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CORRESPONDENCE Essential thrombocythemia (ET) is a chronic myeloproliferative
disorder (MPD) characterized by an elevated thrombocytosis, an
increased number of megakaryocytes with dismegakaryopoiesis in the bone
marrow, and no identifiable underlying primary causes. The disease can
evolve into myelofibrosis and, rarely, into acute leukemia.
According to the current diagnostic criteria of the Polycythemia Vera Study Group (PVSG), ET is lacking in features diagnostic for other MPDs, including Philadelphia chromosome (Ph). Karyotypic anomalies are rare and not specific, and the clonality is
controversial; so this disorder remains a diagnosis of exclusion, and
the identification of subgroups of patients at risk for progression to
leukemia is quite difficult. Recently, some authors reported a BCR-ABL
transcript positivity in about a half of 25 Ph To verify the hypothesis of a new ET variant with possible clinical
implications, we investigated the presence of the molecular counterpart
of the Ph chromosome in a larger series of ET patients with a longer
follow-up. We investigated 112 white patients (44 males, 68 females; median age, 56 years, range 23 to 98 years) diagnosed with ET
following the criteria of the PVSG. The patients were from 3 different
institutions of the same region (Po Valley, northern Italy). At
admission, routine laboratory investigations, including complete blood
film, leukocyte alkaline phosphatase (LAP) score, and serum vitamin
B12 levels, were carried out. Bone marrow aspiration and
biopsy were performed for histological, cytogenetic, and molecular
studies. Bone marrow examinations were repeated at least once, in the
majority of cases. Cytogenetic analyses were performed, at diagnosis
and prior to any treatment, in all patients using conventional banding
methods. Molecular studies for the detection of chimeric messengers
BCR-ABL, coding for p190 and p210 proteins, were performed by
"nested" reverse transcriptase-polymerase chain reaction (RT-PCR)
on total RNAs extracted from Lymphoprep-separated (Nycomed Pharma,
Majorstu, Norway) bone marrow mononuclear cells by a
guanidine-isothiocyanate-phenol-chloroform method.2 cDNA
was synthesized using 1.5 µg total RNA in a 30 µL reaction mixture
as described elsewhere,3 using an antisense primer
specific for the exon a3 of the ABL gene. Nested
PCR was performed as follows: 25 µL cDNA was subjected to 40 cycles
of amplification in a 50 µL reaction mixture containing 0.225 mM dNTPS, 0.5 µM of each primer, 1.0 U of Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany), 1.5 mM MgCl2, 10 mM
Tris-HCl (pH 8.3), and 50 mM KCl. The conditions of amplification were: 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C with 2 minutes of an initial denaturation step at 94°C and 10 minutes
of a final extension step at 72°C. 2.5 µL of the first round of
amplification were subjected to a second round with 2 internal primers
at the same conditions of amplification. As control, 5 µL of the same
retrotranscription reactions were subjected to 35 cycles of PCR using
as primers two oligonucleotides specific for the ABL gene.
The sequence of the primers used in PCR reactions was reported
elsewhere.4,5 The minimal level of the detection of the nested PCR was estimated
adding to 5 × 106 HL60 BCR-ABL In our group of 112 patients, 69 (61.6%) showed a platelet count lower
than 1 × 107/µL (range,
0.62 × 107 to 1 × 107) and 43 (38.3%)
greater than 1 × 107/µL (range,
1.05 × 107 to 2.7 × 107). The white blood
cell count showed a mean count of 8 300/µL, with more than 3%
basophils only in 3 patients and the absence of immature cells, in the
peripheral blood. The hematocrit was less than 40% in all patients.
The LAP score was increased in 54 patients, normal in 23, and not done
in the remaining 35. Serum vitamin B12 level was in normal
range in all patients. The bone marrow examination showed a more or
less important megakaryocytic hyperplasia with dismegakaryocytosis in
all cases; neither myelodysplastic features nor collagen fibrosis were
detected in all cases, at diagnosis. Splenomegaly smaller than 3 centimeters palpable was observed in 13 patients. Thrombotic or
hemorragic events, of variable gravity, were noted in 37 patients. At
cytogenetic examination, no one showed the t(9;22) translocation, at
diagnosis. For all patients, the median follow-up time was 39.70 months
(range, 6 to 242); the 19 newly diagnosed patients were observed for a
mean of 9 months (range, 8 to 11), whereas for 90 patients (80.35%) the median follow-up time was 62.79 months (range, 24 to 144) and for 3 patients was 193 months (range, 161 to 242). The patients followed for
a mean of 108.11 months (range, 60 to 242) were 39. The RT-PCR studies for BCR-ABL transcripts showed the
chimeric product (b3a2 type) only in 1 patient (0.89%). The
clinical and hematologic features remained unchanged in the majority of patients, with few exceptions; 8 patients showed a disease
transformation (Table 1). Two patients
died from blastic crisis and two from cerebral stroke, from 6 to 10 years after diagnosis. To maintain the platelet count below
0.6 × 107/µL, 73 patients underwent a cytoreductive
therapy (hydroxiurea 55, uracil mustard 13, busulfan 4, and interferon
1).
Whether an MPD with marked thrombocythosis and expressing the
BCR-ABL transcripts might be considered a variant form of ET or of CML has raised controversies for several years. Many, including the PVSG, agreed with the latter option because of the high incidence of leukemic transformation of the Ph+ ET,6,7
as well as the similarity of the chimeric transcripts.8 Recently, Blickstein et al1 reported an incidence of 48%
of the BCR-ABL transcript in 25 ET patients, with neither
clinical nor laboratory differences compared with BCR-ABL The results of our observations on a larger series of patients with ET showed the absence of the BCR-ABL rearrangements in this disease. The only BCR-ABL+ patient of our series (0.89%), on which we already reported,10 is probably an unusual case of CML at thrombocythemic onset and long survival, a case that finally progressed to acute leukemia. The longer follow-up of our patients (median 62.79 months for the 80.35% of our series of patients, compared with medians 22.5 and 37 months in Blickstein et al1 and Singer et al,9 respectively) allowed us to document a disease course more consistent with the natural history of ET than that of CML. The discrepancies between the 2 groups of observations, rather than between technical procedures (methodologies and sensitivities appear equivalent) might be due to inaccuracies in ET diagnosis or, at least, to racial differences. Our suggestion is that true ET does not carry the Ph anomaly that, instead, might characterize the CML variant forms with thrombocythemia.
Giovanni Emilia, Roberto Marasca, Patrizia Zucchini, Paola Temperani, Mario Luppi, and Giuseppe Torelli
Francesco Lanza, Cristiano De
Angelis, Domenica Gandini, and GianLuigi Castoldi
Daniele Vallisa, and Luigi Cavanna
Laura del Senno Supported by AIL (G.E., R.M., P.Z., P.T., M.L., and G.T.), by FIRC (C.D.A.), and by AIRC (M.L.). References
1.
Blickstein D, Aviram A, Luboshitz J, et al.
BCR-ABL transcripts in bone marrow aspirates of Philadelphia-negative essential thrombocythemia patients: clinical presentation.
Blood.
1997;90:2768-2771
2.
Diverio D, Riccioni R, Pistilli A, et al.
Improved rapid detection of the PML/RAR
3.
Marasca R, Zucchini P, Galimberti S, et al.
Missense mutation in the PML/RAR
4.
Hughes TP, Morgan HGJ, Martiat P, Goldman JM.
Detection of residual leukemia after bone marrow transplant for chronic myeloid leukemia: role of polymerase chain reaction in predicting relapse.
Blood.
1991;77:874-878 5. Maurer J, Janssen JWG, Thiel E, et al. Detection of chimeric BCR-ABL genes in acute lymphoblastic leukaemia by the polymerase chain reaction. Lancet. 1991;337:1055-1058[CrossRef][Medline] [Order article via Infotrieve]. 6. Morris CM, Fitzgerald PH, Hollings PE, Archer SA, Rosman I, Beard MJ. Essential thrombocythemia and the Philadelphia chromosome. Br J Haematol. 1988;70:13-19[Medline] [Order article via Infotrieve]. 7. Cervantes F, Urbano-Ispizua A, Villamor N, et al. Ph-positive chronic myeloid leukemia mimicking essential thrombocythemia and terminating into megakaryoblastic crisis: report of two cases with molecular studies. Leukemia. 1993;2:327-330. 8. Kwong YL, Chiu EKW, Liang RHS, Chan V, Chan TK. Essential thrombocythemia with BCR/ABL rearrangement. Cancer Genet Cytogenet. 1996;89:74-76[CrossRef][Medline] [Order article via Infotrieve]. 9. Singer IO, Sproul A, Tait RC, Soutar R, Gibson B. BCR-ABL transcripts detectable in all myeloproliferative states [abstract]. Blood. 1998;92:427a.
10.
Marasca R, Luppi M, Zucchini P, Longo G, Torelli G, Emilia G.
Might essential thrombocythemia carry Ph anomaly?
Blood.
1998;91:3084-3085 11. Solé F, Florensa L, Espinet B, Besses C, Lloveras E, Woessner S. Absence of bcr/abl rearrangement in 41 patients with essential thrombocythemia. Haematologica. 2000;85:214-221[Medline] [Order article via Infotrieve].   CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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ET
cases.
fusion gene in acute promyelocytic leukemia.
Leukemia.
1996;10:1214-1216
