J Korean Med Sci.  2012 Oct;27(10):1155-1161. 10.3346/jkms.2012.27.10.1155.

Rapid Detection of Prognostically Significant Fusion Transcripts in Acute Leukemia Using Simplified Multiplex Reverse Transcription Polymerase Chain Reaction

• Cho YU
• Chi HS
• Park CJ
• Jang S
• Seo EJ

• KMID: 1778820
• DOI: http://doi.org/10.3346/jkms.2012.27.10.1155

Abstract

Multiplex reverse transcription polymerase chain reaction (mRT-PCR) has recently emerged as an alternative to cytogenetics. We designed and used simplified mRT-PCR system as a molecular screen for acute leukemia. Fifteen fusion transcripts were included: BCR-ABL1, PML-RARA, ZBTB16-RARA, RUNX1-RUNX1T1, CBFB-MYH11, DEK-NUP214, TCF3-PBX1, ETV6-RUNX1, MLL-AFF1, MLL-MLLT4, MLL-MLLT3, MLL-MLLT10, MLL-ELL, MLL-MLLT1, and MLL-MLLT6. A total of 121 diagnostic acute leukemia specimens were studied, comparing the mRT-PCR system with standard cytogenetics. Fifty-six cases (46.3%) had fusion transcripts revealed by our mRT-PCR assay. The concordance rate between mRT-PCR and cytogenetics was 91.7%. However, false negative results were found in three cases who have inv(16), t(4;11) or t(11;19)(q23;p13.1), respectively. Seven cryptic translocations including ETV6-RUNX1, MLL-MLLT3, MLL-MLLT4, and PML-RARA were detected. This mRT-PCR assay is a useful screening tool in acute leukemia because it provides rapid and reliable detection of clinically important chimeric transcripts. In addition, cryptic translocations provide additional genetic information that could be clinically useful.

Keyword

Acute Leukemia; Multiplex RT-PCR; Cytogenetics; Cryptic Translocations

MeSH Terms

Acute Disease
Adolescent
Adult
Aged
Child
Child, Preschool
Chromosome Inversion
Cytogenetics
Female
Humans
Infant
Leukemia/*diagnosis/genetics/metabolism
Male
Middle Aged
*Multiplex Polymerase Chain Reaction
Oncogene Proteins, Fusion/*genetics
Prognosis
Translocation, Genetic
Young Adult
Oncogene Proteins, Fusion

Figure

• 

  Fig. 1 Flow diagram illustrating the multiplex RT-PCR assay used in this study. (A) Agarose gel with all negative PCR results. (B) Agarose gel with RUNX1-RUNX1T1-positive PCR (591-bp band) in lane 1. Note that there is no need for nested PCR or split-out PCR to determine the specificity of translocation. (C) Agarose gel with a positive band in lane 3. A single PCR analysis was performed using the individual primer sets specific for MLL-ELL (519-bp band) after identification of the positive band in AML reaction tube 3.

• 

  Fig. 2 Examples of fusion transcripts detected by multiplex RT-PCR. Round pointers indicated internal control bands and arrowheads indicated positive PCR bands. (A) BCR-ABL1 b2a2 (401-bp band in lane 1). (B) CBFB-MYH11 isoform A (223-bp band in lane 5). (C) DEK-NUP214 (547-bp band in lane 4). (D) BCR-ABL1 e1a2 (308-bp band in lane 1). (E) ETV6-RUNX1 1B (398-bp band in lane 2). M, 100-bp ladder; N, distilled water (negative control).

• 

  Fig. 3 Fluorescence in situ hybridization with dual color, break apart rearrangement probe on the interphase cells from the patient with inv(16)(p13.1q22) who did not show CBFB-MYH11 fusion gene in the mRT-PCR assay. The green and red signals are separated. It implies the CBFB gene rearrangement.

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