Estela Matutes

Myeloid progenitor cells in the circulation of patients with myelofibrosis and other myeloproliferative disorders

Summary. We have measured the numbers of myeloid progenitor cells in the circulation of patients with myelofibrosis (MF) and other myeloproliferative disorders. In general, progenitor cell numbers were increased in the circulation of patients with MF compared with controls. The mean increases were 9‐fold for the multilineage progenitor cells (CFU‐GEMM), 13‐fold for the erythroid progenitor cells (BFU‐E), 37‐fold for the granulocyte‐macrophage progenitor cells (CFU‐GM) and 167‐fold for the megakaryocyte progenitors (CFU‐MK). Splenectomized patients generally had reduced numbers of circulating progenitor cells. In the CFU‐MK assay, mature megakaryocytes cultured from patients with MF regularly showed large vacuoles in the nucleus and cytoplasm, unlike control cells. The increased colony formation in patients with MK, involving especially CFU‐MK colonies, is consistent with the hypothesis that MF is a primary myeloproliferative disorder in which a megakaryocyte‐derived factor predisposes to the formation of marrow fibrosis.

Characterization of blast cells in chronic granulocytic leukaemia in transformation, acute myelofibrosis and undifferentiated leukaemia. I. Ultrastructural morphology and cytochemistry.

A systematic analysis of the blast cell population was carried out on samples from 50 patients suffering from blast transformation of chronic granulocytic leukaemia (CGL) (31) and of myelofibrosis (4), acute myelofibrosis (AM) (11) and undifferentiated acute leukaemia (4). Transmission electron microscopy (TEM), used in 41 samples, included: morphology and techniques for myeloperoxidase (MPO), platelet‐peroxidase (PPO) and acid phosphatase (AP). The majority of cases were also studied by light microscopy cytochemistry and with a battery of cell markers which are reported in the accompanying paper (San Miguel et al, 1985). The characterization of the type(s) of proliferating blasts was made from the integration of ultrastructural and immunological data. TEM morphology allowed the precise recognition of specific granules in basophil and mast‐cell precursors and of ferritin particles in blasts of erythroid lineage; these rare cell types were not adequately characterized by other methods. The PPO reaction made possible the identification of pure megakaryoblastic proliferations in 38% of cases, including eight of the 11 with AM; megakaryoblasts were also present in nine of 12 cases with mixed blast cell types. The MPO and AP reactions were useful for the characterization of myeloblasts and monoblasts, respectively. Lymphoblasts could be distinguished from other cell types by TEM morphology and negative MPO and PPO reactions. TEM techniques were valuable for diagnosing correctly the type of blast cell in this study in which only four cases (8%) remained unclassifiable.

Increase in Ty Lymphocytes in B-Cell Chronic Lymphocytic Leukaemia

Summary. The proportion of Tγ and Tμ lymphocytes was studied in 40 cases of B‐chronic lymphocytic leukaemia (B‐CLL) and six of B‐prolymphocytic leukaemia (B‐PLL). The significant increase in Ty cells, previously reported in two small B‐CLL series, was confirmed and shown to be directly correlated with the clinical stages of the disease (P < 0.01 to < 0.001). The normal Tμ: Tγ ratio (2.3:1) was reversed in B‐CLL (1:1.4) and B‐PLL (1:1.9). The proportion of Tμ cells was decreased but was not related to stage.

The role of ultrastructural cytochemistry and monoclonal antibodies in clarifying the nature of undifferentiated cells in acute leukaemia

Summary. The nature of the cells in 21 cases of acute leukaemia with blasts which were undifferentiated by light microscopy criteria was investigated by immunophenotyping, ultrastructural cytochemistry and DNA analysis. Two groups of cases were recognized. Fourteen cases were negative with B and T lymphoid markers and expressed one or two myeloid antigens detected by the monoclonal antibodies (McAb) MCS2 (CD13) and MY9 (CD33). Peroxidase activity was demonstrated at ultrastructural level by the method of Roels on unfixed cells in eight out of 10 cases; rearrangement of the immunoglobulin (Ig) genes was demonstrated in one of the three cases investigated. These cases are proliferations of early, MO, myeloblasts which can only be recognized by immunological and ultrastructural cytochemical methods. The remaining seven cases revealed a complex phenotype with expression of myeloid and lymphoid antigens. Peroxidase activity was detected in blasts from two cases with rearrangement of the Ig‐heavy chain gene: in one of them the T cell receptor β and γ chain genes were also found in rearranged configuration. This group comprises cases of biphenotypic and mixed acute leukaemia which probably involve multipotent stem cells. This study demonstrates that the expression of myeloid antigens on blast cells parallels closely the presence of peroxidase activity and that lymphoid markers correlate with gene rearrangements at DNA level. Our findings are reassuring with respect to the specificity of the antimyeloid McAb for the diagnosis of cases which are unclassifiable by conventional methods.

Clinical significance of the presence of myeloid associated antigens in acute lymphoblastic leukaemia

We have analysed the immunological characteristics of blasts from 89 acute lymphoblastic leukaemia (ALL) cases (62 adults and 27 children), by using a panel of antilymphoid and myeloid associated monoclonal antibodies (McAb) and the APAAP method, which detects membrane and cytoplasmic expression of antigens.

Characterization of blast cells in chronic granulocytic leukaemia in transformation, acute myelofibrosis and undifferentiated leukaemia II. STUDIES WITH MONOCLONAL ANTIBODIES AND TERMINAL TRANSFERASE

A panel of 19 monoclonal antibodies (McAb) and the enzyme terminal transferase (TdT) have been applied to the characterization of poorly differentiated blasts from 50 patients with chronic granulocytic leukaemia (CGL) and myelofibrosis in blast crisis (BC), acute myelofibrosis and undifferentiated leukaemia. These cells were also extensively studied by transmission electron microscopy (TEM) (see Polli et al, 1985a). McAb against platelet glycoproteins (GP) showed a high specificity for megakaryoblasts, in particular those reactive with the GPIIb/IIIa complex (J15) and GPIIIa (C15 and C1 7), which were positive in a higher proportion of blasts than the McAb to GPIb (AN51 and FMC25). Findings with these anti‐platelet McAb paralleled those of the platelet‐peroxidase (PPO) reaction in 76% of cases studied simultaneously. The PPO reaction was always positive in cases in which two or more of the McAb were reactive with the blast cells. The differences observed suggest, nevertheless, that PPO is more sensitive for megakaryoblasts than the McAb and that this TEM technique should be reserved for cases which are negative with the platelet specific McAb. Of the McAb against myeloid antigens used in this series OKM1 was positive in 50% of cases but the others failed to demonstrate early features of differentiation in myeloblasts and monoblasts. In only three cases were erythroid precursors demonstrated by TEM and these were the only ones reactive with a McAb to glycophorin‐A (LICR LON/R10). TdT and the McAb J5 helped in the identification of lymphoblasts which were seen as a ‘pure’proliferation in 23% of CGL‐BC and as case that showed frank reactivity with FMC10, 11 and 13 in this study the blasts were mast cell precursors and this may suggest that these leukaemic cells are more mature. The greater sensitivity of OKM1 to detect granulocytic/monocytic differentiation shown here confirms the findings of van der Reijden et al (1983) in AML and of Griffin et al (1983b) in CGL‐BC with a similar McAb, Mol. The demonstration of TdT and the cALL antigen by the McAb J5 showed that blast cells which may be considered undifferentiated or even myeloid on light microscopy morphology were in fact lymphoblasts. In this respect our study confirms a 20–25% incidence of pure lymphoid phenotypes in Ph1‐positive CGL‐BC (Janossy et al, 1980; Greaves et al, 1982; Griffin et al, 1983b). If we consider also the minor populations of lymphoblasts seen in the mixed cases, the incidence of cells with a lymphoid phenotype in CGL‐BC was 40% in this series. The use of McAb against early precursor cells (RFB1, FMC8 and OKIa) did not show a particular advantage for the clarification of the cell lineage of the blasts in the cases under study, although all of them were positive in lymphoblasts. It was of interest, however, that in the mixed cases these reagents usually detected the majority of blasts whilst the lineage specific McAb were reactive with some of the cell populations. A number of mixed blast cell proliferations were demonstrated in this study. TEM analysis has been valuable for distinguishing the morphology and cytochemical markers of the distinct cell populations involved (Polli et al, 1985a). Findings with the various lineage specific McAb were very often suggestive of the presence of more than one type of blast cell (Table VII). Light microscopy studies combining the demonstration of TdT in the cell nucleus with that of an antigen in the cell membrane have allowed us to distinguish megakaryoblasts (TdT negative, AN51 positive) from lymphoblasts (TdT positive, AN51 negative).

Unusual T‐Cell Phenotype in Advanced B‐Chronic Lymphocytic Leukaemia

Summary. T‐lymphocytes isolated by E‐rosetting from 22 patients with B‐cell chronic lymphocytic leukaemia (B‐CLL) showed membrane phenotype features distinct from those of normal T‐lymphocytes. These changes were particularly marked in advanced disease (Rai stages II, III, IV and WBC over 100 × 109/1). The most significant finding was the demonstration, in 10 cases, of a major population of E‐rosette positive cells (40—80%) unreactive with the OKT monoclonal antibodies against mature or immature T‐cells; 15—30% of the cells were unreactive in seven other cases. A significant reduction in OKT4 positive (helper) lymphocytes was seen in 18 cases. The proportion of OKT8 positive cells was increased in two and normal or low in the rest. Only four patients with early disease (stages 0 and 1) had a normal T‐cell phenotype. These findings could explain abnormalities previously described in the T‐lymphocytes of B‐CLL and provide new insights into the pathogenesis of the disease.

Correlation between chromosomal abnormalities and blast phenotype in the blast crisis of Ph-positive CGL

We carried out cytogenetic analysis in 23 patients with Ph-positive chronic granulocytic leukemia in blast crisis. In all cases the type of blast cell was characterized by cytochemistry, immunologic markers, and ultrastructural studies. Twelve cases were classified as myeloid transformation, six as lymphoid, two as mixed (lymphoid and myeloid), and two were unclassifiable. Duplication of Ph was the most frequent abnormality in the whole series. Trisomy 8, i(17q) and trisomy 19 were seen only in patients with myeloid blast crisis (53%, 30%, and 23%, respectively). Our findings suggest that the nature of additional chromosome abnormalities arising in blasts with features of myeloid differentiation are different from those in blasts showing lymphoid differentiation.

Terminal deoxynucleotidyl transferase positive acute myeloid leukaemia: an association with immature myeloblastic leukaemia

Summary. The morphology, membrane markers and ultra‐structural cytochemistry of 39 cases of acute myeloid leukaemia (AML) with variable proportion (10–99%) of terminal deoxynucleotidyl transferase (TdT) positive blasts was compared with that of 134 cases of TdT negative AML. The incidence of TdT positive AML was 22.5% and this was significantly higher in poorly differentiated myeloblastic (M0 and M1) types (54%) than in all other FAB subtypes (10%; P<0.001). Our findings suggest heterogeneity among TdT positive cases. Whilst the majority correspond to genuine TdT positive AML in which evidence for exclusive myeloid nature was demonstrated by phenotypic, cytochemcal and ultrastructural markers, a distinct minority (22%) of cases had mixtures of lymphoid and myeloid blasts. A change in phenotype occurred in three out of six cases studied in relapse. There was no difference in the incidence of immuno‐globulin (Ig) gene rearrangement between TdT positive (two out of 12) and TdT negative (one out of 11) cases, although published data suggests that Ig gene rearrangement is significantly more common in TdT positive cases. The determination of TdT in AML allows the identification of cases of mixed acute leukaemia which probably represent proliferations of multipotent progenitor cells. The majority of TdT positive cases, nevertheless, correspond to immature types of myeloblastic leukaemia which may constitute a clinically distinct subgroup.

α, β and γ T‐cell receptor genes: rearrangements correlate with haematological phenotype in T cell leukaemias

We have studied the arrangement of the α, β and γT cell receptor (TCR) genes in 27 patients with T cell lymphoproliferative disorders. Nine patients had acute lymphoblastic leukaemia (T‐ALL), nine patients had prolympho‐cytic leukaemia (PLL), six patients presented with a T‐CLL/T‐lymphocytosis syndrome, two patients had Sezary syndrome (SS) and one patient had HTLV‐I positive T‐cell leukaemia/ lymphoma (ATLL). α TCR gene rearrangement could be demonstrated by the use of three available probes in only one case. By contrast, both β and γ TCR gene rearrangement could be demonstrated by Southern blot analysis of DNA samples digested with appropriate restriction enzymes in the majority of cases. In general, when rearrangements were present they involved both alleles. The proportion of rearranged chromosomes was lower in T‐ALL than in other forms of T‐cell leukaemia and it was lower in cases with the CD4 —/ CD8+ phenotype than in those with a CD4 +/CD8—phenotype. In three out of 34 cases of B‐cell leukaemia the TCR β‐gene but not the TCR γ‐gene was rearranged, just as in two out of 26 cases of T‐cell leukaemia the immunoglobulin (Ig) heavy chain but not the light chain genes were rearranged.