Laboratory diagnosis of acute myeloid leukaemia.

The modern characterization of acute myeloid leukaemia is a multidisciplinary process. It requires the integration of clinical information, morphology, cytochemistry, immunophenotyping, cytogenetic and molecular genetic diagnostic techniques. It is only by bringing all these modalities together that a clear picture of the disease can be presented. This initial work-up provides essential prognostic information of benefit to the patient. The selection of treatment and the monitoring of treatment response are dependent on the findings at the time of diagnosis.

Lymphocyte subset analysis and glycosylphosphatidylinositol phenotype in patients with paroxysmal nocturnal hemoglobinuria.

Using multicolor flow-cytometry we have examined 19 patients with paroxysmal nocturnal hemoglobinuria (PNH) (18 with active disease and 1 spontaneous remitter) to determine absolute numbers of lymphocyte subsets and the proportion of glycosylphosphatidylinositol (GPI)-deficient clones amongst these subpopulations. Lymphocyte subsets were abnormal in all patients; the most frequent findings were low absolute numbers of natural killer (NK) cells (median, 0.08 x 10(9)/L; normal range, 0.2 to 0.4 x 10(9)/L) and low absolute numbers of B cells (median, 0.05 x 10(9)/L; normal range, 0.06 to 0.65 x 10(9)/L). GPI-deficient B, T, and NK cells were identified in 88%, 84%, and 89% of patients, respectively. The proportion of GPI-deficient cells within individual lymphoid lineages was highly variable, though in most patients the percentage of GPI-deficient NK cells was considerably higher than B or T cells. These observations can be explained when mechanisms of normal lymphopoiesis are considered. Despite these quantitative and qualitative abnormalities, no patients suffered an excessive number or severity of infections. The detection of PNH clones amongst all lymphocyte lineages may provide important information regarding the natural history of the disease and additional insights into kinetics of adult lymphopoiesis.

Two cases of inv(8)(p11q13) in AML with erythrophagocytosis: a new cytogenetic variant.

We describe two patients with acute myeloid leukaemia (AML) associated with erythrophagocytosis and a pericentric inversion of chromosome 8, inv(8)(p11q13). The haematological features were indistinguishable from those of patients with the t(8;16) syndrome and its variants. Our observations emphasize the importance of the breakpoint at 8p11 and the possible involvement of the MOZ gene in all these cases.

T-cell lymphomas in v-Myb transgenic mice.

v-Myb, the transforming protein of avian myeloblastosis virus, causes acute myeloid leukemia in chickens. Similarly, truncation and rearrangement of the c-myb proto-oncogene to yield a v-Myb-like protein leads to myeloid and B cell lymphomas in chickens and mice, and may be a factor in a number of human cancers. To study the effects of deregulation of v-Myb on T cell development, we have generated lines of transgenic mice in which the v-Myb oncoprotein is expressed in a T-cell-specific fashion. Analysis of T cell development in the v-Myb transgenic mice shows that ectopic expression of v-Myb affects the ratio of helper to cytotoxic T cells, by increasing the number of CD4+ helper cells, and inhibits thymic involution, such that mature animals have elevated numbers of thymocytes and circulating mature T cells. In a significant proportion of older animals, high grade T cell lymphomas develop, demonstrating that v-Myb is oncogenic in T cells.

Quantification of PML-RAR alpha transcripts in acute promyelocytic leukaemia: explanation for the lack of sensitivity of RT-PCR for the detection of minimal residual disease and induction of the leukaemia-specific mRNA by alpha interferon.

The RT-PCR technique for the identification of the PML-RAR alpha fusion mRNA is widely used for the detection of minimal residual in acute promyelocytic leukaemia (APL). A positive result after remission induction is highly predictive of early relapse, but the vast majority of patients have no detectable disease by this technique after chemotherapy consolidation, despite the fact that many later relapse. We report a quantitative PCR technique for the PML-RAR alpha cDNA which was used to show that less than 1000 PML-RAR alpha molecules are obtained from 1 microgram of diagnostic bone marrow RNA derived from approximately 1 million APL blasts. The lack of sensitivity of currently employed RT-PCR methods may therefore be explained by their poor yield of PML-RAR alpha cDNA. Minor modifications to the reverse transcription procedure improved this yield 3 fold. Furthermore, expression of the leukaemia-specific transcript increased by approximately one order of magnitude after incubation of the patient's cells for 24 h in vitro with 100 iu/ml alpha interferon.

Relapse into blast crisis following bone marrow transplantation for chronic phase chronic myeloid leukaemia: a report of five cases.

A proportion of patients receiving allogeneic bone marrow transplants (BMT) for chronic myeloid leukaemia (CML) in first chronic phase relapse; most of these relapses show features of chronic phase disease. We report here a series of five patients seen at a single institution over a 10 year period who developed blast crisis as the first sign of relapse after BMT for CML in chronic phase. The blast cells were myeloid in three cases and lymphoid in two. In one case the relapse may have occurred in cells of donor origin. The possible explanations for this unusual sequence of events include incipient transformation that was not detected before BMT, undetected relapse into chronic phase proceeding into transformation post-BMT, and transformation occurring de novo post-BMT in small numbers of residual leukaemic stem cells.

Terminal deoxynucleotidyl transferase and HLA-DR expression appear unrelated to prognosis of acute myeloid leukaemia.

Mononuclear cells from peripheral blood or bone marrow from 314 patients with acute myeloid leukaemia were examined for the presence of nuclear terminal deoxynucleotidyl transferase (304 patients), surface membrane expression of HLA-DR (314 patients) and the common acute lymphoblastic leukaemia antigen (281 patients). All patients were treated with identical remission induction chemotherapy, and morphological diagnosis was carried out in a central laboratory. The overall complete remission rate was 70%. There were no significant correlations between the immunological markers and complete remission rate, duration of remission, or survival.

Beneficial effect of heparin in the management of patients with APL.

115 patients with acute promyelocytic leukaemia (APL) were studied retrospectively to evaluate prognostic factors and assess therapeutic approaches, particularly the use of heparin in the management of disseminated intravascular coagulation (DIC). The remission rate was 86% (30/35 patients) in those who received heparin and 49% (39/80 patients) in those who received no heparin (P = 0.0002). This difference in remission rates was accounted for by a marked decrease in the number of haemorrhagic deaths, especially those due to intracranial haemorrhage (ICH), in the heparin treated group. Other factors associated with a poor remission rate were prothrombin ratio (PTR) greater than 1.3 (P = 0.008), fibrinogen less than 1.5 g/l (P = 0.02) and WCC greater than 2.0 x 10(9)/l (P = 0.03).

Features affecting outcome during remission induction of acute myeloid leukaemia in 619 adult patients.

Six hundred and nineteen patients with de novo acute myeloid leukaemia, entered into the Medical Research Council's eighth trial of therapy have been studied. All patients were treated with the same remission induction regimen. Pretreatment variables comprising age, clinical status, haematological status and a detailed marrow cytology and cytochemistry score have been analysed. Poorer remission rates have been found in older patients, in those with lower Karnofsky scores and in patients with a platelet count of less than 25 X 10(9)/l. Leukaemias showing evidence of cytoplasmic maturation along the granulocyte and monocyte lines, as evidenced by granules, Auer rods, a high percentage of Sudan black positive blast cells and morphological and cytochemical abnormalities of neutrophils were associated with a higher remission rate. Marrow eosinophilia was a good prognostic feature. Nuclear features of immaturity, i.e. increasing numbers and prominence of nucleoli were associated with a low remission rate. Abnormalities of the erythroid series, notably Periodic acid-Schiff positivity which was present in 133 cases (22% of the total), was associated with a low remission rate. Patient age and pretreatment Karnofsky score were the most useful predictors of treatment outcome.

Haemopoietic progenitor cell heterogeneity revealed by a single monoclonal antibody, YW 13.1.1.

Rapid segregation and purification of haemopoietic progenitor cells by simple methods is necessary for a proper analysis of the control of early haemopoiesis. In this paper we describe the use of a rat monoclonal antibody, YW 13.1.1, for that purpose. This reagent reacts with more than 90% of foetal liver cells but spares stem cells. A single-step lysis with antibody and complement achieves a tenfold enrichment for early progenitor cells. The marker also shows an increasing level of expression on the three defined subsets of erythroid progenitor cells. This parallels their developmental pathway and erythropoietin responsiveness. Simple quantitative considerations therefore permit separation of cells at different stages of erythropoiesis.

An unusual translocation, t(3;8), and deletion of chromosome 21 in acute myeloid leukemia.

This report describes a case of acute myeloid leukemia (AML) in which there was an unusual karyotypic abnormality involving chromosomes #3, #8, and #21. The clinical and hematologic features are described and cytogenetic findings are discussed in relation to previously reported variants of the translocation t(8;21) in AML. The breakpoint at 8q22 is a consistent feature in these cases.

8;21 translocation in acute granulocytic leukaemia: cytological, cytochemical and clinical features.

Thirty patients with the 8;21 translocation and three with closely related variants have been studied. Ages ranged from 3 to 64 years (mean 28.3). Thirty-one were entered into the MRC's 8th Acute Myeloid Leukaemia Trial. Twenty-nine (88%) achieved complete remission. Marrow smears from most patients showed granulocytic maturation (M2, FAB classification) with characteristic abnormalities, but at least six showed predominantly myeloblastic (M1) morphology. The blast cells were markedly heterogeneous with regard to size and nuclear cytoplasmic ratio. Typical staining patterns were observed in the blast cells using Sudan black B and diaminobenzidine peroxidase stains, and to a lesser extent with periodic acid-Schiff and chloroacetate esterase. Butyrate esterase was negative in all cases. Auer rods were present in the granulocyte precursors in 31 cases and in eosinophil precursors in two cases. In most cases the existence of the translocation was predicted from the cytological and cytochemical findings. Seven patients developed solid leukaemic deposits, principally in the mastoid cavities, orbital cavities or thoracic spine (extradural).

Effects of monoclonal anti-lymphocyte antibodies in vivo in monkeys and humans.

The safety and efficacy in vivo of three rat monoclonal anti-lymphocyte antibodies has been tested in cynomolgus monkeys. One IgM antibody, CAMPATH 1, was found to cause rapid but transient lymphopenia associated with consumption of complement. Two other antibodies with the same specificity, YTH 34.5 (IgG2a) and YTH 86.1 (IgG2c), had little effect. No acute or chronic toxic effects were associated with administration of any of the antibodies. CAMPATH 1 was used in a Phase I clinical trial for immunotherapy of two patients with terminal malignant lymphoid disease. It had no detectable toxic effects although it caused disappearance of circulating lymphocytes and consumption of complement. An inadequate rate of synthesis of complement components was a limitation to therapy in these patients with very large tumour burden, but might not be a major problem if antibody therapy were used at an earlier stage of the disease. CAMPATH 1 may also be useful as an immunosuppressive agent.

The characterisation of monoclonal antibodies against haemopoietic cells: comparison of an immunoperoxidase method with fluorescence activated cell sorting.

Nucleated cells from normal human peripheral blood and bone marrow have been analysed with 2 rat monoclonal antibodies of known specificity, one (YAML 555.6.6) directed against the P28,33 complex (anti-HLA-DR), and the other (YAML 501.4.4) directed against leucocyte common antigen (anti-LCA). The patterns of reactivity with an indirect immunoperoxidase method on fixed smeared cells were in close agreement with those obtained with a fluorescence activated cell sorter. A further new monoclonal antibody of unknown antigen specificity (YAML 537.2) reacts with an intracellular antigen present in neutrophils and their precursors from the promyelocyte stage onwards, megakaryocytes, and a proportion of monocytes, but not with eosinophils, nucleated red cells or lymphocytes. This reactivity could not be demonstrated using the fluorescence activated cell sorter. The immunoperoxidase method allows the identification of individual positive and negative cells and therefore provides a method of identifying minor reactive and non-reactive cell populations in a heterogeneous cell sample such as normal bone marrow. Cytoplasmic binding sites can be differentiated from membrane binding.