1093P - Bcr Abl fusion protein detection by a modern approach: fluroscent immuno bead assay

Date 30 September 2012
Event ESMO Congress 2012
Session Poster presentation II
Topics Biomarkers
Haematologic Malignancies
Presenter Swati Dasgupta
Authors S. Dasgupta1, U.K. Ray2, A. Mukhopadhyay3, .4, S. Mukhopadhyay4
  • 1Dept Of Molecular Biology, Netaji Subhas Chandra Bose Cancer Research Institute, 700016 - Kolkata/IN
  • 2Molecular Biology, Netaji Subhas Chandra Bose Cancer Research Institute, 700016 - Kolkata/IN
  • 3Dept. Medical Oncology, Netaji Subhas Chandra Bose Cancer Research Institute, 700016 - Kolkata/IN
  • 4Dept Of Molecular Biology, Netaji Subhash Chandra Bose Cancer Research Institute, 700016 - Kolkata/IN

Abstract

Introduction

Chronic Myeloid Leukemia (CML) is probably the most extensively studied human malignancy. CML patients (90-95%) harbor the Philadelphia (Ph) chromosome, a shortened chromosome 22, resulting from a reciprocal translocation t (9; 22) (q34; q11) between the long arms of chromosome 9 and 22, fusion ABL proto-oncogene on chromosome 9 with the BCR gene on chromosome 22. BCR-ABL fusion gene plays a key role in pathogenesis of the disease. Approximately 15-30% of adult and 2-5% of childhood ALL and <3% of AML patients are also found with this translocation. The conventional methods such as cytogenetics, FISH, PCR are time consuming and required special facilities. We have used a simple flowcytometric fluorescent immuno bead assay for detection of BCR-ABL fusion protein.

Materials and methods

Study Period: June 2009- February 2012

Test Specimen

Peripheral blood or Bone marrow from 242 CML patient (from hospital registry of a tertiary cancer institute of Eastern India) Normal (as negative control): Peripheral blood from 20 individuals BCR-ABL positive cell line: K562 FISH was performed using (for 100 samples) directly labeled dual color prob for detection of BCR-ABL as standard laboratory protocol. Isolation of WBC were done by lysis buffer. Then lysate treatment were done to release the intracellular BCR-ABL protein. After fluorescent immune bead assay were performed by captured bead and detector reagent. After forming a sandwich complex (if sample contain targeted protein), they were analysed by flowcytometry method.

Result

In 242 cases 166 (68.6%) were positive for BCR ABL fusion protein by Flowcytometric Bead Assay. Approximately 96% cases were observed as positive by both techniques FISH and Flowcytometric Bead Assay.

Conclusion

We conclude that the Flowcytometry Immuno Bead Assay is a faster, easier and reliable technique for detection of BCR ABL protein in leukemic cells and shows promise for serial evaluation of patients undergoing treatment. The main advantage of immune bead assay is not dependence on the breakpoint position in the BCR gene. Moreover this technique is based on protein translation label.

Disclosure

All authors have declared no conflicts of interest.