Diagnoses of leukemias have been improved by classical karyotyping, FISH and PCR analysis [
3]. As numerical chromosomal aberrations and unknown balanced alterations are detectable solely by cytogenetic analysis; this type of conventional methods form an important platform for molecular characterization of genetic aberrations.
In spite of recent advances in classical karyotype analysis, difficulties in obtaining enough number of good quality metaphases have not been completely overcome. Molecular cytogenetic techniques, such as FISH analysis, when compared with cytogenetic techniques, although have some superiority in terms of time consumed, and easiness, these techniques have disadvantages of having false positivity depending on specific probes, in addition to above mentioned difficulty [4]. These limitations gave rise to increased usage of PCR techniques in leukemia diagnosis and prognosis [17-19]. There are two main advantages of this method. First, this technique might be applied in less than two days, and one can perform PCR reactions of different aberrations and different patients at the same time. Second, in detection of known submicroscopic aberrations, usage of this method provides more certain results and ensures detection of one malignant cell among one million normal cells. Moreover, molecular techniques can be used when the material is insufficient for classical karyotyping.
In this study, RT-PCR and nested-PCR methods were applied for detection of fusion genes at mRNA level. In our study group, t(1;19), t(4;11) translocations have not been seen in any patient. Translocation t(1;19) has been shown in 3-6% of B-ALL cases, previously. Additionally, this translocation has been seen in ALL, T-ALL and AML cases, rarely (less than 1%). For this reason, observing no individual with this translocation might be considered as expected. Likewise, our patient group contains only one infancy ALL case, in which, translocation t(4;11) has been mainly observed [20]. Inv[16] chromosomal alteration is related to AML-M4Eo subgroup and this chromosomal aberration is observed in one of 11 AML cases in this study [9].
Translocation t(12;21), which is detected especially in pediatric ALL cases, was shown in 3 of 19 cases. In a previous study, frequency of t(12;21) translocation was reported as 39% among pediatric ALL cases [14]. This translocation is observed, more frequently, at 3-5 years of age [9]. In our study, 2 of t(12;21) positive cases were 3 years old and one case was 10 years old. These findings confirm that t(12;21) translocation should be routinely screened in all pediatric ALL cases.
P210 product of t(9;22) coding BCL-ABL fusion gene with increased tyrosine-kinase activity, was detected in 5 of 7 adult CML cases and 2 of 19 pediatric ALL cases. Positivity of p210 product was reported in almost all adult CML cases and 20-30% of all Philadelphia positive pediatric ALL cases. Translocation t(9;22)p190 product, occurring due to different break points, was observed in adult ALL and in adult AML cases. Our results are consistent with the previous reports’ findings that p190 product was present in 22% of adult ALL cases and in 1% of adult AML cases [10]. Although p190 transcript is specific to ALL this transcript has been rarely detected in CML patients. In our patient group presence of p210 was shown in 2 (out of 7) CML patients one of which has also p190 transcript. In fact, it has been reported that, all CML patients at the time of diagnosis have some p190 products by alternative splicing mechanism in addition to p210 transcript [21,22]. As a consequence, it might be stated that both p190 and p210 transcript status of all pediatric and adult ALL, AML and CML cases should be checked routinely for diagnostic, prognostic and therapeutic purposes.
Each PCR reaction has 10-2 sensitivity, and nested PCR analysis increases this sensitivity to 10-4 [9,19]. Therefore, for detection of leukemic genetic alterations, PCR methods are fast, sufficient and feasible, and PCR methods should be used as supplementary to cytogenetic analysis, for efficiency. PCR method is also suitable for detection of other newly identified translocations by designation of new specific primers [9]. Molecular detection and use of these translocations in treatment has an additional importance especially in cytogenetically problematic cases.
ACKNOWLEDGEMENTS
Authors thank to Prof. Giuseppe Saglio for supplying positive controls. This project is supported by Akdeniz University, Research Foundation (Project no: 20.01.0103.06).
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