Leukemias are diseases characterized by an uncontrolled proliferation of malignant hematopoietic stem cells. The fusion gene BCR-ABL is the result of a reciprocal translocation between chromosome 9 and chromosome 22 t (9;22) and encodes an oncogenic tyrosine kinase protein responsible for the neoplastic transformation observed in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The isoform p210 is the hallmark of CML, detectable in more than 95% of cases, while in LAL, the fusion gene BCR-ABL may be present in both isoforms p190 (60% of cases) and p210 (40 % of cases). The molecular detection of BCR-ABL is essential to diagnose CML and Philadelphia positive ALL, making possible the administration of proper treatment. Moreover, the discrimination of the isoform, only possible using molecular methods, facilitates the choice of the specific quantitative test for the molecular monitoring during the treatment. To date, the most widely used molecular techniques for the detection of the transcripts are based on the RT-PCR method (Reverse Transcription-Polymerase Chain Reaction). It is a time-consuming procedure consisting of several steps, retro transcription - amplification - gel detection, which must be performed by skilled personnel and in equipped laboratories. The risk of cross-contamination, due to the multistep feature of the technique, and the absence of an internal reaction control, may also lead to false positive or false negative signals generation. The PhD project presented in this thesis describes the development and optimization of an innovative molecular diagnostic test, based on RT-QLAMP® technology (Reverse Transcription Loop-Mediated Isothermal Amplification), and its application in the diagnostic field for the simultaneous detection of BCR-ABL p210 and p190 transcripts. The optimization was performed on both plasmid controls and RNA extracted from cell lines. Moreover the final assay was validated on clinical samples. The LAMP technology has proven to be very sensitive and specific, simple and rapid. The amplification and detection of the transcripts occur in real-time, inside a single tube and starting directly from RNA. The negative clinical samples were validated by the amplification of the internal control and the possibility of cross-contamination of the sample was dramatically decreased. In addition, the method has proved to be very robust due to the insensitivity to the major PCR inhibitors. The new diagnostic assay overcomes all the limitations of the currently used diagnostic methods and represents a valid alternative for the molecular diagnosis of chronic myeloid leukemia and acute lymphoblastic leukemia.

(2016). Development of an innovative molecular assay for the simultaneous detection of the BCR-ABL Major and Minor fusion transcripts by the use of Loop Mediated Isothermal Amplification reaction (Q-LAMP). (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).

Development of an innovative molecular assay for the simultaneous detection of the BCR-ABL Major and Minor fusion transcripts by the use of Loop Mediated Isothermal Amplification reaction (Q-LAMP)

TETTAMANZI, VERONICA
2016

Abstract

Leukemias are diseases characterized by an uncontrolled proliferation of malignant hematopoietic stem cells. The fusion gene BCR-ABL is the result of a reciprocal translocation between chromosome 9 and chromosome 22 t (9;22) and encodes an oncogenic tyrosine kinase protein responsible for the neoplastic transformation observed in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The isoform p210 is the hallmark of CML, detectable in more than 95% of cases, while in LAL, the fusion gene BCR-ABL may be present in both isoforms p190 (60% of cases) and p210 (40 % of cases). The molecular detection of BCR-ABL is essential to diagnose CML and Philadelphia positive ALL, making possible the administration of proper treatment. Moreover, the discrimination of the isoform, only possible using molecular methods, facilitates the choice of the specific quantitative test for the molecular monitoring during the treatment. To date, the most widely used molecular techniques for the detection of the transcripts are based on the RT-PCR method (Reverse Transcription-Polymerase Chain Reaction). It is a time-consuming procedure consisting of several steps, retro transcription - amplification - gel detection, which must be performed by skilled personnel and in equipped laboratories. The risk of cross-contamination, due to the multistep feature of the technique, and the absence of an internal reaction control, may also lead to false positive or false negative signals generation. The PhD project presented in this thesis describes the development and optimization of an innovative molecular diagnostic test, based on RT-QLAMP® technology (Reverse Transcription Loop-Mediated Isothermal Amplification), and its application in the diagnostic field for the simultaneous detection of BCR-ABL p210 and p190 transcripts. The optimization was performed on both plasmid controls and RNA extracted from cell lines. Moreover the final assay was validated on clinical samples. The LAMP technology has proven to be very sensitive and specific, simple and rapid. The amplification and detection of the transcripts occur in real-time, inside a single tube and starting directly from RNA. The negative clinical samples were validated by the amplification of the internal control and the possibility of cross-contamination of the sample was dramatically decreased. In addition, the method has proved to be very robust due to the insensitivity to the major PCR inhibitors. The new diagnostic assay overcomes all the limitations of the currently used diagnostic methods and represents a valid alternative for the molecular diagnosis of chronic myeloid leukemia and acute lymphoblastic leukemia.
VANONI, MARCO ERCOLE
BCR-ABL, LAMP, Molecular Diagnostic, CML
BIO/11 - BIOLOGIA MOLECOLARE
English
BIOTECNOLOGIE INDUSTRIALI - 15R
28
2014/2015
(2016). Development of an innovative molecular assay for the simultaneous detection of the BCR-ABL Major and Minor fusion transcripts by the use of Loop Mediated Isothermal Amplification reaction (Q-LAMP). (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/103108
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