Myeloid Leukemia

PRV-1 Overexpression in PV and ET 271
Fig. 2. Nonspecific band generated by the PRV-1 amplification procedure. PRV-1
mRNA in patients with PV (lanes 1–4). Lanes 1 and 3 show a nonspecific band (approx
450 bp in length). Negative control (water) and positive control (previously tested and
sequenced cDNA) are indicated with – and +, respectively. MW indicates molecularweight
marker.
Fig. 3. PRV-1 overexpression in several types of leukocytosis. PRV-1 mRNA in
patients with acute postsurgical secondary leukocytosis (lanes 1–4), inflammatory/
infectious secondary locytosis (lanes 5–6), chronic myeloid leukemia (lane 7),
infectious mononucleosis (lane 8), and myelodysplastic syndrome (lanes 9–10). Negative
control (water) and positive control (previously tested and sequenced cDNA) are
indicated with – and +, respectively. MW indicates molecular-weight marker.
assay.
4. In our original paper, all cases that were negative for PRV-1 expression by UV
detection were further analyzed and validated by hybridization with a specific
radiolabeled PRV-1 probe in order to exclude false-negative results (10). Because
samples were never found to be negative at UV examination but positive with
radioactive hybridization, we have discontinued this procedure. In conclusion,
UV detection represents an easy, sensitive, and reproducible method to detect
PRV-1 overexpression in highly purified peripheral blood granulocytes.
5. We have also used an alternative, and more specific, pair of oligoprimers to detect
PRV-1 overexpression. These primers (forward 5�-CCA CAG ACG GGT CAT
GAG CG-3�; reverse 5�-GGG CTG TGG GGC CCA AAG-3�), were used under
the following conditions: 95°C for 40 s, 55°C for 40 s and 72°C for 45 s; this
produced a 395-bp fragment. The PCR buffer and the oligoprimer, dNTP, and
MgCl 2 concentrations for this amplification were the same as described in the standard
method (see Subheading 3.4.). The results of PRV-1 overexpression obtained
with these alternative primers were the same as those obtained with the original primers;
however, the alternative primers produced a weaker PCR product.
6. We have found PRV-1 overexpression in several patients affected by secondary
leukocytosis (10). The clinical history and laboratory data generally permit discrimination
of these false-positive cases from PV and ET (Fig. 3).