Ph1-positive Chronic Myelocytic Leukemia Research Articles

Constitutional chromosomal aberration, t(4;12) (q23;p11), in a patient with Ph1 positive chronic myelocytic leukemia

A 54-year-old man with Ph1 positive chronic myelocytic leukemia and two balanced reciprocal translocations in bone marrow cells is described. One was the typical translocation between chromosomes No. 9 and 22, t(9;22), while the other was a constitutional translocation between chromosomes No. 4 and 12, t(4;12)(q23;p11), which was also present in all PHA-stimulated peripheral blood lymphocytes. Two brothers, one sister, one son, and one daughter also had the same constitutional translocation between chromosomes No. 4 and 12. None of them had signs of hematological disorder. The follow-up investigation of the family may clarify whether this constitutional translocation involves increased risk of developing CML.

Amplification of the BCR/ABL fusion gene clustered on a masked Philadelphia chromosome in a patient with myeloblastic crisis of chronic myelocytic leukemia

Although the chronic phase of chronic myelocytic leukemia (CML) is characterized by the Philadelphia (Ph) chromosome creating a hybrid BCR/ ABL gene, additional genetic changes involved in blast crisis are poorly understood. We report a 4–8-fold amplification by tandem duplication of the BCR/ ABL fusion gene clustered on a masked Ph chromosome in a 61-year-old male patient with CML in myeloblastic crisis. Our finding suggests that the BCR/ ABL amplification may play a role as a novel mechanism in the progression to an aggressive blast transformation in some cases of Ph-positive CML.

Ph-negative acute lymphocytic leukemia occurring after interferon therapy for Ph-positive chronic myelocytic leukemia

We report a unique case of chronic myelocytic leukemia (CML) with the Philadelphia (Ph) chromosome. The patient obtained cytogenetic complete remission (CR) after treatment with interferon (IFN). When he transformed to acute lymphocytic leukemia (ALL), cytogenetic analysis showed that the karyotype was normal and fluorescence in situ hybridization (FISH) indicated that blast cells were Ph-negative. Immunophenotyping showed that the cells were CD34-positive and CD38-negative. Blast cells in his final stage were BCR/ABL rearrangement negative by reverse transcription polymerase chain reaction (RT-PCR).

Identical abnormality of the short arm of chromosome 18 in two Philadelphia-positive chronic myelocytic leukemia patients with erythroblastic transformation, resulting in duplication of BCR-ABL1 fusion

Two patients with Ph-positive chronic myelocytic leukemia in erythroblastic transformation and rearrangement of the short arm of chromosome 18 are reported. Fluorescence in situ hybridization studies showed that the 18p rearrangement resulted from translocation of the main part of chromosome 22 long arm to 18p, including BCR-ABL1 fusion. The 18p abnormality resulted, thus, in loss of 18p and duplication of BCR-ABL1 in both patients. The possible relation to the erythroblastic type of blastic phase is briefly discussed. In addition an apparently intact germline ABL1 gene was duplicated and inserted into chromosome 6 at band p21 in one of these patients.

Ph-positive CML in blastic phase with monosomy 7 in a down syndrome patient: Monitoring by interphase cytogenetics and demonstration of maternal allelic loss

We report a case of Ph-positive chronic myelocytic leukemia in blastic phase in an 11-year-old boy with Down syndrome. Monosomy 7 was the only additional chromosomal anomaly in the blastic clone. Fluorescence in situ hybridization analysis on interphase nuclei with a centromeric probe specific to chromosome 7 proved to be efficient in disease monitoring, and showed, together with the results of chromosome analysis on metaphases, that B-lymphocytes at the origin of an EBV-established line were not part of the leukemic clone. The study of DNA polymorphisms showed that the origin of the constitutional trisomy 21 was a maternal anaphase I nondisjunction, that the chromosome 7 lost in the blastic marrow clone was the maternal one, and led us to postulate that the mother's chromosomes are prone to impairment of normal disjunction. The study of allelic losses of chromosome 7 loci proved to be a further possibility for disease monitoring.

Some observations on fluorescence in situ hybridization evaluation of chronic myelocytic leukemia

Cosmid probes specific for the major BCR/ABL rearrangement (commercially available probes) were employed by us to evaluate 134 patients with the clinical diagnosis of chronic myelocytic leukemia (CML). The following represent our main observations: 1) No significant statistical difference regarding a false positive fusion signal was seen in the distribution pattern between the Ph negative (−) cases (110) and the normal controls (3.23 ± 1.97 vs 2.8 ± 1.92, respectively). 2) The mean percentage of cells with a positive fusion signal in the Ph positive (+) CML cases at presentation was 70 ± 12.3 in bone marrow samples (12 cases) and 45 ± 12.4 in blood samples (3 cases). 3) A significant discrepancy was seen between the interphase FISH and cytogenetics data in the monitoring of four out of eight patients post-therapy. The clinical significance of this observation needs to be clarified. 4) Three Ph(−) patients with significant leukocytosis had closely adjacent but not fused signals (20%, 16%, and 20% of cells, respectively, vs. the control: 4%). This may indicate a temporal and spatial association between chromosomes 9 and 22 prior to the BCR/ABL rearrangement (Ph chromosome formation).

Clonal origin of Philadelphia chromosome negative cells with trisomy 8 appearing during the course of α-interferon therapy for Ph positive chronic myelocytic leukemia

The transient appearance of a Philadelphia chromosome (Ph) negative clone with trisomy 8 was found in the bone marrow cells from a patient with Ph positive CML during the course of a-interferon (IFN) therapy. To determine whether this clone was derived from a Ph positive clone or from some other cell lineage, we performed molecular cytogenetic studies on bone marrow cells from the patient by fluorescence in situ hybridization (FISH). No fusion of BCR-ABL could be detected in cells with trisomy 8, clearly indicating that the Ph negative trisomy 8 clone was not derived from the Ph positive CML population.

A variant Philadelphia chromosome (Ph1) positive chronic myelocytic leukemia.

A rare case of variant Philadelphia (Ph1) chromosome positive [46, XX, t (9; 22) (q34; q11), inv (9) (9q22; 22q13)] chronic myelocytic leukemia (CML) was described. The patient, 73 years old female, was hospitalized to our hospital because of leukocytosis. Hematological findings corresponded to those of CMLs. However, this case lacked hepatosplenomegaly. Southern blot analysis using a 3 breakpoint cluster region (bcr) probe revealed a bcr rearrangement. The patient has been in the chronic phase for sixteen months without treatment. Clinical and chromosomal changes are under observation in order to get accumulate data for a pathophysiological analysis of variant Ph1 positive CMLs.

Application of Log-Linear Model in Inference on Karyotypic Evolution in Chronic Myelocytic Leukemia

Relationships among additional chromosome abnormalities in chronic myelocytic leukemia (CML) with translocation 9;22 [Philadelphia chromosome (Ph1)-positive CML] were analyzed by log-linear models on 709 karyotypes reported in the literature. Additional abnormalities, such as the gain of chromosome 8 (+8), gain of Philadelphia chromosome (+Ph1), isochromosome of the long arm (q) of chromosome 17 [i(17q)], and the gain of chromosome 19 (+19), were frequently observed. A four-way 2 x 2 x 2 x 2 contingency table was considered with respect to the appearance of these four abnormalities, then the hierarchical log-linear models having at least four main effects were fitted to the observed contingency table. Akaike's information criteria of the models reflected the fitness of the model very well. Parameter estimates of the interaction terms indicated that the combinations of two abnormalities, '+8 and +19', '+Ph1 and +19', and '+8 and i(17q)' were positively associated, while '+Ph1 and i(17q)', and '+19 and i(17q)' were negatively associated. Based on the results of the data analysis, an inference was made on the route of karyotypic evolution in Ph1-positive CML; it statistically supports the hypothesis presented by Heim and Mitelman.

Application of log-linear model in inference on karyotypic evolution in chronic myelocytic leukemia.

Relationships among additional chromosome abnormalities in chronic myelocytic leukemia (CML) with translocation 9;22 [Philadelphia chromosome (Ph1)-positive CML] were analyzed by log-linear models on 709 karyotypes reported in the literature. Additional abnormalities, such as the gain of chromosome 8 (+8), gain of Philadelphia chromosome (+Ph1), isochromosome of the long arm (q) of chromosome 17 [i(17q)], and the gain of chromosome 19 (+19), were frequently observed. A four-way 2 x 2 x 2 x 2 contingency table was considered with respect to the appearance of these four abnormalities, then the hierarchical log-linear models having at least four main effects were fitted to the observed contingency table. Akaike's information criteria of the models reflected the fitness of the model very well. Parameter estimates of the interaction terms indicated that the combinations of two abnormalities, '+8 and +19', '+Ph1 and +19', and '+8 and i(17q)' were positively associated, while '+Ph1 and i(17q)', and '+19 and i(17q)' were negatively associated. Based on the results of the data analysis, an inference was made on the route of karyotypic evolution in Ph1-positive CML; it statistically supports the hypothesis presented by Heim and Mitelman.

Inv (3) (q21q26) Associated with Abnormal Thrombopoiesis in a Ph-positive Chronic Myelocytic Leukemia

We report a rarely occurred paracentric inversion of the long arm of chromosome 3, i. e. inv (3) (q21q26), observed in patient of Ph-positive chronic myelocytic leukemia with abnormal thrombopoiesis. To our knowledge, this is the sixth case reported in English literature. This additional chromosome marker usually appears in the late chronic phase or blastic phase of chronic myelocytic leukemia, and indicates a poor prognosis. Its relationship to abnormal thrombopoiesis is also discussed. (J Genet Mol Biol 1990; 1: 14-21)

Lymphoid Blast Crisis in a Patient with Philadelphia-Chromosome-Negative Chronic Myelocytic Leukemia

A 24-year-old man with Philadelphia-chromosome (Ph)-negative chronic myelocytic leukemia (CML) developed lymphoid blast crisis. In the chronic phase, karyotype was normal and the clinical and hematological features were indistinguishable from those of Ph-positive CML. Rearrangement of the breakpoint cluster region (bcr) was observed. In the blast phase, blast cells showed early B-cell phenotype (CALLA+, Ia+, TdT+) with a rearranged immunoglobulin heavy-chain gene joining region (JH). By using an immunoblotting method and antiphosphotyrosine sera, P210bcr-abl protein was detected. The patient responded well to vincristine and prednisolone (VP) therapy. These findings support the concept that Ph-negative bcr+ CML can behave in a very similar fashion to Ph-positive CML, not only in the clinical features of the chronic phase but also in the manner of the blast crisis.

The Philadelphia chromosome: Considerations Based on Studies of Variant Ph Translocations

The nature of the Philadelphia (Ph) translocation and the process of its formation were studied by attempting various chromosome bonding analyses of variant Ph translocations among 210 patients with Ph-positive chronic myelocytic leukemia examined at the National Institute of Radiological Sciences, Chiba. The following assumptions could be drawn from the results of the analyses: 1) The involvement of specific regions of chromosomes #9 and # 22. q34 and q11, respectively, is an indispensable condition of the Ph translocation. 2) The so-called variant Ph translocations are all complex and are derived from a standard Ph translocation. 3) The Ph translocations, both standard and complex ones, are not always stable. The complex translocations are subject to further chromosome evolution, as is the conversion of the standard translocation to complex translocations. There seems to be no fundamental difference between the standard and complex Ph translocations, with the latter being merely a more progressed form of the former. Analyses at the molecular level of the same cases employed in this study are yielding results that support the above assumptions.

Sequential karyotype study on Ph-positive chronic myelocytic leukemia. Significance of additional chromosomal abnormalities during disease evolution.

Twenty-eight patients with Ph-positive chronic myelocytic leukemia (CML), who all died of the disease, had cytogenetic studies throughout the progression of the disease: at diagnosis, during chronic phase (CP), accelerated phase (AP), and blastic transformation (BT). The aim of this sequential study was to appreciate the frequency and the significance of additional chromosomal abnormalities (ACA) during CML evolution, especially in the CP. In our series ACA were rare (five of 28 patients) and simple (four of five) in CP. They were much more frequent and complex in AP (11 of 16) and in BT (22 of 24) with complex abnormalities (13 of 24). In CP, ACA predictive value for metamorphosis was poor: only three of 13 patients had ACA within 1 year before BT, and only two of 11 within 1 year before AP. ACA were mainly observed during the last period before BT: ten of 17 patients studied within 6 months prior BT had ACA, but by then two of three were in AP. ACA, especially when complex, appear to be a hallmark of CML metamorphosis.

T Lymphocytes Lack Rearrangement of the bcr Gene in Philadelphia Chromosome-Positive Chronic Myelocytic Leukemia

To study the possible involvement of T lymphocytes in Philadelphia chromosome (Ph)-positive chronic myelocytic leukemia (CML) we analyzed the arrangement of the bcr gene in T cell and non-T cell samples of 12 CML patients. Although all the patients showed bcr rearrangements in non-T cell fractions, T cell populations lacked respective gene recombinations. Moreover, by Southern blot analyses using T cell receptor beta chain sequences our data indicate polyclonality of T cell samples from 11 of 12 cases; in one patient a clonal T cell population could be identified. These results suggest that T lineages of most Ph-positive CML patients are not derived from pluripotent stem cells involved in leukemogenesis and thus confirm previous investigations based on cytogenetic or glucose-6-phosphate dehydrogenase analyses. The demonstration of polyclonal T cell populations may reflect persistence of stem cells committed to differentiate only into T cells.

Philadelphia chromosome-positive chronic myelocytic leukemia with a supplementary t(4;9)(q21;p22) and long survival

A Ph-positive chronic myelocytic leukemia (CML) patient was clinically and cytogenetically evaluated during a 12-year period. She acquired a supplementary chromosome abnormality, t(4;9)(q21;p22), at least 5 years prior to transformation to blastic phase. Her blast crisis was accompanied by characteristic chromosome changes, such as trisomy 1, trisomy 17, and multiple Ph chromosomes.

The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript.

In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.

The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript.

In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.

Near-triploid Ph-positive leukemia

Tetraploid populations have been observed in various types of leukemia, but relatively few reports exist of triploid cell populations in acute or chronic leukemia. We report two cases of Ph-positive leukemia with a modal triploid cell population. Examination of peripheral blood from a 3-year-old boy with Ph-positive acute lymphoblastic leukemia (ALL) and a 68-year-old male with Ph-positive chronic myelocytic leukemia (CML) in blastic crisis revealed modal populations of 72 and 63 chromosomes, respectively. G-banding analysis of both cases revealed the following: karyotypic instability (no clonality), dominant trisomy, and the random association of the Ph chromosome with gains and losses of chromosomes involved in this translocation. The cytogenetic evidence obtained suggests that the triploid cell populations were not derived from a duplication of a hypodiploid cell population, but resulted from random loss of chromosomes from tetraploid cell populations derived from duplication of pseudodiploid cells.

Differences between labeling index and DNA histograms in assessing S-phase cells from a homogeneous group of chronic phase CML patients.

The reliability of DNA histogram analysis in accurately estimating S-phase cells from human tumors was tested by comparing the results to those of simultaneously obtained tritiated thymidine labeling index (LI) studies. Patients with chronic myelocytic leukemia (CML) during chronic phase were selected for study because the Philadelphia chromosome (Ph) was the only cytogenetic abnormality in each case and, since it is a balanced translocation, the frequently encountered problem of aneuploidy in human neoplastic cells was avoided. Unfortunately, when 30 CML patients were studied simultaneously by DNA histogram analysis and LI studies, the correlation coefficient between the two results was only r = 0.611. A comparison of three different mathematical programs for DNA histogram analysis showed that none was completely satisfactory. We conclude that DNA histogram analysis does not provide the same data as autoradiographically processed labeling index studies even in patients with Ph-positive CML during the chronic phase when the situation is not complicated by additional aneuploidy.