Faculty Bibliography

A patient who was diagnosed with chronic myeloid leukemia remained in chronic phase for 14 years before progressing into a lymphoid blast crisis in 1983. The acute phase was successfully treated, and the patient has remained in an indolent chronic phase to date. Cytogenetic and molecular analysis during this second chronic phase confirm the presence of the Philadelphia chromosome and its transcribed BCR-ABL mRNA. The breakpoint within M-bcr occurred in the 3' portion of the region and expressed a hybrid joining the b3 exon of BCR to the a2 exon of ABL.

In order to study which hemopoietic precursor cells express the hybrid BCR/ABL fusion mRNA we have developed a technique based on the polymerase chain reaction (PCR) for the examination of single hemopoietic colonies grown on semi-solid agar. The technique was developed by examining single CFU-GM colonies grown from newly diagnosed patients with chronic myeloid leukaemia (CML). RNA was isolated from individual 14 day colonies and reverse transcribed to a complementary DNA (cDNA) copy which formed the substrate for a PCR. We have studied 3 cases of CML using this method and have found that 5 out of 5, 9 out of 10 and 20 out of 23 colonies examined were positive. Thus we describe a simple and useful technique for the study of gene expression in a limited number of hemopoietic precursor cells.

Three major types of mRNA can be expressed as a result of the Philadelphia translocation, dependent on the position of the break within the BCR gene on chromosome 22. In addition, alternative splicing of the mRNA transcribed from the BCR/ABL fusion gene has been reported and it has been suggested that this may play a role in the generation of the acute phase of Philadelphia positive chronic myeloid leukaemia (CML). We have examined the fusion RNA present in 24 cases of chronic phase CML and 21 cases of patients with CML in blast crisis using the polymerase chain reaction. In no case was it possible to detect the presence of the e1a2 junction which encodes the p190 hybrid protein product. We conclude that the acquisition of the p190 does not play a significant role in the generation of the blast crisis of CML. Neither could we detect a significant difference in the number of cases which simultaneously express both b2a2 and b3a2 junction products in samples isolated from chronic phase and blast crisis. In the series analysed by ethidium bromide stained gels, there was, however, an increase in the percentage of cases expressing the b3a2 junction in the mononuclear cells of blast crisis patients as compared to the white blood cells of patients in chronic phase.

The occasional finding of cells positive for the Philadelphia (Ph) chromosome months or years after bone-marrow transplantation for chronic myeloid leukaemia raises the possibility that the Ph-positive clone may never be eradicated. The polymerase chain reaction with probes able to detect the transcript of the bcr/abl hybrid gene at very low levels was used to study marrow cells from seven patients in continuing haematological and cytogenetic remission 5-7 years after allogeneic bone-marrow transplantation for chronic myeloid leukaemia. No evidence of the leukaemic mRNA was found. Thus, it seems that all leukaemic cells were eradicated in these patients and that they are truly cured.

The Philadelphia (Ph) chromosome is a small chromosome 22, which results from a reciprocal translocation between the long arms of chromosome 9 and 22, designated t (9;22) (q34;q11). It was first described in association with chronic myeloid leukaemia (CML), where 90% of cases examined are Ph-positive. A similar cytogenetic abnormality has also been identified in the acute leukaemias but in a much lower percentage. The ubiquitous nature of the translocation in CML suggested that it was causally implicated in the pathogenesis of the disease. Recent work at the molecular level has corroborated this idea. As a consequence of the translocation, the Abelson protooncogene (ABL), located on chromosome 9 is moved to chromosome 22 where it is joined to a truncated gene, known as BCR. The result of this genomic reorganisation is a hybrid gene encoding a novel chimaeric protein product with enhanced protein tyrosine kinase activity. It is thought that it is this activity which is necessary for the generation of the leukaemic phenotype. The t(9;22) has provided a model to illustrate how cellular proto-oncogenes can be activated by chromosomal translocation and has stimulated interest in investigating other chromosomal translocations in human malignancies.

The relationship between platelet size and platelet count was investigated in 41 patients with Crohn's disease. A high platelet count was associated with a decrease in platelet size, but an overall increase in the platelet crit. There was also a significant correlation between the patient's platelet count and serum orosomucoids, which have traditionally been used to assess disease activity.

Seven patients with Philadelphia (Ph) chromosome positive essential thrombocythemia (ET) were investigated for the presence of a rearrangement within the major breakpoint cluster region (M-bcr) using the Southern blot technique and, in six cases, for the presence of the hybrid bcr-abl mRNA using the polymerase chain reaction (PCR). The molecular studies showed rearrangement of M-bcr in all cases; there was evidence of the b2a2 mRNA junction in one case and of b3a2 junction in five cases. These findings are identical to what might have been expected in Ph-positive chronic myeloid leukemia. These features may explain the poor prognosis of Ph-positive ET in comparison with cytogenetically normal cases. Conversely, the differences in clinical presentation may be due to other genetic changes.