Acute Lymphoblastic Leukemia (ALL): Detection of Prognostically Significance Fusion-Genes By Multiplex-Polymerase Chain Reaction (M-PCR)
Abramson Cancer Center of the University of Pennsylvania
Ultima Vez Modificado:: 23 de marzo de 2007
Presenter: Manrique, G.
Presenter's Affiliation: ASESP, Uruguay
Type of Session: Scientific
- Acute lymphoblastic leukemia (ALL) is associated with a very poor prognosis in adults compared to the pediatric population.
- In children, the TEL/AML1 translocation is associated with improved response to therapy and is considered a good prognostic factor. In children and adults, the BCR/ABL translocation (Philadelphia chromosome) is associated with resistance to treatment and has a worse prognosis.
- The identification of specific chimeric genes related with ALL is relevant for clinical assessment and may allow therapeutic implementation, monitoring minimal residual disease (MRD) and risk-stratification assignment.
- Classic cytogenetic techniques requires isolation and fixation of cells in metaphase to determine karyotype. PCR analysis can be performed on extracted DNA directly, thereby making analysis of specific translocations easier with higher throughput.
- The goal of this study was to use real time PCR (RT-PCR) to standardize a rapid, specific and sensitive molecular method to simultaneously detect the most prognostic relevant chromosomal translocations in ALL.
- Design: Single-institution, retrospective laboratory-based study
- 82 patients with B-cell ALL (61 pediatric, 21 adults)
- Clinical samples:
- Bone marrow and peripheral blood samples previously analyzed by flow cytometry and cytogenetic techniques were used for reverse transcriptase multiplex-PCR (RT-M-PCR) analysis.
- RT-M-PCR was used to characterize the following fusion genes:
- The results from RT-M-PCR were compared to results from cytogenetic techniques.
- B-cell clonality was demonstrated by flow cytometry in all patients
- 26 of 82 samples (32%) were PCR (+) at diagnosis for one of the following specific fusion genes:
- BCR/ABL (6 adults/3 children)
- MLL/AF4 (8 children)
- E2A/PBX1 (1 child).
- All of the above patients showed the corresponding cytogenetic translocation.
- The TEL/AML fusion gene was undetectable by cytogenetic methods, but was identified in samples from 8 children.
- During follow-up to evaluate treatment effectiveness, MRD was established in 8 patients predicting hematological relapse.
- The RT-M-PCR method is a very useful tool for a fast and sensitive identification of prognostic significance fusion genes and early diagnosis of relapse risk in leukemia.
- This assay shows a good correlation with the cytogenetic findings, allowing the detection of the t(12,21) translocation (TEL/AML), which was undetectable by conventional cytogenetics.
- The development and use of multiplexed RT-PCR techniques compared to standard cytogenetic techniques offers the advantages of sensitivity and rapidity of detection of specific chromosomal translocations. The disadvantage is that unexpected translocations will be missed, and there is a theoretical possibility of lower sensitivity if primer sites are mutated.
- It appears that at least for the TEL/AML translocation, RT-PCR is more sensitive than the classic cytogenetic technique, and can detect patients with a more favorable prognosis.
- It is unclear how this technique would compare to advanced cytogenetic techniques, such as spectral karyotyping with fluorescent stains, compared with the standard Giemsa-stained karyotyping technique.