Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Translocations and Chromosomal Aberrations in Acute Leukemia

Abstract

We have developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) reaction, which enables us to detect 29 translocations/chromosomal aberrations in patients with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). Through the construction and optimization of specific primers for each translocation, we have been able to reduce the set-up to 8 parallel multiplex PCR reactions, thus greatly decreasing the amount of work and reagents. We show the value of our set-up in a retrospective analysis on cryopreserved material from 102 AML and 62 ALL patients. The multiplex RT-PCR detected a hybrid mRNA resulting from a structural chromosomal aberration in 45 of 102 (44%) of the AML and in 28 of 62 (45%) of the pediatric ALL cases. Importantly, in 33% of AML and in 47% of the ALL cases with cytogenetic data, submicroscopic chromosomal aberrations or masked translocations were shown that were not detected in the cytogenetic analysis either for structural reasons or because of an insufficient number of metaphases obtained. This multiplex RT-PCR system, which can handle up to 10 patients with a response time of 2 working days, is thus an important tool that complements cytogenetic analysis in the up-front screening of acute leukemia patients and should provide a rapid and efficient characterization of leukemia cells, even in situations with sparse patient material.

Prognosis and outcome of patients with acute myeloid leukemia based on FLT3‑ITD mutation with or without additional abnormal cytogenetics

“…R6616-02; Omega Bio-Tek, Inc., Norcross, GA, USA). Nested RT-PCR detection method and reaction system were used as previously reported (8,9). AML-associated mutated genes were detected using high-throughput sequencing technology.…”

Clinical-biological characteristics and treatment outcomes of pediatric pro-B ALL patients enrolled in BCH-2003 and CCLG-2008 protocol: a study of 121 Chinese children

“…Fusion transcript analysis was performed by a multiplex reverse transcription polymerase chain reaction (RT-PCR), as described previously [11, 14]. This system was able to simultaneously detect 29 fusion transcripts associated with B-ALL including BCR/ABL1 , E2A - PBX1 , E2A - HLF , ETV6/RUNX1 , KMT2A/AFF1 , KMT2A - MLLT1 , KMT2A - MLLT3 and KMT2A - MLLT10 which are related to B-ALL.…”

Prognostic significance of various 11q23/KMT2A rearrangements in infants with acute lymphoblastic leuekemia

“…Анализ хромосом выполняли в соответствии с Международной номенклатурой хромосом человека, принятой на момент выполнения стандартного цитогенетического исследования [25][26][27]. Выявление перестроек 11q23/KMT2A проводили в образцах костного мозга методами гнездной полимеразной цепной реакции (ПЦР) с предшествующей обратной транскрипцией (ОТ-ПЦР) и флуоресцентной гибридизации in situ (FISH) с зондами LSI MLL dual color break apart rearrangement probe (Abbott Molecular, США) и XL MLL plus (MetaSystems, Германия) по ранее описанным протоколам [28][29][30][31]. В качестве контроля выделения РНК оценивали экспрессию гена ABL в ходе одностадийной ОТ-ПЦР.…”