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Diagnosis of CBFB-MYH11-Positive AML 169<br />
2. The control PCR (e.g., RAR) of all samples is positive.<br />
3. The sensitivity is more than 1 in 100.<br />
4. All negative controls are negative.<br />
5. Both duplicate analyses are positive; i.e., a clear CBFB-MYH11 PCR product is<br />
visible after UV exposure of the agarose gel.<br />
6. The specificity of the CBFB-MYH11 PCR product has been confirmed by<br />
sequencing.<br />
A sample is considered to be negative when:<br />
1. The quality of the RNA is good as determined by agarose gel electrophoresis (see<br />
Note 6).<br />
2. The control PCR (e.g., RAR PCR) of all samples is positive.<br />
3. The sensitivity is more than 1 in 100.<br />
4. All negative controls are negative.<br />
5. Both duplicate analyses are negative.<br />
3.2. Diagnosis of CBFB-MYH11-Positive AML by Quantitative MYH11<br />
RT-PCR<br />
Real-time quantitative PCR (qPCR) is currently being used for routine identification<br />
and quantification of fusion genes and transcripts associated with<br />
hematological malignancies. The value for an individual sample as obtained<br />
with real-time PCR is represented by the cycle at which a signal is detected<br />
above the background. This value is called the threshold cycle (Ct) value. When<br />
applying a calibrator series with known amounts of input (e.g., diluted cells or<br />
molecules yielding a calibration curve with Ct values) the amount of a given<br />
sample can be calculated. Naturally, individual values should be corrected for<br />
variations in PCR efficiency, cDNA input, and so on, using a reference gene.<br />
3.2.1 Quantitative MYH11 PCR to Identify CBFB-MYH11-Positive Cases<br />
Among Newly Diagnosed AML<br />
The amplicon size used in qPCR should be less than 300 bp to guarantee<br />
efficient amplification. Because the distance between the smallest and longest<br />
CBFB-MYH11 fusion transcript is over 1200 bp, the efficient detection of all<br />
fusion transcripts requires at least four different qPCRs. The MYH11 gene is<br />
very weakly expressed in normal bone marrow cells and CBFB-MYH11-negative<br />
leukemia cells. However, as a result of the fusion to CBFB, the involved<br />
MYH11 moiety is strongly expressed (approx 100- to 1000-fold higher) (13). A<br />
single MYH11 real-time PCR can be applied to function as an initial screen to<br />
identify CBFB-MYH11-positive cases based upon the relatively high MYH11<br />
expression at diagnosis. To completely rule out false-positive results, the presence<br />
of CBFB-MYH11 should subsequently be confirmed with qualitative PCR<br />
as described under Subheading 3.1.1. The MYH11 qPCR is not suitable to
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