The Molecular Screening Using Multiplex Reverse Transcription Polymerase Chain Reaction and the Comparison with Cytogenetic Analysis in the Prognostic Stratification of Acute Leukemia
Authors: Cho Young-Uk , Chi Hyun-Sook , Jang Seongsoo , Seo Eul-Ju , Lee Jung-Hee , Lee Je-Hwan , Seo Jong-Jin , Lee Kyoo-Hyung , Park Chan-Jeoung 
Published: April 2020

INTRODUCTION: The detection of chromosomal aberrations is essential for the diagnosis and prognostic stratification of acute leukemia. Classical cytogenetic analysis has the advantage of comprehensive screening of all types of abnormalities such as numerical aberrations and deletions as well as translocations. However, this technique is time-consuming and labor-intensive, and cannot detect cryptic translocations. Recently, a multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) has emerged as a tool for overcoming disadvantages of cytogenetic analysis with some degree of comprehensiveness. We designed multiplex RT-PCR system for acute leukemia based on sets of chromosomal translocations rationally selected with cost-effectiveness and clinical relevance. Herein, we tried to use molecular screening of acute leukemia with a multiplex RT-PCR system we designed, and compared with the results of cytogenetic analysis in terms of prognostic stratification.

MATERIALS AND METHODS: A total of 121 consecutive patients with acute leukemia were included from November 2006 to July 2007. Multiplex RT-PCR system (Seegene, Korea) for molecular screening was carried out to detect 16 fusion transcripts: BCR-ABL, PML-RARα, PLZF-RARα, AML1-ETO, CBFβ-MYH11, DEK-CAN, E2APBX1, TEL-AML1, and MLL rearrangements (MLL-AF4, MLL-AF6, MLL-AF9, MLL-AF10, MLL-ELL, MLL-ENL, MLL-AF17, MLL-PTD). Cytogenetic analysis was performed by standard GTL-banding technique. The discordant findings were validated by fluorescence in situ hybridization (FISH) or conventional single RT-PCR.

RESULTS: Eighty-nine (73.6%) adults and 32 (26.4%) children were included in this study. A total of 77 (63.6%) were diagnosed as acute myeloid leukemia (AML) and the remaining 44 (36.4%) were acute lymphoblastic leukemia (ALL) including 7 biphenotypic acute leukemia. Cytogenetic analysis was available in 118 (97.5%) of the patients. Sixty-four cases of fusion transcripts were detected in 60 patients (49.6%). Within the AML group, the following fusion transcripts were detected: 14 AML1-ETO, 8 PML-RARα, 3 CBFβ-MYH11, 1 DEK-CAN, 1 BCR-ABL, 6 MLL-PTD, 4 MLL-AF6, 3 MLL-AF9, 1 MLL-ELL. Within the ALL group, the following fusion transcripts were detected: 12 BCR-ABL, 4 TEL-AML1, 2 E2A-PBX1, 3 MLL-AF4, 1 MLL-AF9, 1 MLLPTD. The concordance rate between two tests was 88.3%. In 14 cases, the results of two methods did not agree. Seven cytogenetically unrevealed abnormalities were detected with the multiplex RT-PCR: 3 TEL-AML1, 2 MLL-AF9, 1 MLL-AF6, 1 PML-RARα. The frequency of cryptic translocation was 5.8%. MLL-PTD which is impossible to be observed by cytogenetic analysis was detected in remaining seven patients. On the other hand, the corresponding fusion transcripts were not detected by multiplex RT-PCR in three cases of inv(16), t(4;11), and MLL rearrangement suggested by FISH, respectively. Molecular screening could preferentially assign 32.5% of AML patients to favorable-risk group, and did 34.1% of ALL patients to adverse-risk group. Although the concordance rate between two methods as a prognostic factor was 89.5% in risk-determined group by multiplex RT-PCR, six patients (3 AML and 3 ALL) disclosed different risk group by molecular screening from cytogenetic analysis.

CONCLUSIONS: We conclude that molecular screening using multiplex RT-PCR was useful in providing informations about genetic abnormalities more rapidly and giving appropriate therapeutic decision in acute leukemia patients. It was also useful in the detection of cytogenetically cryptic translocations. Although the multiplex RT-PCR system could not be a whole substitute for cytogenetic analysis, we expect that two methods are complementary at the time of diagnosis of acute leukemia, by which more genetic aberrations would be detected efficiently.