Highly purified primitive hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia

Abstract

The hierarchical level of stem cell involvement in acute promyelocytic leukemia (APL) characterized by the pathognomonic PML-RARA fusion gene is unknown. To determine if the cells of the primitive hematopoietic stem cell compartment are involved in the leukemic process, we have used molecular and cell sorting techniques in peripheral blood and bone marrow (BM) cells at diagnosis from three patients with APL and t(15; 17). In two of them, clonality analysis was also possible using the BstXI polymorphic site of the PGK gene. The PML-RARA fusion gene was readily identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of BM cells obtained at diagnosis in all three patients. These same samples were then used to sort CD34+ cells and their CD38+ and CD38-subsets by fluorescence-activated cell sorting. In both female patients, CD34+/CD38+ and CD34+/CD38- cell fractions were polyclonal using PCR, whereas a monoclonal pattern was identified at the BM sample obtained at diagnosis either by Southern blotting or by PCR. Because of the high sensitivity of the PCR analysis, the polyclonal pattern of these cell populations could mask the presence of a minor clone. To detect this clone, we preformed RT-PCR analysis for t(15; 17). In one female patient, the abnormal PML-RAR fusion gene was found only in the more mature CD34+/CD38+ cell fraction using a nested PCR approach, whereas the polyclonal CD34+/CD38- fraction was PML-RARA negative. These findings were confirmed in a third patient with APL in whom the PML-RARA transcripts were absent in the CD34+/CD38- cell fraction. To study the clonality at the level of clonogenic progenitors, we used in one patient PGK analysis by PCR of individual burst-forming units-erythroid and colony-forming units-granulocyte- macrophage obtained from the CD34+/CD38- and CD34+/CD38+ cell populations at diagnosis and from the BM sample obtained during remission. The two highly purified cell populations gave rise to morphologically normal colonies clonal for both the BstXI site containing (A) and the BstXI site lacking (B) PGK allelles, indicating their polyclonal content, a pattern that was also found in clonogenic progenitors obtained at remission. These findings strongly suggest that the primitive hematopoietic stem cells as defined by the CD34+/CD38- antigens are not involved by the neoplastic process in APL. These results may have important implications for autografting strategies of retinoic acid/chemotherapy-resistant or relapsed patients.

Cell lineage specific involvement in acute promyelocytic leukaemia (APL) using a combination of May‐Grünwald‐Giemsa staining and fluorescence in situ hybridization techniques for the detection of the translocation t(15;17)(q22;q12)

“…This was postulated before by Takatsuki et al (1993 ) who used a two‐step PCR and demonstrated PML/RARA‐positive clones in colonies of progenitor cells including CFU‐GM. Turhan et al (1995 ) performed PCR after fluorescence‐activated cell sorting on APL cells and strongly suggested that the haemopoietic stem cells defined as more mature by the CD34 + /CD38 + antigens were positive for PML/RARA but more primitive progenitors defined by CD34 + /CD38 − antigens were found to be PML/RARA negative. So far our technique cannot address this question, because we were not able to examine a subset of the most immature progenitor cells by FICTION with two different antibodies searching for cells that are CD34 positive but CD38 negative.…”

Retinoic acid regulatory pathways, chromosomal translocations, and acute promyelocytic leukemia

“…A recent interesting finding is that PML-RARA is present not only in the granulocytemonocyte colony forming unit (CFU-GM) but also in the erythroid burst forming unit (BFU-E), suggesting that this genetic event takes place at a differentiation stage preceding the promyelocytic progenitor cells (Takatsuki et al, 1994). However, the primitive hematopoietic stem cells as defined by the CD34+/CD38-phenotype are PML-RARAnegative (Turhan et al, 1995).…”

Assessment of Clonality and Its Relevance in Acute Myeloid Leukaemia and Bone Marrow Transplantation

“…Results for the HUMARA PCR compared well with samples analysed with PGK, HPRT and M27~ by Southem blotting. This system has been used to investigate the donal status of highly purified FACS-sorted haemopoietic stem cells in myeloid malignancies [19,20] and individual haemopoietic colonies grown in semi-solid culture [21] where cell numbers are small.…”