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Leukemias Identification using Biochips

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Identification of Chromosomal Translocations in Major Leukemias using Oligonucleotide Microarrays

Tech Area / Field

  • MED-DID/Diagnostics & Devices/Medicine
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology

Status
3 Approved without Funding

Registration date
26.02.2003

Leading Institute
Engelhardt Institute of Molecular Biology, Russia, Moscow

Supporting institutes

  • State Research Center of Virology and Biotechnology VECTOR, Russia, Novosibirsk reg., Koltsovo

Collaborators

  • Argonne National Laboratory (ANL), USA, IL, Argonne\nHumboldt Universität / Charité Universitätsklinikum, Germany, Berlin\nSt. Jude Children's Research Hospital, USA, TN, Memphis\nFriedrich-Schiller-Universitat / Institute of Human Genetics and Anthropology / Core Unit Chip Applications, Germany, Jena\nFriedrich-Schiller-Universitat / Institute of Human Genetics and Anthropology, Germany, Jena

Project summary

The aim of the project is to develop a microarray-based approach for rapid and sensitive identification of chromosomal translocations occurring in hematological malignancies.

Chromosomal rearrangements, such as translocations, deletions, inversions and amplifications – can perturb genes intimately involved in carcinogenesis. The identification of consistent chromosomal abnormalities in tumor cells, the isolation of genes affected by these changes and the elucidation of their mechanisms of action and clinical correlations is a major concern in cancer research. In leukemias, the identification of chromosomal aberrations allows to classify precisely the disease according to genetic changes, to risk-stratify patients and to choose an adequate therapy.

The identification of chromosomal translocations will be performed using the combination of RT-PCR and hybridization with microarrays. The activation of protooncogenes very often results from the formation of fusion genes and in this case detection of chimerical mRNA is a key element in molecular analysis of the disease. Because of a great number of genes, involved in structural rearrangements and a variety of breakpoints inside one gene, the application of microchips is a very promising approach, allowing to facilitate strongly the analysis and to make it more precise and reliable.

The biochips developed in the Center for Biological Microchips represent arrayed blocks of polyacrylamide gel about 100 mkm in diameter and about 1 nl in volume attached to a hydrophobic glass surface. The biochips have been demonstrated to be very efficient for identification of genetic mutations, detection of gene polymorphism etc. In our preliminary studies in EIMB we used the combined approach including the multiplex reverse transcription-polymerase chain reaction (RT-PCR) and hybridization with oligonucleotide microarrays for the identification of 3 selected risk-stratifying translocations occurring in leukemias, such as t(9;22), t(4;11), and t(15;17). This approach was successfully applied for the clinical diagnostics of about 10 patients from the Russian Children’s Clinical Hospital (RCCH) and 11 patients from the Hospital of the University of Chicago.

For the purpose of prospective studies we plan to perform the following work:

1. Synthesis of oligonucleotide probes specific to certain chromosome aberrations. Oligonucleotide probes for the detection of chromosome aberrations: t(9;22), t(4;11), t(12;21), t(1;19) most important in Acute Lymphoblastic Leukemia; t(15;17), t(8;21), inv(16) in Acute Myelogenous Leukemia and the variant of t(9;22) in Chronic Myeloid Leukemia will be chosen. The above-mentioned translocations represent a group of major risk-stratifying rearrangements in leukemias. Since there exist a number of breakpoint variants for each type of translocation, special attention will be paid to the identification of these variants and verification of breakpoint sequence. As a whole, a set of up to 60 oligonucleotide probes will be designed, synthesized and modified for further immobilization in gel.

2. Development of microchips containing identifying oligonucleotides. A microarray will be designed bearing a set of oligonucleotide probes, each copolymerized in a separate gel pad, which are complementary to the sequences of genes involved in these translocations. The composition of gel and polymerization conditions will be selected to provide the efficiency of hybridization and high reproducibility of the results. Different variants of oligonucleotide modification needed for copolymerization in a gel will be tested.

3. Checking of oligonucleotide probes in experiments with microchips. Each oligonucleotide probe will be tested in model experiments in order to find it hybridization efficiency in experiments with a)self-complementary oligonucleotides, b) cell culture lines carrying the translocations, and c) selected patient samples with known genotype. Different conditions of hybridization will be tested.

4. Choosing and synthesis of primers for multiplex PCR analysis. As identification is based upon an analysis of fluorescently labeled RT-PCR products of the fusion genes, corresponding primers will be synthesized and tested in the multiplex PCR. The multiplex PCR protocols will be optimized. Different procedures for fluorescent probe labeling will be compared to choose the most simple, reproducible and inexpensive.

5. Development of an inexpensive microchip reading device. A specialized microchip reader based on wide field microscope optics combined with a CCD camera, and a specialized software, will be fabricated in EIMB. A specific software for analysis of leukemia-microarrays will be designed. The reader will be ready for transferring to SRG VB Vector to test clinical leukemia specimens. The sensitivity of the microarray-based approach will be estimated, and the possibility of minimal residual disease monitoring will be studied.

6. The microchips will be used in clinical screening of at least two hundred of patients with leukemia, treated in the RCCH, Moscow and in Siberia clinics, which samples will be collected by SRC VB Vector. The type of translocation and the breakpoint variant will be determined. All patient samples will be tested also with the conventional PCR analysis. The full coincidence of these two approaches is expected. It is anticipated that 30-40% of leukemia patients will receive the molecular diagnosis after the identification of PCR products from fusion genes. A computerized database will be established to accumulate data on the efficiency of biochips application in leukemia treatment. The samples, received from SRC VB Vector will be analyzed cytogenetically and DNA libraries of abnormal chromosomal regions will be established using microdissection and DOP-PCR. This information can be important for searching of new translocations.

7. Additionally, the innovation of biochip technology will be tested: PCR amplification of fusion gene sites directly in biochip gel pads using fluorescent labeled primers. The approach will hopefully decrease the time of analysis.

In case of positive results, the accumulated experience in the biochip technology use for rapid leukemia diagnostics is planned to be introduced to the hospitals for more wide testing to meet all requirements of clinical diagnostics.


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