Charles Dowding

Rearrangement of the bcr Gene in Philadelphia-Chromosome-Negative Chronic Myeloid Leukemia

The majority of patients with chronic myeloid leukaemia (CML) have a characteristic deletion of a portion of the long arm of one chromosome 22, the Philadelphia (Ph 1) chromosome, in their myeloid cells. The missing material is reciprocally translocated to chromosome 9 such that the usual karyotype is described as t(9;22)(q34;q11). In Ph 1-positive CML, the oncogene (c-abl) normally present on chromosome 9 is translocated to chromosome 22 [1, 2] where it comes into juxtaposition with a region named the “breakpoint cluster region” (bcr) [3]. A chimeric abl-related mRNA has been identified in cells from patients with CML [4] and is associated with the presence of a fusion protein that has tyrosine kinase activity [5]. Thus, patients with Ph 1-positive CML show evidence of rearrangement of DNA within the bcr region.

The position of the M‐BCR breakpoint does not predict the duration of chronic phase or survival in chronic myeloid leukaemia

It has been reported that patients with chronic myeloid leukaemia (CML) with 5’breakpoints within the major breakpoint cluster region (M‐BCR) of the BCR gene have somewhat better prognoses than those with 3’breakpoints. We studied the position of the breakpoint in 67 patients with CML in chronic phase using conventional Southern blotting. Using restriction enzymes BglII, BamHI and HindIII and two genomic probes, a 0.6 kb (3’M‐BCR) probe hybridizing to a part of the intron between exons b3 and b4 and a 2.0 kb (5’M‐BCR) probe hybridizing to sequences including exon b1, we localized the breakpoint in M‐BCR as occurring 5’(n= 38) or 3’(n= 28) of the HindIII restriction site located just downstream of exon b3. We failed to localize the breakpoint in one patient. The median durations of chronic phase (37 versus 44 months respectively) and of survival (50 versus 51 months respectively) for patients with 5’and 3’breakpoints were not significantly different. When we analysed only patients whose DNA was collected within 4 weeks of diagnosis (5’breakpoints, n= 30; 3’breakpoints, n= 19), there was again no significant difference in duration of chronic phase or survival. The median survivals of patients divided into good, intermediate and poor prognosis categories in accordance with the prognostic index developed by Sokal and colleagues were 54, 50 and 26 months respectively. This study confirms the value of the Sokal prognostic index but provides no support for the notion that the precise genomic position of the breakpoint in M‐BCR correlates with prognosis.

Complete remission after autografting for chronic myeloid leukaemia

A previously untreated 31-yr old female with Ph-positive chronic myeloid leukaemia (CML) received busulphan and melphalan at high dosage followed by an autograft of peripheral blood stem cells collected 4 weeks earlier. Though recovering haemopoiesis was at first mainly Ph-positive, Ph-negative haemopoiesis predominated at 12 months and has persisted until the most recent study (24 months post-autograft). Her haemopoietic cells now show no rearrangement of the bcr gene, unlike the cells collected at diagnosis. Autografting carried out soon after diagnosis could be valuable for obtaining cytogenetic remissions in other patients with CML.

Purification of haemopoietic progenitor cells from patients with chronic granulocytic leukaemia using Percoll density gradients and elutriation

Purification of haemopoietic progenitor cells from chronic granulocytic leukaemia buffy coat preparations requires a multistep approach using complementary cell separation techniques. In this study Percoll density gradient centrifugation and centrifugal elutriation were used to isolate large numbers of immature progenitor cells. Percoll density gradients were valuable as a first separation step: CFU‐GM and CFU‐GEMM could be enriched 75‐fold in a light density fraction of d< 1mD056 g/ml and the technique could be adapted to cope with more than 1010 buffy coat leucocytes. Progenitors cells were concentrated 3‐fold by elutriation used as single method to separate buffy coat cells or when used to purify further light density Percoll fractions. When Percoll gradients and elutriation were used sequentially, undifferentiated mononuclear cells were enriched to more than 90% purity arid between 5% and 40% of these cells formed CFU‐GM or BFU‐E colonies consisting of more than 40 cells. The enriched fractions were further characterized with monoclonal antibodies. The density and elutriation profiles of these colony forming cells resembled corresponding profiles of cells that reacted with the monoclonal antibody BI‐3C5, which recognizes an antigen on primitive haemopoietic progenitor cells. Physical separation methods are a valuable first stage in the attempt to procure relatively pure myeloid progenitor cell populations, whose characteristics can then be further studied at a cellular or molecular level.

Adhesive defects in chronic myeloid leukemia.

Normal human bone marrow contains a population of primitive haemopoietic progenitor cells that adhere to cultured bone marrow-derived stromal layers in vitro. These progenitor cells can be enumerated by a blast colony assay which is set up in several stages (Fig. 1; Gordon et al 1985a, b; 1987a).

Inactivation of the retinoblastoma susceptibility gene in a human high grade non-Hodgkin's lymphoma cell line.

Summary We studied the expression of the retinoblastoma (RB) gene product (p105) in a B‐cell line established from a patient with non‐Hodgkins lymphoma (large cell type). The karyotype of this cell line, named Ri‐1, showed amongst other changes an apparent deletion of one chromosome 13 on band q14. No p105 could be detected by immunoprecipitation analysis and Western blotting in Ri‐1 cells. Northern blotting revealed that RB mRNA is not expressed in Ri‐1. Southern blotting confirmed the loss of one RB allele but showed a normal gross structure of the remaining allele. This suggests that the inactivation of the RB gene in Ri‐1 cells is due to deletion of one allele and point mutations or small deletions in the other, as is often the case in retinoblastomas. Our findings imply that inactivation of the RB gene may play a role in the pathogenesis of high grade malignant lymphomas and that studies of RB in primary lymphoma samples would be of interest.

Antigenic determinants on myeloid leukaemia colony‐forming cells resemble those of normal myeloid progenitor cells and differ from those of circulating blast cells

Summary. We studied the antigenic characteristics of leukaemic colony‐forming cells (CFU‐L) from the blood of patients with chronic granulocytic leukaemia (CGL) in blastic transformation (BT) and acute myeloid leukaemia (AML) by in vitro culture techniques after complement‐mediated lysis with one anti‐DR and 10 selected myeloid monoclonal antibodies (McAbs), all of which were cytotoxic in the presence of complement. At the same time we studied the antigenic characteristics of the circulating blast cells from the same patients using in addition one non‐complement fixing antibody (BI.3C5) with standard immunofluorescence and immunoalkaline phosphatase techniques. We also used myeloid progenitor cell assays in conjunction with cytotoxic McAbs to investigate the antigenic determinants on Day 7 CFU‐GM, Day 14 CFU‐GM and BFU‐E from the blood of patients with CGL in chronic phase (CP) and from normal bone marrow. We found that two of the McAbs, S4–7 and WGHS29.1, recognized a higher proportion of CFU‐L from the blood of AML patients than from patients with CGL‐BT. However, the patterns of reactivity for CFU‐L from CGL‐BT and AML patients with the other McAbs quite closely resembled those observed in CFU‐GM and BFU‐E from normal individuals and patients with CGL in CP. A McAb with DR specificity and one of the myeloid McAbs, 54/39, recognized both CFU‐L from CGL‐BT and AML and reacted also with circulating blast cells from the same patients. In contrast, six of the other myeloid McAbs that recognized CFU‐L failed to label the corresponding blast cells. We conclude

Parasiticidal effect of 16α-bromoepiandrosterone (EpiBr) in amoebiasis and cysticercosis

The effect of the dehydroepiandrosterone analog 16alpha-bromoepiandrosterone (EpiBr) was tested on the tapeworm Taenia crassiceps and the protist Entamoeba histolytica, both in vivo and in vitro. Administration of EpiBr prior to infection with cysticerci in mice reduced the parasite load by 50% compared with controls. EpiBr treatment induced 20% reduction on the development of amoebic liver abscesses in hamsters. In vitro treatment of T. crassiceps and E. histolytica cultures with EpiBr, reduced reproduction, motility and viability in a dose- and time-dependent fashion. These results leave open the possibility of assessing the potential of this hormonal analog as a possible anti-parasite drug, including cysticercosis and amoebiasis.

Phenotypic Characterization of Normal and CML CD34–Positive Cells: Only the Most Primitive CML Progenitors Include Ph-neg Cells

We studied the sequence of acquisition of CD33, CD38 and HLA-DR antigens on CD34+ cells from marrow and blood of Ph-chromosome positive CML patients and normal marrow. We examined the Ph status of the various CML cell populations. The mean proportions of normal and CML CD34+ cells expressing CD33 and CD38 were not significantly different. However, a significantly greater proportion of CML CD34+ cells expressed HLA-DR antigens compared with normal CD34+ cells and the level of HLA-DR expression per CML cell was abnormally high. When the sequence of acquisition of these antigens on normal and CML CD34+ cells was evaluated using 3-colour fluorescence analysis, the results suggested that HLA-DR was expressed earlier than CD38 or CD33 and these findings were confirmed by following the acquisition of CD38 and CD34+/DR+/CD38-subpopulation during liquid culture. We performed cytogenetic studies on CD34+ subpopulations in 6 cases. In 4 cases there were some Ph-negative metaphases detectable in the CD34+/DR-subpopulation (range 12.5 to 60%). In the CD34+/DR+ fractions, however, all 6 patients had only Ph-positive metaphases and only 1/5 patients had detectable Ph-negative metaphases in the CD34+/CD38-subpopulation. We conclude that expression of HLA-DR antigens may precede the expression of CD38 on CD34+ cells during normal stem cell differentiation. In CML DR may be expressed aberrantly and Ph-negative cells are found predominantly in the DR negative subpopulation.

A New Parasiticidal Compound in T. solium Cysticercosis

The effect of 16α-bromoepiandrosterone (EpiBr), a dehydroepiandrosterone (DHEA) analogue, was tested on the cysticerci of Taenia solium, both in vitro and in vivo. In vitro treatment of T. solium cultures with EpiBr reduced scolex evagination, growth, motility, and viability in dose- and time-dependent fashions. Administration of EpiBr prior to infection with T. solium cysticerci in hamsters reduced the number and size of developed taenias in the intestine, compared with controls. These effects were associated to an increase in splenocyte proliferation in infected hamsters. These results leave open the possibility of assessing the potential of this hormonal analogue as a possible antiparasite drug, particularly in cysticercosis and taeniosis.