Analysis of CD34 populations in mobilised peripheral blood stem cell harvests and in bone marrow by fluorescent in situ hybridisation for the bcr/abl gene fusion in patients with chronic granulocytic leukaemia

Abstract

For those patients ineligible for allogeneic bone marrow transplant and who are non-responsive to interferon, autotransplant with peripheral blood stem cells (PBSC) mobilised after intensive chemotherapy, may provide a novel approach to improve prognosis in patients with chronic granulocytic leukaemia. PBSC harvests are assessed for CD34-positive cell numbers, which serve as an indicator of engraftment potential, and are also analysed cytogenetically to ascertain tumour cell contamination. However, a more accurate assessment of PBSC harvest contamination requires investigation of the Philadelphia (Ph) status of the CD34 pos population, in which the cells that provide long-term engraftment are contained. In this study, we have analysed these levels in mobilised PBSC and also in bone marrow (BM) harvests, taken several weeks prior to mobilising chemotherapy. Using fluorescent in situ hybridisation for the bcr/abl gene fusion, we have shown that the median number of Ph negative cells in CD34 pos isolated populations was 14.95% in BM compared to 79.05% in PBSC harvests and that in all PBSC samples tested, Ph positivity in CD34 pos populations was always detectable either by FISH or one round PCR methods. In paired assessments of both PBSC and BM harvests, higher levels of Ph negative CD34 pos cells (у14%) isolated from BM harvests, taken prior to intensive chemotherapy, correlated with higher levels of Ph negative CD34 pos cells (у78.5%) in PBSC harvests. These data may aid in the selection of patients for whom PBSC harvesting, after mobilisation, is more likely to achieve an autograft product containing predominantly Ph negative CD34 pos cells and may exclude those patients for whom the risk, morbidity and expense of stem cell harvesting may have no apparent benefit over a chronic phase BM harvest.

Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate

“…Similar cell types with the Ph chromosome are enhanced in human marrow from CML patients (51). After multiparameter FACS separation, cells with the surface phenotype of pluripotential stem cells isolated from CML marrow show the presence of the Bcr-Abl rearrangement at the chromosomal level by in situ chromosome analysis (7,8). Reverse transcription-PCR analysis on small numbers of such cells produces Bcr-Abl junction products (9,52).…”

“…The primary affected cell appears to be the pluripotent hematopoietic stem cell and closely related immature committed progenitors (6). Highly enriched stem cell elements from CML patients are Bcr-Abl-positive at the genomic level by in situ chromosome analysis (7,8). Analysis of multilineage, myeloid, erythroid, and other colony forming cells demonstrates the Bcr-Abl gene and its chimeric mRNA (9,10).…”