Rapid quantitative detection of TEL-AML1 fusion transcripts in pediatric acute lymphoblastic leukemia by real-time reverse transcription polymerase chain reaction using fluorescently labeled probes.
OBJECTIVE
We report a new real-time reverse transcription polymerase chain reaction (RT-PCR) method for quantification of TEL-AML1 transcripts. The method is based on hybridization probe (HybProbe) chemistry applied in LightCycler equipment. The study group comprised 44 successive cases of pediatric acute lymphoblastic leukemia (P-ALL).


METHODS
The quantitative estimation of TEL-AML1 transcripts was performed in 10 P-ALL TEL-AML1-positive patients. The PCR was performed in capillary tubes in 10 microL final volumes using two sets of primers: M1, which detects the long (L-form) and short (S-form) transcripts, and M2, specific for the L-form. The fluorescently labeled HybProbes (TEL 3FL and TEL 5LC) hybridize to the TEL region. TEL-AML1 expression was normalized relative to the levels of AML1 transcripts. Standard curves were prepared using serial dilutions of plasmids with TEL-AML1 or AML1 inserts.


RESULTS
The sensitivity attained allowed the detection of TEL-AML1 transcripts at a 10-4 dilution of a cDNA sample from a patient at diagnosis. The within-assay coefficient of variation (CV) for TEL-AML1 was 7.0% and the between-assay CV was 13%. Levels of TEL-AML1 transcript and the TEL-AML1/AML1 ratio decreased by more than four log units (p <0.001) during or after the course of initial treatment. Most of the patients who achieved complete remission after 5-6 months of initial treatment were TEL-AML1 negative, although some positive samples with negligible amounts of TEL-AML1 transcripts were still detected.


CONCLUSIONS
This method has the sensitivity and reliability required to monitor the presence of minimal residual disease, and could be a powerful tool in monitoring the efficacy of the response to chemotherapy.
Bolufer
Pascual
P
Laboratorio de Biología Molecular, (Lab Hormonas), C Maternal Hospital Universitario La Fe, Avda Campanar 21, 46009 Valencia, Spain. bolufer_pas@gva.es
Barragán
Eva
E
Verdeguer
Amparo
A
Cervera
José
J
Fernández
José María
JM
Moreno
Isabel
I
Lerma
Enrique
E
Esquembre
Carlos
C
Tasso
María
M
Fuster
Vicente
V
Bermúdez
Mar
M
Sanz
Miguel A
MA
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D001773:Blood Cells / Q000737:chemistry; D001854:Bone Marrow Cells / Q000737:chemistry; D002648:Child; D002675:Child, Preschool; D002881:Chromosomes, Human, Pair 12 / Q000235:genetics / Q000648:ultrastructure; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics / Q000648:ultrastructure; D003199:Computer Systems; D050676:Core Binding Factor Alpha 2 Subunit; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D019793:Fluorescein; D005456:Fluorescent Dyes; D005500:Follow-Up Studies; D006801:Humans; D007223:Infant; D008297:Male; D018365:Neoplasm, Residual; D015345:Oligonucleotide Probes; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis*; D012074:Remission Induction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014178:Translocation, Genetic; D000097764:ETS Translocation Variant 6 Protein
D050676:Core Binding Factor Alpha 2 Subunit; D018076:DNA, Complementary; D019793:Fluorescein; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C102421:TEL-AML1 fusion protein; D000097764:ETS Translocation Variant 6 Protein