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)

Abstract

Summary. Acute promyelocytic leukaemia (APL) is strongly associated with the translocation t(15;17) which therefore provides a reliable marker to assess the potential involvement of different cell lineages. Six cases with morphologically, cytogenetically and molecularly proven APL were analysed at diagnosis or relapse by combining fluorescence in situ hybridization (FISH) with standard May-Grü nwaldGiemsa (MGG) staining at the single cell level on bone marrow and blood smears. With the FICTION technique, combining immunophenotyping with FISH, haemopoietic precursor cells were identified using monoclonal antibodies against CD34, B-and T-lymphocytes could be identified with CD19 and CD3. In addition, HLA-DR-positive cells were studied for the presence of t(15;17).Morphologically identified myeloblasts were relocated on the smear after FISH and were found to be PML/RARA positive in 91%, abnormal promyelocytes in 97%. In contrast, a positive signal was obtained in only 18% of PMN. Erythroblasts, lymphocytes and plasma cells did not show a PML/RARA rearrangement. Accordingly, all cells expressing CD3 or CD19 were PML/RARA negative. CD34 positive precursor cells identified by FICTION were PML/ RARA positive in 97%. HLA-DR-positive cells contained a PML/RARA rearrangement in 24% of cells in one case and were negative in the two other cases investigated. These data indicate that APL appears to originate from a level of haemopoietic precursor cells but is restricted to the myeloid lineage. The low proportion of PML/RARA-positive PMN points to the impairment of blast cell differentiation beyond the promyelocyte stage but also emphasizes the existence of normal residual haemopoietic stem cells from which PML/ RARA-negative PMN must be derived.

Acute promyelocytic leukemia: where does it stem from?

“…Indeed cases of hypergranular M3 that may be considered to represent the most differentiated form of the disease, which are CD34 negative according to conventional immunophenotype criteria, have nevertheless been shown to harbor the PML-RARA fusion in the majority of CD34 þ cells. 66 This is consistent with the existence of a hierarchy in APL as well as other subsets of AML. 20 Furthermore, it would suggest that the current transgenic models of APL may not be successfully targeting expression of PMLRARa at an appropriate level to the progenitors that are subject to leukemic transformation in man.…”

“…30 A subsequent study, which employed morphological analysis in conjunction with the FICTION technique, combining FISH and immunophenotype analysis, was in accordance. 66 In each of the six cases of hypergranular APL examined, which all happened to lack expression of lymphoid-affiliated antigens, the PML-RARA fusion was restricted to the myeloid lineage, with no evidence of involvement of erythroblasts, plasma cells, or B or T lymphocytes. 66 Similarly, the t(15;17) was found to be restricted to the granulocytic series in an earlier study that utilized the MAC method (morphology-antibody-chromosomes).…”

“…66 In each of the six cases of hypergranular APL examined, which all happened to lack expression of lymphoid-affiliated antigens, the PML-RARA fusion was restricted to the myeloid lineage, with no evidence of involvement of erythroblasts, plasma cells, or B or T lymphocytes. 66 Similarly, the t(15;17) was found to be restricted to the granulocytic series in an earlier study that utilized the MAC method (morphology-antibody-chromosomes). 67 However, two further studies yielded results with quite different implications regarding the origins of APL.…”

The incidence of acute promyelocytic leukemia appears constant over most of a human lifespan, implying only one rate limiting mutation

“…In contrast, mutations from more differentiated cells appear to be approximately constant throughout life, 12,38 implying the event causing APL is restricted to cells committed to differentiation, a conclusion for which there is other experimental evidence. [34][35][36][37] Presumably, the mutation rate for the PML/RAR␣ translocation is so low that its occurrence is negligible in cells as infrequent and slowly dividing as stem cells. Only the much greater number of cell divisions that occur during differentiation allow a sufficient probability of mutation to cause disease at the observed rates.…”

A comprehensive leukemia database: integration of cytogenetics, molecular genetics and microarray data with clinical information, cytomorphology and immunophenotyping

“…Cytomorphological findings were supplemented as well as fluorescence in situ hybridization. 16,17 Immunophenotyping was applied according to standard procedures. 18 The integrated leukemia database A multi-user database with a web frontend consisting of eight main modules was established:…”