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 ET cases.1 

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 cells a progressive lower amount of K562 cells expressing the b3a2 form of theBCR-ABL chimeric mRNA. After RNA extraction and nested PCR as reported above, the detection level of the method was 1 BCR-ABL+ K562 cell in 105 BCR-ABLHL60 cells.

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).

Table 1.

Disease transformation

CaseDisease conversionTime to conversion (y)BCR-ABL+Outcome after conversion
AML 12 yes Death in 3 mo  
AML no Death in 2 mo 
IM no Alive at 3 y  
IM 10 no Alive at 2 y  
IM no Alive at 2 y 
MDS no Alive at 6 mo 
MG no Alive at 2 y  
MG no Alive at 2 y 
CaseDisease conversionTime to conversion (y)BCR-ABL+Outcome after conversion
AML 12 yes Death in 3 mo  
AML no Death in 2 mo 
IM no Alive at 3 y  
IM 10 no Alive at 2 y  
IM no Alive at 2 y 
MDS no Alive at 6 mo 
MG no Alive at 2 y  
MG no Alive at 2 y 

Cytogenetics at conversion for case 1: t(7;13)(q33;q13), inv(11)(p12;q24). Cytogenetics at conversion for cases 2-8 was unchanged.

AML, acute myeloid leukemia; IM, idiopathic myelofibrosis; MDS, myelodisplastic syndrome; MG, monoclonal gammopathy.

Whether an MPD with marked thrombocythosis and expressing theBCR-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,6,7as well as the similarity of the chimeric transcripts.8Recently, 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-ABLpatients, suggesting, with others, the possibility of a new ET variant. Subsequently, Singer et al9 detected the molecular rearrangement in 63% of their 16 patients. Nevertheless, these observations were not confirmed in a further small series of ET patients, recently reported.10 Most recently, some investigators reported the absence of BCR-ABL rearrangement in all 41 of their ET patients, studied by fluorescence in situ hybridization (FISH).11 

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.

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

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