Faculty Bibliography

Two cases are described with the rare combination of inv(16)(p13q22), strongly associated with acute myelomonocytic leukemia with eosinophilia, M4Eo, and the Philadelphia translocation, t(9;22)(q34;q11), hallmark of chronic myeloid leukemia (CML) and rarely found, (less than 1%), in acute nonlymphocytic leukemia. The patients were: case 1, a 9-year-old girl presenting with a white blood cell count (WBC) 42 x 10(9)/L with 32% blasts and bone marrow with blasts and eosinophil precursors consistent with M4Eo, and case 2, a 25-year-old man with WBC 34.7 x 10(9)/L with 13% blasts and bone marrow with features of M4Eo and basophilia. Both patients achieved remission but died following bone marrow transplantation in first remission (case 1) or in relapse (case 2). Cytogenetic findings were: case 1, at diagnosis, 46,XX,inv(16)(p13q22)(21)/46,XX,t(9;22) (q34;q11),inv(16)(8)/46,XX(10), and case 2, at diagnosis, 46,XY,t(9;22) (q34;q11),inv(16)(p13q22) (16) and in remission, 46,XY,t(9;22)(q34;q11) (1)/46,XY (24). Investigation of the breakpoint on 22 in case 1 with Southern blotting and the polymerase chain reaction demonstrated the presence of a p190 mRNA and a breakpoint typical of acute leukemia. Thus a diagnosis of M4Eo was supported by clinical and cytogenetic sequelae in each case; the Ph in case 1 was apparently secondary to inv(16), in case 2 the Ph probably preceded inv(16) in the etiology of the leukemia.

Altered behaviour or the transformation of a cell can result from the abnormal expression of some oncogene products. Elevated or inappropriate expression can result from (i) mutations in the regulatory region of the gene, (ii) aberrant expression of a transcription factor involved in the regulation of the gene, (iii) gene amplification, or (iv) the insertion of a viral promoter upstream of the gene. In addition, an alteration in the product of a proto-oncogene can lead to the acquisition of a transforming activity. Such changes have been shown to include (i) point mutation, (ii) deletion, and (iii) the formation of fusion genes. Finally, the loss of activity of a gene product can contribute to transformation. This can come about by (i) small or large deletions, (ii) point mutations which abolish function or expression of an intact protein, or (iii) mutations which lead to a protein with an activity which can inhibit the suppressor activity of the normal allele.

We used the polymerase chain reaction (PCR) to detect residual leukemia-specific mRNA in blood and marrow from 37 patients in complete hematologic and cytogenetic remission after allogeneic bone marrow transplant (BMT) for chronic myeloid leukemia (CML). Our two-step PCR method involved the use of "nested primers" in the second step and could detect one K562 cell diluted into 10(5) normal cells. Elaborate measures were taken to exclude false-positive and false-negative results. In nine patients whose blood and marrow were studied simultaneously the results were concordant (two positive and seven negative). Twenty-three patients transplanted in chronic phase (CP) with unmanipulated donor marrow were studied. Blood cells from nine of these patients were studied 3 to 6 months post-BMT and six were PCR positive; three were negative on subsequent studies. Blood cells from 18 patients studied between 8 months and 8 years post-BMT were all PCR negative. Nine patients transplanted in CP with T-cell-depleted marrow cells were studied. Blood from five was positive 3 to 24 months post-BMT; blood from five was negative 3 to 6 years post-BMT. Four patients no longer in first CP were studied after BMT with unmanipulated donor marrow. Blood from all four was positive 5 to 19 months post-BMT. Based on the known clinical results of transplant in these three cohorts we conclude that PCR may be positive within 6 months of BMT in patients who can expect long-lasting remission, whereas PCR positivity later after BMT may indicate that the probability of cure is reduced. Thus, the technique may prove useful for early assessment of new transplant protocols that might inadvertently increase the risk of relapse.

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.

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.