Fay Katz

The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9.

The t(7;11)(p15;p15) translocation is a recurrent chromosomal abnormality associated primarily with acute myeloid leukaemia (FAB M2 and M4). We present here the molecular definition of this translocation. On chromosome 7 positional cloning revealed the consistent rearrangement of the HOXA9 gene, which encodes a class I homeodomain protein potentially involved in myeloid differentiation. On chromosome 11 the translocation targets the human homologue of NUPP98, a member of the GLFG nucleoporin family. Chimaeric messages spliced over the breakpoint fuse the GLFG repeat domains of NUP98 in-frame to the HOXA9 homeobox. The predicted NUP98–HOXA9 fusion protein may promote leukaemogenesis through inhibition of HOXA9-mediated terminal differentiation and/or aberrant nucleocytoplasmic transport.

Diagnosis of acute promyelocytic leukaemia by RT-PCR: detection of PML-RARA and RARA-PML fusion transcripts

Summary. Acute promyelocytic leukaemia (APL; AML M3) is identified by a unique t(15;17) translocation which fuses the PML gene to the retinoic acid receptor alpha gene (RARA). Reverse transcription coupled with the polymerase chain reaction (RT‐PCR) has been used to develop a diagnostic test for APL based on the PML‐RARA fusion message. Separate PCR assays were designed to amplify either PML‐RARA (15q+ derived) or RARA‐PML (17q‐ derived) chimaeric transcripts. PML‐RARA transcripts were detected in every case from a series of 18 APL patients with cytogenetically confirmed t(15;17) translocations, whereas RARA‐PML messages were detected in only 67% (12/18) of these patients. This suggests that it is the 15q + derivative which mediates leukaemogenesis. Furthermore the PCR approach (or Southern analysis) may be used to identify in which of the alternative PML introns the breakpoint occurs; 52% of cases (15/29 patients) utilize a 5′ PML intron and 48% the 3′ intron (14/29 cases). Neither the choice of PML intron nor the expression of the 17q‐derivative could be correlated with the microgranular variant of APL (M3V), overall survival rate, age, sex or presence of coagulopathy. Finally, the fusion message is undetectable in five remission samples. This indicates a possible use for RT‐PCR in monitoring remission patients for evidence of relapse.

The use of DNA probes to monitor minimal residual disease in childhood acute lymphoblastic leukaemia

DNA probes to both the joining region (JH) of the immunoglobulin heavy chain gene (IgH) and to the β chain of the T‐cell antigen receptor complex (TCR) have been used as tumour‐specific markers to monitor the rearrangements of the IgH chain gene and the TCR β gene in the blast cells of children presenting with acute lymphoblastic leukaemia (ALL) of B or T cell origin. Blast cells from 68 children with early B cell ALL and eight with T‐ALL were examined at presentation, at day 28 after commencement of therapy and at varying times thereafter. An additional 43 patients (42 with B cell ALL, one with T‐ALL) were studied both at presentation, at completion of their 2‐year treatment course and 3 months later. Twelve patients, drawn from both these groups, were studied at relapse as were a further eight patients in whom an extramedullary relapse had occurred. Persistence of clonally‐derived cells as a predictor of early relapse was seen in the day 28 bone marrows of 11/76 newly‐diagnosed children (nine early B and two T‐ALL) followed by rapid, overt relapse in four of the early B ALL cases. No minimal residual disease (MRD) was detected in bone marrows from any of the 43 patients completing their 2‐year treatment course, but six of these subsequently relapsed at varying time periods thereafter. Identical patterns of rearrangement at both presentation and relapse were seen in most cases. Oligoclonality, or multiple IgH chain gene rearrangements was seen in the blast cells of 15% of patients with early B cell ALL. No correlation between oligoclonality, high white count, unfavourable phenotype, or abnormal karyotype could, however, be ascertained.

Possible evidence for genomic imprinting in childhood acute myeloblastic leukaemia associated with monosomy for chromosome 7

Monosomy or deletion of chromosome 7 is a frequent finding in both de novo and secondary acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). Based on analysis of deletions of chromosome 7 in such patients, it has been suggested that there is a critical region of the chromosome lying within bands q21‐q31. We have examined bone marrow and peripheral blood samples from 10 patients with MDS, AML and biphenotypic acute leukaemia who had monosomy for or rearrangement of chromosome 7, seeking evidence of non‐random allele loss that might suggest the presence of imprinted genes on the chromosome.

Infant acute lymphoblastic leukaemia with t(11;19)

Seven cases of infant acute lymphoblastic leukaemia with t(11:19) (q23;p13) are described. They are characterized by a high white cell count, organomegaly, early central nervous system (CNS) disease, and a poor prognosis. Blasts are usually of an immature early B‐cell lineage although monocytoid features are present in some cases. The characteristics of infant acute leukaemia with t(11;19) are very similar to those found with t(4;11), and the presence of t(11;19) may indicate the same poor prognosis.

Leukaemia arising in donor cells following allogeneic bone marrow transplantation for β thalassaemia demonstrated by immunological, DNA and molecular cytogenetic analysis

Summary Combined DNA analysis, molecular cytogenetic and immunological techniques have been used to identify the donor origin of common acute lymphoblastic leukaemia (cALL) which arose in a male patient with β thalassaemia major, 5 years after an allogeneic bone marrow transplant from his HLA‐matched sister. The necessity of using multiple techniques in this and similar cases is emphasized and the possible mechanisms for the development of donor leukaemia and the leukaemic transformation of donor cells are discussed.

Analysis of ras gene mutations in childhood myeloid leukaemia

Previous studies have shown that approximately 30% of adult acute myeloid leukaemias and 20% of adult acute lymphoid leukaemias contain point mutated ras oncogenes. In order to assess whether ras oncogenes are also involved in childhood leukaemias, we have used polymerase chain reaction (PCR) amplification and synthetic oligonucleotide probes to study the nature and frequency of ras gene mutations in childhood leukaemias, concentrating largely on the acute myeloid leukaemias (AML). Thirty‐four childhood presentation AML DNAs were screened for mutations in and around codons 12, 61 and 117 of N‐. K‐ and H‐ras. Eight of these samples (24%) contained ras mutations. As in the adult disease, the gene predominantly involved was N‐ras (6/8), with occasional activation of K‐ras (2/6). The most common base change was a G→A transition at codon 12 or 13 (4/8). Of the patients with mutant ras. 4/8 were diagnosed as AML FAB subtype M5. Five of the 34 childhood AMLs analysed displayed abnormalities of chromosome 7. However, none of these cases contained a mutant ras gene.

Elimination of T cells from human peripheral blood and bone marrow using a cocktail of three anti‐T cell immunotoxins

Summary. Four anti‐T cell monoclonal antibodies were coupled to ricin‐A and tested for their ability to kill T cells in peripheral blood and bone marrow using a clonogenic assay to quantify T cell survival. The immunotoxins (IT) prepared from RFT11 (CD2) and WT1 (CD7) antibodies were the most toxic to peripheral blood T cells. The immunotoxin prepared from RFT1 (CD5) was the next most efficient toxin and the immunotoxin prepared from RFT8 (CD8) was the least toxic. When these reagents were applied to peripheral blood cells at 3×10–8 M the number of T cell colonies was reduced by an average of 95%, 94%, 84% and 50%, respectively. Peripheral blood T cells from different donors showed marked variability in their sensitivity to ITs. However, a cocktail of three ITs prepared from RFT11, WT1 and RFT1 gave superior and consistent killing (mean 99.9%; range 99.8–100%) of peripheral blood T cells from six donors. When this cocktail was applied to bone marrow cells from six donors, an average of 99.6% (range 99.5–99.8%) of the T cells were killed. Under the same conditions there was little or no reduction in the number of normal haematopoietic progenitors (CFU‐GM, CFU‐GEMM, CFU‐Meg and BFU‐E).

Co-ordinate expression of BI.3C5 and HLA-DR antigens on haemopoietic progenitors from chronic myeloid leukaemia.

Haemopoietic cells isolated from the peripheral blood of patients with chronic myeloid leukaemia (CML), have been extensively purified and enriched using either Percoll density gradients or Percoll density gradients combined with elutriation. The quantitative expression of the BI.3C5 associated antigen and the co-expression of BI.3C5 and HLA-DR antigens on these two populations has been studied using either single or simultaneous two colour FACS sorting, following by in-vitro culture for single and multilineage haemopoietic progenitors thus obtained. The data show that the CFU-GEMM are always found in the most strongly BI.3C5 positive fraction, irrespective of the separation procedure and that the bulk of the CFU-GEMM co-express BI.3C5 and HLA-DR. The cell types initiating these CFU-GEMM are morphologically immature blasts. The more mature cells of the myelomonocytic and erythroid lineages forming single lineage colony types show variable BI.3C5 expression, although most are HLA-DR positive. Such enriched populations of malignant progenitors could provide a useful source of material to study both gene expression and the molecular mechanisms underlying malignant transformation.

Expression of HPCA‐1 and HLA‐DR antigens on growth factor‐ and stroma‐dependent colony forming cells

The expression of HLA‐DR and HPCA‐1 antigens (recognized by the L243 and BL3C5 antibodies respectively) on adult human bone marrow cells was examined by fluorescence activated cell sorting and colony assays. Nearly all the (day 14) lineage restricted and multipotential colony forming cells analysed in methylcellulose cultures in the presence of added growth factors express HLA‐DR and HPCA‐1 determinants. Two colour cell sorting reveals that the lineage restricted HLA‐DR positive progenitors express variable levels of BI.3C5 positivity whereas most of the multipotential progenitors, the multi‐CFC or CFU‐GEMM, are highly BI.3C5 positive. The isolated HLA‐DR and BI.3C5 positive populations also contain haemopoietic precursors which adhere to and form colonies on pre‐formed stromal layers. Thus, haemopoietic progenitors assayed in both types of culture system can be analysed and enriched by simultaneous two‐colour sorting using anti‐HLA‐DR and BI.3C5 monoclonal antibodies. Similarities in the antigenic phenotype of such cells, however, precludes the use of these reagents for segregating growth factor‐dependent from stroma‐dependent progenitors.