Myeloid Leukemia

118 Rossi, Levati, and Biondi
associated coagulopathy, the yield and quality of RNA from diagnostic samples
are frequently poor (see Note 1).
Several studies have attempted to correlate the type of PML-RARA transcript
either with clinico-biological features at diagnosis, or with treatment response
and outcome (reviewed in ref. 9). Because bcr2 and bcr1 are located in
PML exon 6 and intron 6, respectively, sequencing of all apparent L transcript
cases would be needed to clearly distinguish these two isoforms. Such distinction
is usually not reported in clinical studies with a large number of patients.
Accordingly, the vast majority of analyzed series have compared the two major
PML-RARA isoforms (i.e., bcr1 plus bcr2 cases vs bcr3 cases). At diagnosis,
no correlation was found with respect to sex, platelet count, presence of
coagulopathy, or retinoic acid syndrome, when comparing patients with L-type
or S-type PML-RARA transcripts. However, patients with S-type transcripts
had significantly higher white blood cell counts and more frequently M3v
morphology. Although S-type transcripts correlated with established adverse
prognostic features (i.e., hyperleukocytosis, M3v), this association did not
translate into poorer outcome when compared to patients with L-type transcripts
in the context of combined all-trans retinoic acid (ATRA) and chemotherapy
treatment (9–11).
Among the different methods (conventional karyotyping, FISH, PML
immunostaining with specific antibodies, and RT-PCR detection of the PML-
RARA fusion gene), RT-PCR appears to be the most suitable for MRD detection.
For a variety of reasons and because of the relatively low sensitivity of RT-PCR
for PML-RARA, diagnosis and monitoring of bone marrow (BM) samples is preferred
to the use of peripheral blood (PB), especially when the circulating blast
cell (or leukemic promyelocyte) count is low. The recently introduced real-time
quantitative RT-PCR (RQ-PCR) can also be applied for MRD monitoring.
Overall, there is general agreement that a positive PML-RARA test after consolidation
is a strong predictor of subsequent hematological relapse, whereas
repeatedly negative results are associated with long survival in the majority of
patients (9–11). On that basis, positivity at two consecutive tests was suggested
to be sufficient for clinical intervention even before clinical relapse occurs (14).
However, these correlations are not absolute, as cases have been reported who
either remain PCR-positive in long-term remission or, more frequently, ultimately
relapse after negative tests (10,11).
2. Materials
2.1. RNA Extraction
1. Guanidinium-isothiocyanate solution (GTC): 4 M guanidinium-isothiocyanate,
0.5% N-lauroylsarcosine, 25 mM Na citrate (pH 7.0), and 0.1 M βmercaptoethanol.