Myeloid Leukemia

Diagnosis of CBFB-MYH11-Positive AML 171
1. Prepare the PBGD qPCR mix by pipetting in the following order for one PCR
reaction (see Notes 2 and 3): 25.25 µL H 2O, 10.0 µL 25 mM MgCl 2, 0.50 µL
dNTPs, 5.0 µL 10X PCR buffer A, 1.5 µL primer PBGDfor (Table 1), 1.5 µL
primer PBGDrev (Table 1), 1.0 µL probe PBGDpro, and 0.25 µL Taq gold DNA
polymerase (total volume of 45.0 µL).
2. Mix the PBGD qPCR mix and add per reaction 5.0 µL of cDNA (50 ng).
3. Perform the PBGD qPCR using an initial denaturing step of 10 min at 94°C to
activate the Taq gold DNA polymerase, followed by 45 cycles of 15 s at 94°C
(denaturation) and 1 min at 60°C (primer and probe annealing and extension).
4. Collect the Ct values of the unknown samples generated by the real-time PCR
equipment.
3.2.3. Validation of Results, Normalization of Quantities,
and Identification of Positive Samples
Check whether the required sensitivity is observed (see Note 8) and check
the quality of the MYH11 and PBGD PCRs according to Note 9. Check the
cDNA quality according to Note 10.
Normalize the MYH11 expression quantity of unknown samples (as generated
by TaqMan software) for PCR and cDNA input variations by multiplying
the quantity with the correction factor 2 ∆cycle threshold(Ct) , in which ∆Ct = Ct
PBGD of a sample minus the average PBGD Ct (see also Note 10 for details).
After normalization, a maximal variation of fourfold in the quantity between
duplicate samples is allowed.
Samples with normalized MYH11 quantities within a 25-fold range of a standard
positive control (either higher or lower) are considered to be CBFB-
MYH11 positive (see Note 11 for more details). For these samples, the presence
of the CBFB-MYH11 fusion and transcript type should be confirmed with qualitative
CBFB-MYH11 PCR. Negative samples have normalized MYH11 quantities
that are at least 1000-fold lower than that of the standard control. So far we
have observed occasional samples with normalized MYH11 quantities between
25- and 1000-fold lower compared to the standard control. All of these samples
appeared negative in qualitative CBFB-MYH11 PCR. Nevertheless, we recommend
screening these samples for the presence of CBFB-MYH11 by qualitative
PCR.
3.3. Monitoring of CBFB-MYH11-Positive AML by Quantitative RT-PCR
With the development of real-time PCR, it is possible to monitor CBFB-
MYH11-positive disease with a sensitivity of at least 1 malignant cell among
10,000 normal cells (3,4). Recent studies have shown that the CBFB-MYH11
expression at diagnosis is similar among patients, and CBFB-MYH11 expression
levels can be used as an indicator of the percentage of CBFB-MYH11positive
cells (3,4,15). As mentioned previously, at least 10 different