Acute promyelocytic leukemia (APL) accounts for 10% of cases of acute myeloid leukemia and is characterized by a particularly aggressive disease progression. Its peculiarity is the presence at diagnosis, 80% of cases, of a severe hemorrhagic syndrome. This dysfunction is often manifested in the form of hemorrhage at brain level that, before the advent of the revolutionary therapy with ATRA (All-Trans Retinoic Acid) caused the early death of almost 20% of patients. At the genetic level, the disease is caused in 99% of cases by a balanced translocation between chromosomes 15 and 17, leading to the formation of the chimeric protein PML-RAR. In RARA gene, the breakpoint constantly occurs in intron 2, while is variable in PML gene, resulting in three different isoforms (bcr1, bcr2 and bcr3). Due to the fast and dramatic progression of the disease and to exploit the availability of life-saving drug it is necessary to have a very quick method of diagnosis, therefore the object of this work is the development of a molecular assay for the ultra-rapid diagnosis of APL, based on the molecular technology LAMP (loop mediated isothermal amplification), a non-PCR method for the amplification of nucleic acids, very rapid and highly specific. LAMP is a quite recent technology, developed in Japan, originally based on the detection of the amplification signal with turbidimetry. The detection method has been greatly improved at DiaSorin, exploiting the use of fluorescent probes. This innovation allowed the detection of different targets in real-time and in multiplex format, thanks to the use of probes emitting at different wavelenghts. A further point of improvement has been the introduction of an engineered polymerase can perform the reverse transcription of RNA in a single step and in a single tube. Two assays have been developed, based on RT-Q-LAMP technology, ultra-rapid and simple to perform, which, starting directly from RNA, are able to identify and discriminate in 15 minutes, the three transcripts bcr1, bcr2 and bcr3, together with an internal control for the validation of negative results. Sensitivity and specificity of the assays have been evaluated on plasmids and validated on positive (n = 88) and negative (n = 94) clinical samples. The results obtained with RT-Q-LAMP assays have been compared with the reference method (PCR or sequencing), giving 100% agreement on the final result. The assays have proven to be highly specific on RNAs negative for the PML-RARA translocations (n = 282) and on water samples (NTC, n = 627). Moreover, both assays demonstrated to be very robust, even when used on suboptimal RNA, with low concentration or poor purity and quality. Compared to current methods, such as PCR, expensive in terms of time, reagents, and which require specialized staff, these new RT-Q-LAMP assays, thanks to the characteristics mentioned above, prove to be extremely competitive and suitable for actual clinical needs.

(2016). Development of a Novel Molecular Assay for the Ultra-Rapid Diagnosis of Acute Promyelocytic Leukemia by RT-Q-LAMP Technology. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).

Development of a Novel Molecular Assay for the Ultra-Rapid Diagnosis of Acute Promyelocytic Leukemia by RT-Q-LAMP Technology

RIGO, FRANCESCA
2016

Abstract

Acute promyelocytic leukemia (APL) accounts for 10% of cases of acute myeloid leukemia and is characterized by a particularly aggressive disease progression. Its peculiarity is the presence at diagnosis, 80% of cases, of a severe hemorrhagic syndrome. This dysfunction is often manifested in the form of hemorrhage at brain level that, before the advent of the revolutionary therapy with ATRA (All-Trans Retinoic Acid) caused the early death of almost 20% of patients. At the genetic level, the disease is caused in 99% of cases by a balanced translocation between chromosomes 15 and 17, leading to the formation of the chimeric protein PML-RAR. In RARA gene, the breakpoint constantly occurs in intron 2, while is variable in PML gene, resulting in three different isoforms (bcr1, bcr2 and bcr3). Due to the fast and dramatic progression of the disease and to exploit the availability of life-saving drug it is necessary to have a very quick method of diagnosis, therefore the object of this work is the development of a molecular assay for the ultra-rapid diagnosis of APL, based on the molecular technology LAMP (loop mediated isothermal amplification), a non-PCR method for the amplification of nucleic acids, very rapid and highly specific. LAMP is a quite recent technology, developed in Japan, originally based on the detection of the amplification signal with turbidimetry. The detection method has been greatly improved at DiaSorin, exploiting the use of fluorescent probes. This innovation allowed the detection of different targets in real-time and in multiplex format, thanks to the use of probes emitting at different wavelenghts. A further point of improvement has been the introduction of an engineered polymerase can perform the reverse transcription of RNA in a single step and in a single tube. Two assays have been developed, based on RT-Q-LAMP technology, ultra-rapid and simple to perform, which, starting directly from RNA, are able to identify and discriminate in 15 minutes, the three transcripts bcr1, bcr2 and bcr3, together with an internal control for the validation of negative results. Sensitivity and specificity of the assays have been evaluated on plasmids and validated on positive (n = 88) and negative (n = 94) clinical samples. The results obtained with RT-Q-LAMP assays have been compared with the reference method (PCR or sequencing), giving 100% agreement on the final result. The assays have proven to be highly specific on RNAs negative for the PML-RARA translocations (n = 282) and on water samples (NTC, n = 627). Moreover, both assays demonstrated to be very robust, even when used on suboptimal RNA, with low concentration or poor purity and quality. Compared to current methods, such as PCR, expensive in terms of time, reagents, and which require specialized staff, these new RT-Q-LAMP assays, thanks to the characteristics mentioned above, prove to be extremely competitive and suitable for actual clinical needs.
CHIARADONNA, FERDINANDO
PML-RARA, LAMP, APL
BIO/11 - BIOLOGIA MOLECOLARE
English
BIOTECNOLOGIE INDUSTRIALI - 15R
28
2014/2015
(2016). Development of a Novel Molecular Assay for the Ultra-Rapid Diagnosis of Acute Promyelocytic Leukemia by RT-Q-LAMP Technology. (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/10281/103093
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