To the Editor:
We read with interest the article by Blickstein et al,1 in which marrow aspirates from 25 patients with Ph−essential thrombocythemia (ET) were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of BCR-ABL transcripts: the authors found that 48% of patients were PCR+. In a subsequent letter, Marasca et al2were unable to confirm these observations: only 1 of the 20 (5%) patients tested in their series showed BCR-ABL expression. These contrasting experiences prompted us to examine our own patients with ET, and we wish to report our results.
We investigated prospectively marrow aspirates referred to our institution for cytogenetic analysis from 18 consecutive patients with ET (5 men, 13 women; median age, 71 years; range, 38 to 92 years). All patients fulfilled the criteria for diagnosis of ET defined by the Polycythemia Vera Study Group (PVSG).3 Median platelet count was 1,140 × 109/L (range, 756 to 1,462). All patients were Ph− by standard cytogenetic analysis: none of the patients had received cytoreductive treatment at the time of marrow sampling. RT-PCR was performed according to the method of Cross et al,4 with a sensitivity of 1:105. All assays were performed with appropriate controls and were repeated at least twice. None of our patients was found to express BCR-ABL.
How can we reconcile our results with those of Blickstein et al?1 The patient population we have studied is slightly different from Blickstein’s, particularly in regard to previous treatment: only 3 of 27 patients in their study were newly diagnosed, with the remaining patients having received hydroxyurea at time of study, whereas all of our patients were newly diagnosed. However, it is hard to explain the discrepancy between our results and those of Blickstein’s on this basis, although the possibility of further mutations, including BCR-ABL rearrangement, occurring with follow-up cannot be excluded. Blickstein et al do not state whether any of the three untreated patients they studied were BCR-ABL+, but acquisition of the Ph chromosome has been described as a late change in other myeloproliferative disorders,5 and it would certainly be interesting to perform a sequential study on patients with ET to determine whether BCR-ABL rearrangements can be acquired as a result of the natural history of the disorder or secondary to treatment.
Our results are much more in keeping with those of Marasca’s group,2 although more stringent criteria for the diagnosis of ET were used in this series (patients with platelet counts <1,000 × 109/L were excluded). Technical differences also seem unlikely to explain the discrepancy: precise details of the RT-PCR methodology used may vary slightly between the studies, but the assay sensitivities appear similar. We suggest that a larger study of BCR-ABL expression in sequential marrow samples from Ph−ET patients is performed, with further alternative methodologies such as fluorescence in situ hybridization and “quantitative” PCR being applied to samples yielding positive results.
Response
We thank Stuart Hackwell and colleagues for bringing to our attention their observation. Hackwell et al found that none of their 18 newly diagnosed ET patients expressed BCR-ABL transcripts in their bone marrow. The discrepancies between our observation and that of the Salisbury group is quite puzzling since another group recently provided support to our previous data: Singer et al1-1 reported that 63% of their ET patients carried BCR-ABL transcripts in peripheral blood. These data are in accord with our updated findings that 48% of 40 bone marrow aspirates of hydroxyurea-treated Ph− ET patients were positive for BCR-ABL.1-2 In peripheral blood, 18% expressed BCR-ABL transcripts.1-2 1-3 It should be noted that our RT-PCR analysis in bone marrow aspirates and peripheral blood was done according to the clinical protocol, ie, screening 1.25 × 106 cells.
It seems there is a spectrum of observations. On one end are healthy adults who carry BCR-ABL transcripts in 1 to 10 in 108peripheral blood leukocytes, presumed to be lost through normal cell differentiation and death.1-4 On the other end are Ph− chronic myeloproliferative treated patients who express the transcripts in bone marrow and/or peripheral blood.1-1-1-3 The role of hydroxyurea in generation of translocation and gene fusion is not clear because, on the one hand, BCR-ABL transcripts were detected in healthy individuals,1-4and on the other hand the potential effect of hydroxyurea on genomic stability has not been excluded.
We agree with Hackwell et al that a larger group of sequential marrow samples is needed to elucidate the natural history of BCR-ABL transcripts in ET Ph− patients, using fluorescence in situ hybridization and quantitative PCR analysis as well.
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ACKNOWLEDGMENT
This work was undertaken by Salisbury Health Care, who received funding from the NHS executive; the views expressed in this publication are those of the authors and not necessarily those of the NHS executive.
