevant samples, genes, genotyping methods, links to appropriate databases, handling of samples, sending of samples, and appropriate user-specific security is imposed.

Experimental Haplotyping

Investigator: William Evans

We have developed and applied a novel method for determining whether SNPs within a gene up to 30 kb in length are on the same or different alleles. We have used this method to experimentally assigning haplotypes for several genetic targets, including thiopurine methyltransferase, IL-4 and CYP3A4. In addition to the PAAR website, please see http://www.stjude.org/leukemia.

Hepatocyte Inducibility

Investigator: Steve Strom

We have established a system for primary culture of human hepatocytes at the University of Pittsburgh. We are using the hepatocytes to test the effect of various inducers on enzymes of interest to the PAAR group. We have also established methods for assessing the level of transcripts for the following targets, CYP1A1, CYP1A2, CYP3A4, UGT1A1, and UGT2B7, and these data are shared with PAAR group investigators.

Comparative Genomics as a Tool to Identify Putative Functional Regions

Investigator: Anna Di Rienzo

Comparative genomics techniques are used to assess sequence conservation and divergence among species including human, baboon, dog, mouse and rat. Genet targets that are being investigated include the UGT1A, UGT2B, CYP3A gene clusters, and the thiopurine methyltransferase, carboxylesterase 1 and 2 genes.

Pharmacogenetics of ALL

Investigator: Mary V. Relling

Using DNA samples from consecutively treated children on St. Jude Children's Research Hospital and Children's Oncology Group acute lymphoblastic leukemia (ALL) protocols, whose therapy is uniform. We are assessing the importance of pharmacogenetic determinants to pharmacodynamic responses, in particular, determinants of event-free survival risk of therapy related acute myelocytic leukemia, risk of avascular necrosis, methotrexate induced neurotoxicity, hepatotoxicity, and other important adverse effects will be assessed. In addition, etoposide pharmacokinetics and pharmacodynamics are assessed and the pharmacokinetic genetics determinants of pharmacokinetic parameters are a part of this project. Loci examined include vitamin D receptor, the glucocorticoid receptor, UGT1A1, CYP3A4, CYP3A5, the glutathione transferases GSTM1, GSTP1, and GSTT1, thiopurine methyltransferase, the reduced folate carrier, MDR1, thymidylate synthase, and methylene-tetrahydrofolate reductase.

Pharmacogenetics of ALL

Investigator: Cheng Cheng, William Evans, Mary V. Relling

In addition to the target gene approaches indicated above, genetic predictors of phenotypes of interest are being discovered using microarray expression analysis of ALL blasts and of normal blood cell RNA. Gene expression profiles are being used to differentiate patients of differing germline polymorphisms status, to differentiate patients who do vs do not develop secondary tumors, and to identify genes whose expression is related to DNA rearrangements caused by topoisomerase II inhibitors. These data provide the basis for the discovery of polymorphisms with functional consequence in ALL.

Pharmacogenetics of Treatment-induced Tumors

Investigator: Janet Rowley, Ching-Hon Pui, Mary V. Relling

Topoisomerase II inhibitors can cause a fatal secondary leukemia in a subset of patients. Polymorphisms in TPMT, CYP3A4, CYP3A5, NQO1, MLL, and the G-CSF receptor have been tested for links to secondary leukemia in well-controlled comparisons of similarly treated patients with identical primary diagnoses who do vs do not develop therapy-related leukemia. Additional genetic targets based on expression array differences between patients who do vs do not develop secondary leukemia are being explored.