A multiplex reverse transcriptase-polymerase chain reaction strategy for the diagnostic molecular screening of chimeric genes: a clinical evaluation on 170 patients with acute lymphoblastic leukemia.
OBJECTIVE
In the last few years molecular methods have allowed the identification of leukemia-associated genetic lesions, which may represent the most accurate predictors of clinical outcome. These considerations strengthen the need for rapid identification of the abnormalities. Our aim was to demonstrate whether a modified multiplex reverse transcription polymerase chain reaction (RT-PCR) system might be successfully used to screen a large number of patients with acute lymphoblastic leukemia (ALL).


METHODS
In this study we adapted the multiplex RT-PCR assay, previously described by Pallisgaard et al., to detect all the most frequent genetic lesions with their characteristic splicing variants occurring in acute lymphoblastic leukemia, such as the MLL/AF4, MLL/ENL, BCR/ABL p190 (e1a2) and p210 (b2a2,b3a2) isoforms, E2A/PBX1, TEL/AML1, SIL/TAL1 and the novel NUP98/RAP1GDS1 transcript, recently described in a T-ALL leukemic subtype.


RESULTS
We used the multiplex RT-PCR assay to screen 170 ALL patients (70 children and 100 adults). PCR positivity was detected in 67 (39%) of the 170 ALL patients studied. The comparison between cytogenetic and molecular analyses showed complete correspondence between the two assays in all patients with an evaluable karyotype. Finally, the observed incidence of genetic lesions in our ALL patients was similar to the frequency usually reported both in children and in adults with ALL.


CONCLUSIONS
These results show that, compared to single RT-PCR reactions, our multiplex RT-PCR system allows rapid, specific, simultaneous as well as a less expensive, laborious and time-consuming detection of the most frequent fusion transcripts in ALL patients. Therefore, it might be recommended for rapid diagnostic molecular screening of large numbers of patients, such as those enrolled in multicenter, co-operative studies. Furthermore, we have shown that multiplex RT-PCR is an open system that can easily be adapted to detect new leukemic genes.
Elia
Loredana
L
Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Ematologia, University La Sapienza, via Benevento 6, 00161 Rome, Italy.
Mancini
Marco
M
Moleti
Luisa
L
Meloni
Giovanna
G
Buffolino
Sonia
S
Krampera
Mauro
M
De Rossi
Giulio
G
Foà
Robin
R
Cimino
Giuseppe
G
D000328:Adult; D002648:Child; D020732:Cytogenetic Analysis; D006801:Humans; D007621:Karyotyping; D025202:Molecular Diagnostic Techniques; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics*; D011379:Prognosis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016896:Treatment Outcome
D015514:Oncogene Proteins, Fusion