RTPCR for Leukemia Specific Translocations

PCR analysis of fusion genes is based on the design of oligonucleotide primers within exons at the opposite sides of the breakpoint fusion regions, so that the PCR product contains the tumor-specific fusion sequence. The precise breakpoint recombination site at the DNA level is specific for each patient and difficult to determine. In the majority of the ALL translocations, the breakpoint regions span greater than 2 kilobases (kb; limit of amplification by conventional PCR), making DNA PCR difficult. However, since the breakpoints are mostly intronic, and spliced out at the mRNA level, the preferred target for detection of translocations is the chimeric messenger RNA (mRNA).43

Reverse transcription-PCR (RT-PCR) requires extraction of total RNA or mRNA from mononuclear cells, reverse transcription of RNA into cDNA, and then PCR, followed by electrophoresis. The sensitivity of the method is specific for each target and can be assessed by amplification of serial dilutions of RNA from diagnostic specimens or cell lines containing the specific translocation into RNA from healthy individuals that do not have the translocation. The presence of a very small number of abnormal cells, in the range of 1 in 105 to 1 in 106, has been consistently detected using appropriate conditions.53 This sensitivity is approximately 1000 to 10,000 times more than Southern blot analysis. A PCR test that is sufficiently sensitive to detect one leukemia cell in 102 to 103 normal cells is acceptable for diagnostic testing. A higher sensitivity is required for MRD assessment during follow-up analyses and should achieve a sensitivity of one leukemia cell in 104 to 105 normal cells.18 More than one primer set may need to be used to detect all fusion transcripts when the translocation breakpoints can be in different introns of one or both of the fusion genes, as shown in Figure 31-3.

Junctional ("N") IgH regions

5' VH + DH i JH 3'

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VH primer DH JH primer primer

Figure 31-2. Schematic representation of an IGH VDJ recombination region and the strategy used to amplify the N regions. Probes for the N regions can be used as patient-specific probes (shaded boxes with arrows above = N regions).

Figure 31-2. Schematic representation of an IGH VDJ recombination region and the strategy used to amplify the N regions. Probes for the N regions can be used as patient-specific probes (shaded boxes with arrows above = N regions).

Fusion transcripts

IZ1 234 2 3 45

Figure 31-3. RT-PCR detection of different fusion transcripts generated by the same translocation. The presence of different fusion transcripts requires the use of multiple primer pairs to detect fusion transcripts formed by breakpoints in different introns of the genes, depending on the size of the transcript.

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