The t(12;13)(p13;q12) is a rare, recurrent translocation reported in a range of hematological malignancies. We have analyzed the molecular basis of this lesion in three patients with acute myeloid ...leukemia (AML), two of whom were known to have chromosome 12 breakpoints within the ETV6 gene. Fluorescence in situ hybridization (FISH) with ETV6 cosmids indicated that this gene was also disrupted in the third patient, while the normal ETV6 allele was retained. 3′ rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) from bone marrow mRNA of this individual identified a novel sequence fused to ETV6 that was homologous to a region just upstream of the mouse CDX2 homeobox gene, the human homologue of which has previously been mapped to chromosome 13q12. PCR primers designed to amplify an ETV6-CDX2 fusion identified two major transcripts from this patient. First, a direct in-frame fusion between exon 2 of ETV6 and exon 2 of CDX2, and second, a transcript that had an additional sequence of unknown origin spliced between these same exons. Surprisingly, apparently normal CDX2 transcripts, usually expressed only in intestinal epithelium, were also detectable in cDNA from this patient. Neither normal nor fusion CDX2 mRNA was detectable in the two other patients with a t(12;13), indicating that this translocation is heterogeneous at the molecular level. Reverse transcription-PCR analysis showed that CDX2 mRNA, but not ETV6-CDX2 mRNA, was strongly expressed in 1 of 10 patients with chronic myeloid leukemia in transformation, suggesting that deregulation of this gene may be more widespread in leukemia. CDX2 is known to regulate class I homeobox genes and its expression in hematopoietic cells may critically alter the balance between differentiation and proliferation.
We studied the kinetics of EBV-transformed B-cell lines from patients with chronic myeloid leukaemia (CML) using RT-PCR for BCR-ABL transcripts and immunoglobulin heavy chain (IgH) gene ...rearrangements. Peripheral blood mononuclear cells, obtained from four patients with CML in chronic phase and from one in accelerated phase, were incubated with supernatant from the B95-8 EBV producing cell line. In 11/25 (44%) B-cell cultures established we demonstrated the presence of BCR-ABL transcripts at intervals ranging from 32 to 125 d post EBV transformation. In all but two cases, evidence of BCR-ABL transcripts disappeared with time. Cultures were initially polyclonal with respect to IgH rearrangements but became progressively oligoclonal, suggesting the longer-term survival of fewer clones, all of which were BCR-ABL negative. We conclude that BCR-ABL-positive lymphoid cultures can be established in the short term from the majority of patients with CML but they have limited capacity to survive in the longer term. Therefore, in lymphoid cells the presence of the BCR-ABL chimaeric gene appears to confer no survival advantage.
Recent developments in the understanding of the process of antigen presentation by major histocompatibility complex (MHC) molecules and their recognition by T-lymphocytes has led investigators to ...speculate that the hybrid bcr/abl fusion protein P210 present in chronic myeloid leukemia (CML) cells may generate leukemia-specific antigens recognized by T-cells. We used synthetic peptides representing the fusion region of P210 to study MHC class I and class II pathways of antigen recognition in normal subjects and patients with CML. We found that most normal individuals have a low proliferative response to 18mer fusion peptides representing the two alternative splicing variants b2a2 and b3a2, and a T-lymphocyte precursor frequency (HTLPf) characteristic of unprimed responders. No increase in HTLPf was found in CML patients after bone marrow transplantation (BMT), suggesting that peptide recognition does not form part of the graft-versus-leukemia process. In contrast, untransplanted patients with CML had very high HTLPf, suggesting an autologous but immunologically ineffective recognition of leukemia-specific peptides through HLA class II. Preliminary studies using the T2 cell line (which expresses HLA class I only in the presence of peptides binding to HLA-A2) indicate that nonapeptides spanning the breakpoint of the b2a2 and b3a2 variants of P210 do not bind to this particular class I molecule and are therefore unlikely to initiate class I mediated lymphocyte responses.
We used fluorescence in situ hybridization (FISH) to metaphase chromosomes with BCR and ABL cosmid probes in conjunction with the polymerase chain reaction (PCR) to study the mechanism by which the ...ABL proto-oncogene is inserted into a morphologically normal chromosome 22 in patients with Ph-negative chronic myeloid leukaemia characterized by the BCR-ABL chimeric gene. In control patients with Ph-positive CML the ABL probe localized to 22q- and the 3' BCR probe localized to 9q+. In nine Ph-negative CML patients the ABL probe localized to one normal chromosome 9 and to one 'normal' chromosome 22. Both 5' and 3' BCR probes localized exclusively to the chromosomes 22. By PCR all had evidence of BCR-ABL transcripts, but none had evidence of the reciprocal ABL-BCR gene product that is seen in 70% of the Ph-positive CML patients. These data confirm the view that Ph-negative CML results from insertion of ABL-containing DNA sequences into a normal-appearing chromosome 22 without reciprocal translocation of sequences from chromosome 22 to chromosome 9.
CML has served as a model for study of pathogenesis, evolution and treatment of malignancy for nearly 150 years and will continue to do so. The increasing interest worldwide in biology and medicine ...and the explosion of activity in molecular biology make it probable that many forms of leukaemia, lymphoma and solid tumours that are lethal today will be readily prevented or cured within the next 20 or 30 years.
Using IgH DNA fingerprinting we have previously demonstrated clonal immunoglobulin heavy chain (IgH) gene rearrangements during chronic phase (CP) chronic myeloid leukemia (CML) in patients destined ...to develop lymphoid blast crisis (L-BC). In view of this we decided to follow a cohort of CP CML patients to determine the frequency with which abnormal IgH fingerprints are found and their relationship, if any, to treatment regimen. Thirty three, initially CP, CML patients were studied on 111 occasions over a 16 month period using consensus PCR amplification of the third complementarity determining region (CDR3) of the IgH gene and high resolution polyacrylamide gel electrophoresis (IgH DNA fingerprinting). Of these 33 patients, thirteen received interferon-alpha (IFN) containing regimens and 15 non-IFN containing regimens throughout the study period. Five patients received variable therapy. During the period of observation 7 patients experienced disease progression: 5 accelerated phase, 1 L-BC and 1 myeloid blast crisis (M-BC). Abnormal IgH fingerprints were seen in 29 of the 111 (26%) specimens analysed. The 28 patients who received uniform therapy (IFN or non-IFN) over the 16 months were classified as "normal" (n=18, normal pattern on all occasions) or "abnormal" (n=10, abnormal on 1 or more occasions). Analysis by patient group (normal vs abnormal) showed that fingerprint abnormalities were associated with an elevated peripheral blood lymphocyte count (p=0.0001) but not with changes in the total white cell count. Comparison of the IFN vs non-IFN groups showed the former all had normal patterns whereas 10 of 15 non-IFN therapy patients were abnormal (p=0.00023). The peripheral blood lymphocyte counts in the normal vs abnormal patients within the non-IFN group were not significantly different. The patient who developed L-BC demonstrated a persistent IgH fingerprint pattern abnormality from 7 months prior to the diagnosis of L-BC. The M-BC patient had a normal pattern at all times. We conclude that: (1) abnormal IgH fingerprints are found in a significant number of CP CML patients; (2) in this cohort the use of IFN was associated with normal CP CML IgH fingerprints, and (3) detection of abnormal IgH fingerprints may be highly predictive for the lineage of impending blast crisis.
There is remarkable recent progress in our understanding of the biology of chronic myelogenous leukemia (CML). First, the BCR/ABL rearrangement was identified as the molecular basis of the disease. ...Second, animal models support the notion that the BCR/ABL gene product causes a syndrome similar to CML. Third, recent advances in understanding the functions of the normal ABL protein have given clues to the mechanism(s) of ABL-induced leukemias and approaches to blocking this process. Extrapolating these findings to humans seems reasonable. The challenge now is to determine how the BCR/ABL gene product causes chronic phase CML. Also unresolved is whether BCR/ABL also plays a role in the acute phase of the disease. Finally, the relationship between the two common forms of BCR/ABL, the P190 and P210 configurations, and different disease phenotypes, like CML and Philadelphia (Ph1)-chromosome positive acute lymphoblastic leukemia (ALL), needs to be clarified. There is also substantial progress in treating CML. Bone marrow transplants have emerged as the preferred therapy. These result in long-term leukemia-free survival in more than one-half of appropriately selected subjects. How transplants cure CML is complex and controversial. Some data suggest high-dose treatment is the dominant factor whereas other data implicate antileukemia effects of the immune system. Interferon treatment has also proven effective in CML. Whether it prolongs survival of persons with CML remains to be determined, as does its mechanism of action. Certainly the most important and difficult challenge in CML therapy is determining how to use knowledge about the causes CML to treat the disease. These and other issues in the biology and therapy of CML were the subject of a recent meeting of basic and clinical scientists. The meeting, third in a series begun in 1987, was held on Martha's Vineyard, Cape Cod, Massachusetts, USA from 4-7 April, 1992. Four major topics were considered in five sessions: molecular biology, cell biology, Ph1-chromosome positive ALL, and therapy of CML. This report summarizes meeting highlights.
Some patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) may achieve partially or completely Ph-negative hemopoiesis after treatment with high doses of cytotoxic drugs ...or interferon-alpha at standard dosage. This observation leads to three important questions: 1) are Ph-negative myeloid cells in such patients strictly normal? 2) can such Ph-negative cells be identified in all newly diagnosed patients or only in a minority? and 3) what is the basis for the proliferative advantage manifested by CML cells and why might it be temporarily lost as a result of treatment? The mechanisms that might prevent the proliferation of normal cells in an environment of Ph-positive cells and the relevance of these questions to the design of a strategy aimed at obtaining complete remission in the majority of patients are considered. Such a strategy might incorporate autografting with Ph-negative stem cells harvested during the recovery phase of high dose chemotherapy.
1,25-dihydroxyvitamin D3 induces monocyte-macrophage differentiation and inhibits proliferation of cells from the human promyelocytic leukaemia cell line HL60. Similarly human bone marrow progenitor ...cells differentiate preferentially along the monocyte-macrophage pathway when incubated in the presence of 1,25-dihydroxyvitamin D3. We suggest that the inhibition of growth which occurs after addition of the vitamin to HL60 might be paralleled in vivo by inhibition of proliferation of leukaemic cells; also we speculate that the vitamin may be involved in the control of both monocyte-macrophage and osteoclast production in vivo.
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