Megakaryocytes carry the fused bcr-abl gene in chronic myeloid leukaemia: a fluorescence in situ hybridization analysis from bone marrow biopsies

Abstract

Histological examination of bone marrow biopsies shows that about one-third of chronic myeloid leukaemia (CML) patients exhibit an increase of megakaryocytes. The megakaryocytic predominance may be so striking that differentiation from other chronic myeloproliferative disorders (CMPD) may be difficult in some CML patients. Megakaryocytes in CML are clonal as demonstrated by loss of glucose-6-phosphate dehydrogenase isoenzymes. The Ph translocation, fusing the abl and bcr genes on chromosomes 9 and 22, however, obviously occurs as a second step in tumour development. So far, the Ph translocation has not been assigned explicitly to megakaryocytes. The question is whether the megakaryocytic cell lineage could harbour the bcr/abl fusion in those CML cases with striking proliferation of megakaryocytes but lack this genetic defect in cases with normal or decreased megakaryocyte counts. We therefore performed triple-colour fluorescence in situ hybridization (FISH) for portions of the bcr and abl genes flanking the breakpoint in CML in paraffin sections of CML cases with normal and with increased numbers of megakaryocytes. This method allows identification of the bcr/abl fusion in single, morphologically intact cells, whereas conventional cytogenetics requires lysis and thus destruction of the cell. Among the 21 CML patients examined by FISH, 10 were informative for bcr and abl genes and displayed distinct hybridization signals within nuclei of bone marrow cells. Besides the granulopoietic cells, megakaryocytes of all those patients (4 without and 6 with varying grades of megakaryocytic increase) displayed bcr/abl fusion signals indicative of a Ph translocation. The lack of hybridization signals in the remaining 11 cases indicates that this technique is not of value diagnostically and should be reserved for scientific questions. Positive controls consisted of conventional chromosome preparations from bone marrow aspirates demonstrating the Ph chromosome in all patients examined, and negative controls of paraffin sections of bone marrow biopsies from non-CML patients. These showed no fusion signals in bone marrow cells, including megakaryocytes, using FISH. Our results demonstrate clearly that not only the transforming event but also the Ph translocation leading to the bcr/abl fusion happens prior to the differentiation of the pluripotent stem cell into different myeloid lineages. The megakaryocytic proliferation evident in some CML cases is probably a consequence of the disease progress.

Improvement of platelet dysfunction in chronic myelogenous leukemia following treatment with imatinib: a case report

“…[10] showed that not only the transforming event but also the Ph + translocation leading to BCR-ABL + fusion happens prior to the differentiation of the pluripotent stem cell into different myeloid lineages.…”

Simple and reliably sensitive diagnosis and monitoring of Philadelphia chromosome-positive cells in chronic myeloid leukemia by interphase fluorescence in situ hybridization of peripheral blood cells

“…2,[42][43][44][45] There is some controversy over whether lymphocytes are of leukemia origin in CML. Garicochea et al 46 reported that T lymphocytes had no bcr/abl fusion gene, while Tefferi et al 42 demonstrated that t(9;22) was detectable in 34% of CD3-positive T lymphocytes, in 32% of CD19-positive B lymphocytes and in 82% of CD34-positive precursor cells.…”

Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH)

“…Biotin is covalently linked to the C-5 position of dUTP (Langer et al 1981), but can also be chemically coupled to other nucleotides such as adenosine and cytosine (Gebeyehu et al 1987). Biotinylated probes are detected by the avidin or streptavidin method or less frequently by anti-biotin antibodies (Cremer et al 1986;Hopman et al 1986b;Nolte et al 1996a). The sensitivity of the former method can be further enhanced by the use of a bridging anti-avidin antibody followed by a second round with fluoresceinated avidin (Waldman et al 1991;Kim et al 1993;Nasarek et al 1995).…”

“…If proteolytic pretreatment is used to demask the genomic DNA, this step has to be optimized for different tissues and fixation times. Perhaps more reliable and reproducible results, especially for the detection of single copy genes in thin paraffin sections, have been obtained by boiling the sections in citric buffer prior to FISH (Nolte et al , 1996a. This pretreatment, that is easily performed in a microwave oven, can be standardized, in contrast to enzymatic digestion, which has to be titrated (Werner et al 1996).…”