MEGAKARYOCYTES CARRY THE FUSED BCR-ABL GENE IN CHRONIC MYELOID-LEUKEMIA - A FLUORESCENCE IN-SITU HYBRIDIZATION ANALYSIS FROM BONE-MARROW BIOPSIES

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 i soenzymes. The Ph translocation, fusing the abl and bcr genes on chrom osomes 9 and 22, however, obviously occurs as a second step in tumour development. So far, the Ph translocation has not been assigned explic itly to megakaryocytes. The question is whether the megakaryocytic cel l lineage could harbour the bcr/abl fusion in those CML cases with str iking proliferation of megakaryocytes but lack this genetic defect in cases with normal or decreased megakaryocyte counts. We therefore perf ormed triple-colour fluorescence in situ hybridization (FISH) for port ions of the bcr and abl genes flanking the breakpoint in CML in paraff in sections of CML cases with normal and with increased numbers of meg akaryocytes. This method allows identification of the bcr/abl fusion i n single, morphologically intact cells, whereas conventional cytogenet ics requires lysis and thus destruction of the cell. Among the 21 CML patients examined by FISH, 10 were informative for bcr and abl genes a nd displayed distinct hybridization signals within nuclei of bone marr ow cells. Besides the granulopoietic cells, megakaryocytes of all thos e patients (4 without and 6 with varying grades of megakaryocytic incr ease) displayed bcr/abl fusion signals indiciative of a Ph translocati on. The lack of hybridization signals in the remaining 11 cases indica tes that this technique is not of value diagnostically and should be r eserved for scientific questions. Positive controls consisted of conve ntional chromosome preparations from bone marrow aspirates demonstrati ng the Ph chromosome in all patients examined, and negative controls o f paraffin sections of bone marrow biopsies from non-CML patients. The se showed no fusion signals in bone marrow cells, including megakaryoc ytes, using FISH. Our results demonstrate clearly that not only the tr ansforming event but also the Ph translocation leading to the bcr/abl fusion happens prior to the differentiation of the pluripotent stem ce ll into different myeloid lineages. The megakaryocytic proliferation e vident in some CML cases is probably a consequence of the disease prog ress.