20Jan 2017

Optimization of allele specific PCR (AS-PCR) for the early detection of FLT3 (D835Y) mutation in Acute Myeloid Leukemia patients at Tabuk, Saudi Arabia.

  • Prince Fahd Bin Sultan Research chair, Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, Kingdom of Saudi Arabia.
  • King Fahd specialist Hospital, Ministry of Health, Tabuk, Saudi Arabia.
Crossref Cited-by Linking logo
  • Abstract
  • Keywords
  • References
  • Cite This Article as
  • Corresponding Author

Activating mutations in FLT3 occur in ~30% of adult AML, primarily consisting of internal tandem duplication (ITD) mutations (~25%) and point mutations in the tyrosine kinase domain (~7%), at the activation loop (residue D835). The early detection of FLT3- D835Y in AML patients is clinically important for AML patients in this small region of Saudi Arabia especially for determining drug responses, prognosis, and selection of therapy., The present study was conducted on 47 specimens among which 37 were from leukemia patients (5 were newly diagnosed AML, 7 ALL and 25 MPD ) and 10 healthy controls. Genomic DNA from blood was isolated using DNA QIAamp DNA Blood Mini Kit from Qiagen and stored at -200C until PCR. The Allele Specific-PCR based assay for FLT3 (D835Y) mutation was optimized in the Lab .The mutation was detected in 2/5 cases of AML, 0/25 of MPD and 0/5 of ALL. It was observed that patients with AML possessed significantly higher frequency of FLT3 D835 mutation (2/5) than other leukemia cases and healthy controls. The D835Y Flt3-TKD mutation was not detected in any of the healthy controls (0/10). It was concluded that allele specific PCR technique permits the direct detection of FLT3 c.2503G>T (D835Y) point mutation in AML patients and acts as useful genetic technique that is very valuable for molecular diagnosis, prognosis, drug response, and predisposition to AML patients. In addition, the test was simple, fast, and inexpensive procedure that does not require any special equipment other than a thermo cycler.


  1. Cancer Registry Report, 2006, Ministry of Health, Riyadh, Saudi Arabia.
  2. Koc Y, Miller KB, Schenkein DP. Extramedullary tumors of myeloid blasts in adults as a pattern of relapse following allogeneic bone marrow transplantation. Cancer 1999;85:8-15.
  3. Chandra KC (2011) Genetic abnormalities and challenges in the treatment of acute myeloid leukemia.Genes and Cancer2(2):95-107
  4. Whitman S.Pet al.?(2001) Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study.?Cancer Research?61(19):7233-7239.
  5. Levis?M,?Small?D.?Novel FLT3 tyrosine kinase inhibitors.?Expert Opin Investig Drugs2003;12:1951-1962.
  6. Yamamoto?Y,?Kiyoi?H,?Nakano?Y,?et al.?Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies.?Blood?2001;97:2434-2439.
  7. Thiede?C,?Steudel?C,?Mohr?B,?et al.?Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis.?Blood?2002;99:4326-4335.
  8. Armstrong SA,?Mabon ME,?Silverman LB,?Li A,?Gribben JG,?Fox EA,?Sallan SE,?Korsmeyer SJ. FLT3 mutations in childhood acute lymphoblastic leukemia. 2004 May 1;103(9):3544-6.
  9. Abu-Duhier?FM,?Goodeve?AC,?Wilson?GA,?et al.?Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.?Br J Haematol2001;113:983-988.
  10. Moreno?I,?Martin?G,?Bolufer?P,?et al.?Incidence and prognostic value of FLT3 internal tandem duplication and D835 mutations in acute myeloid leukemia.?Haematologica?2003;88:19-24.
  11. Lee BHet al.?(2007) FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia.?Cancer Cell, 12 (4), 367-380
  12. Abu-Duhier FM,et al.?(2001) Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.?British Journal of Haematology?113:983-988.
  13. Yanada M, Matsuo K, Suzuki T, Kiyoi H, & Naoe T (2005) Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta-analysis.Leukemia19:1345-1349
  14. Jiang J,et al.?(2004) Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML.?Blood?104(6):1855-1858
  15. Cordell JL, Pulford KA, Bigerna B, Roncador G, Banham A, Colombo E, Pelicci PG, Mason DY, Falini B Detection of normal and chimeric nucleophosmin in human cells. 1999 Jan 15; 93(2):632-42.
  16. Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine..Exp Mol Pathol. 2008 Aug; 85(1):50-8.
  17. Abu-Duhier?FM,?Goodeve?AC,?Wilson?GA,?et al .?Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.?Br J Haematol2001;113:983-988
  18. Zheng?R,?Small?D .?Mutant FLT3 signaling contributes to a block in myeloid differentiation.?Leuk Lymphoma?2005;46:1679-1687.
  19. Bagrintseva?K,?Schwab?R,?Kohl?TM,?et al.?Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of acquired drug resistance to PTK inhibitors in FLT3-ITD-transformed hematopoietic cells. Blood?2004;103:2266-2275
  20. Gilliland?DG .?Hematologic malignancies.?Curr Opin Hematol?2001;8:189-191.
  21. Abu-Duhier?FM,?Goodeve?AC,?Wilson?GA,?et al.?Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.?Br J Haematol2001;113:983-988
  22. Rosnet?O,?Buhring?HJ,?Marchetto?S,?et al.?Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells.?Leukemia?1996;10:238-248.
  23. Thiede?C,?Steudel?C,?Mohr?B,?et al.?Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis.?Blood2002;99:4326-4335
  24. Moreno?I,?Martin?G,?Bolufer?P,?et al.?Incidence and prognostic value of FLT3 internal tandem duplication and D835 mutations in acute myeloid leukemia.?Haematologica?2003;88:19-24.
  25. Fr?hling?S,?Schlenk?RF,?Breitruck?J,?et al.?Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm.?Blood?2002;100:4372-4380
  26. Auewarakul?CU,?Sritana?N,?Limwongse?C,?Thongnoppakhun?W,Yenchitsomanus?PT.?Mutations of the FLT3 gene in adult acute myeloid leukemia: determination of incidence and identification of a novel mutation in a Thai population.?Cancer Genet Cytogenet?2005;162:127-134
  27. Meshinchi?S,?Woods?WG,?Stirewalt?DL,?et al.?Prevalence and prognostic significance of Flt3 internal tandem duplication in pediatric acute myeloid leukemia.?Blood?2001;97:89-94.
  28. Bali?P,?George?P,?Cohen?P,?et al.?Superior activity of the combination of histone deacetylase inhibitor LAQ824 and the FLT-3 kinase inhibitor PKC412 against human acute myelogenous leukemia cells with mutant FLT-3.?Clin Cancer Res?2004;10:4991-4997
  29. Choudhary?C,?Schwable?J,?Brandts?C,?et al.?AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations.?Blood?2005;106:265-273.
  30. Neben?K,?Schnittger?S,?Brors?B,?et al.?Distinct gene expression patterns associated with.?Oncogene?2005;24:1580-1588.

[Abu-Duhier FM, Rashid Mir, Jamsheed Javid, Fawzia Sharaf and Nabil Burrow. (2017); Optimization of allele specific PCR (AS-PCR) for the early detection of FLT3 (D835Y) mutation in Acute Myeloid Leukemia patients at Tabuk, Saudi Arabia. Int. J. of Adv. Res. 5 (1). 817-822] (ISSN 2320-5407). www.journalijar.com


Dr Faisal AbuDuhier
Prince Fahd Bin Sultan Research chair, Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, Kingdom of Saudi Arabia.

DOI:


Article DOI: 10.21474/IJAR01/2819       DOI URL: http://dx.doi.org/10.21474/IJAR01/2819


Share this article