Blast crisis in a murine model of chronic myelogenous leukemia.

The P210bcr/abl protein is produced in cells from patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML). Retroviral transfer of the gene encoding P210bcr/abl into murine bone marrow induces a granulocytic leukemia that models the chronic phase of human CML. We have transferred the leukemic clone to syngeneic animals, albeit with surprising inefficiency, and have observed CML and clonally related acute leukemias of lymphoid or myeloid phenotype in some transplant recipients. These data show that murine CML can result from retroviral transfer of the bcr/abl gene into pluripotent hematopoietic stem cells, that infected clones repopulate poorly after adoptive transfer, and that these clones can give rise to acute leukemia, reflecting evolution to a phase resembling blast crisis in the human disease.     

The correlation of breakpoint cluster region rearrangement and p210 phl/abl expression with morphological analysis of Ph-negative chronic myeloid leukemia and other myeloproliferative diseases

The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from a reciprocal translocation t(9;22) (q34;q11) and is associated with chronic myeloid leukemia (CML). The translocation can be identified at the DNA level in Ph-positive CML by using a probe to the breakpoint cluster region (bcr). In addition, as a result of this translocation an abl-related 210-kd protein with protein tyrosine kinase (PTK) activity is produced. We analyzed 28 cases of Ph-negative CML for rearrangement of the chromosome 22 sequences and found that eight of the 28 show rearrangement of the bcr. When 12 of the Ph-negative cases were independently reviewed, five were indistinguishable from Ph-positive CML on the basis of morphology, peripheral blood film and clinical details. These five also showed bcr rearrangement. The other seven were reclassified as six atypical CML (aCML) and one chronic myelomonocytic leukemia (CMML). None of these seven showed bcr rearrangement. In addition 11 cases of bcr- CML were assayed for abl-related PTK, and no detectable activity was present, whereas p210 phl/abl PTK was observed both in Ph-positive (three cases examined) and Ph-negative, bcr + (four cases examined) CML. Therefore, bcr + CML, whether or not the Ph chromosome is cytogenetically apparent, involves a similar molecular alteration and produces the 210-kd protein with enhanced PTK activity. Furthermore, these cases can be distinguished from Ph-negative bcr- CML by careful evaluation of clinical and hematologic data.     

Expression of c-abl in Philadelphia-positive acute myelogenous leukemia

The identical cytogenetic marker, t(9;22)(q34;q11) (Philadelphia [Ph] translocation), is found in approximately 90%, 20%, and 2% of adult patients with chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myelogenous leukemia (AML), respectively. In CML, the molecular events resulting from the Ph translocation include a break within the bcr locus on chromosome 22, transfer of the c-abl protooncogene from chromosome 9 to 22, and formation of an aberrant 210- kD bcr-abl fusion protein (p210bcr-abl). Recently, the absence of bcr rearrangement and expression of a distinct aberrant 190-kd abl protein (p190c-abl) has been described in Ph-positive ALL, with the suggestion that the two abl variants may be pathogenetically associated with myeloid v lymphoid leukemogenesis. Here we report that the genomic configuration and translation product of Ph-positive AML can be similar to that of Ph-positive ALL: the break at 22q11 may occur outside the 5.8 kb bcr region and result in expression of a 190-kD abl protein lacking these bcr sequences. Phosphokinase enzymatic activity, a fundamental property of p210bcr-abl, was also associated with AML- derived p190c-abl. Our current observations indicate that p190c-abl can be found in cells of lymphoid or myeloid lineage and is therefore unlikely to play a specific role in the development of lymphoid leukemias. Formation of p190c-abl instead of p210bcr-abl appears to be a characteristic of the acute rather than the chronic Ph-positive leukemic state.     

Significance and correlations of molecular analysis results in patients with Philadelphia chromosome-negative chronic myelogenous leukemia and chronic myelomonocytic leukemia

PURPOSE: Several investigators have documented a rearrangement of the breakpoint cluster region (bcr) in selected patients with a morphologic diagnosis of chronic myelogenous leukemia (CML) but no abnormality of the Philadelphia chromosome (Ph) by cytogenetic studies. Our intention was to systematically investigate the incidence of the bcr rearrangement in such patients, and to correlate the findings with patient characteristics, response to therapy (especially alpha interferon treatment), and overall prognosis. PATIENTS AND METHODS: Molecular analysis studies were performed in 40 patients with Ph-negative CML (23 patients) and myelomonocytic leukemia (CMML; 17 patients). RESULTS: Rearrangement of the breakpoint cluster region (bcr) was detected in 11 of the 23 patients with Ph-negative CML (48 percent), indicating the presence of the abnormal molecular events in Ph-positive CML without documentation of the Ph cytogenetic abnormality. None of the 17 patients with CMML had the bcr rearrangement. Patients with Ph-negative CML and the bcr rearrangement had characteristics similar to those of patients with Ph-positive disease. These included a younger age, higher white blood cell counts, a higher incidence of thrombocytosis and basophilia, and a lower occurrence of thrombocytopenia. The leukocyte alkaline phosphatase score was not a helpful distinguishing feature. Among 21 patients receiving alpha interferon-based regimens, response to therapy was significantly better among patients with Ph-negative disease and the bcr rearrangement (seven of seven, 100 percent), compared with those without the bcr rearrangement (one of six, 17 percent), or patients with CMML (two of eight, 25 percent) (p less than 0.01). At this time of follow-up, only one of the 11 patients with Ph-negative CML and the bcr rearrangement had died from complications of allogeneic bone marrow transplantation, compared with three deaths among the 12 patients with Ph-negative CML and no bcr rearrangement, and 11 deaths among the 19 patients with CMML. CONCLUSION: We conclude that molecular studies help in better understanding the nosology of Ph-negative CML, and define a subgroup of patients with clinical, therapeutic, and prognostic correlations similar to those of patients with Ph-positive CML.     

Molecular biology of chronic myelogenous leukemia

In this review we have described molecular consequences of the t(9;22) translocation typical of CML and some cases of ALL. This data indicates an important role for abl and bcr and suggest some common mechanisms of activation of c-abl related tyrosine kinase activity. This data also provides insight into the relationship between Ph1 positive, Ph1 negative CML and Ph1 positive ALL. Although this data answers some questions, they raise others; eg, what is the molecular basis of the t(9;22) translocation? How does increased abl related kinase activity eventuate in CML? Finally, this data reviewed suggests that factors other than abl and bcr must play a role in CML. Definition of these factors will be important in the future.     

Detection of minimal residual leukemia after bone marrow transplantation in patients with chronic myeloid leukemia using the polymerase chain reaction

We have utilized the polymerase chain reaction (PCR) to sensitively detect the minimal residual leukemia in 24 patients who were hematologically normal and were negative for Ph1 chromosome after bone marrow transplantation (BMT). None of the patient showed breakpoint cluster region (BCR) gene rearrangement by Southern blot analysis, however, PCR technique revealed the bcr/abl mRNA in 13 of 24 patients. In patients who received CY + TBI as a conditioning regimen, 6 out of 8 patients were detected bcr/abl mRNA by PCR. On the other hand, patients who received high dose AraC+ CY + TBI or busulfan + CY as a conditioning, bcr/abl mRNA was detected in 4 out of 9 patients and 1 out of 5 patients, respectively. There was no clear association between the presence or absence of graft versus host disease and the detection of bcr/abl mRNA by PCR.     

A possible correlation between the type of bcr-abl hybrid messenger RNA and platelet count in Philadelphia-positive chronic myelogenous leukemia.

The Philadelphia (Ph1) chromosome, in which the hybrid bcr-abl gene is formed, is thought to be the initial event in chronic myelogenous leukemia (CML). The position of the breakpoint within the breakpoint cluster region (bcr) on Ph1 chromosome and the splicing pattern determine the species of the fused bcr-abl messenger RNA (mRNA). We tried to detect the two types of fused mRNAs in 57 chronic-phase cases of Ph1-positive CML using the polymerase chain reaction procedure (RT-PCR). The bcr exon 2/abl exon 2 fused mRNA (b2-a2) was detected in 17 patients, the bcr exon 3/abl exon 2 fused mRNA (b3-a2) was detected in 34 patients, and both types of mRNA were detected in six patients. The platelet counts of patients who expressed b3-a2 mRNA or both types were significantly higher than those of patients who expressed only b2-a2 (841.5 v 373.5 x 10(9)/L; P less than .015), although there was no significant difference in the white blood cell counts or hemoglobin. This finding suggests a possibility that the type of bcr-abl mRNA may affect the thrombopoietic activity in CML.     

Calcium signaling induces acquisition of dendritic cell characteristics in chronic myelogenous leukemia myeloid progenitor cells.

Effective host T lymphocyte sensitization to malignant cells depends on successful antigen presentation. In this study, we examined the capacity of malignant myeloid progenitor cells of patients in the chronic phase of chronic myelogenous leukemia (CML) to acquire characteristics of activated dendritic cells (DCs) after intracellular calcium mobilization, thereby bypassing a need for third-party antigen-presenting cells. Treatment of purified CD33(+) CML cells from 15 patients with calcium ionophore (CI) consistently resulted in de novo expression of the costimulatory molecules CD80 (B7.1) and CD86 (B7.2), CD40 and the DC-specific activation marker CD83, as well as marked up-regulation of MHC class I and II molecules and the adhesion molecule CD54. Most of these changes occurred within 24 hr of treatment. Morphologically, CI-treated CML cells developed long dendritic projections similar to those seen in mature DCs. Functionally, CI-treated CML cells provided stimulation of allogeneic T lymphocytes 10- to 20-fold that of untreated CML cells or untreated monocytes. Fluorescent in situ hybridization of CI-activated CML cells confirmed their leukemic origin by displaying the typical bcr/abl fusion signal. No difference in bcr/abl translocation percentages between untreated and CI-treated CML nuclei was observed. These observations indicate that calcium mobilization may constitute a valuable approach for rapidly and reliably generating CML-derived DCs for immunotherapy of CML.     

The coiled-coil domain and Tyr177 of bcr are required to induce a murine chronic myelogenous leukemia-like disease by bcr/abl

The bcr/abl fusion in chronic myelogenous leukemia (CML) creates a chimeric tyrosine kinase with dramatically different properties than intact c-abl. In P210 bcr/abl, the bcr portion includes a coiled-coil oligomerization domain (amino acids 1-63) and a grb2-binding site at tyrosine 177 (Tyr177) that are critical for fibroblast transformation, but give variable results in other cell lines. To investigate the role of the coiled-coil domain and Tyr177 in promoting CML, 4 P210 bcr/abl-derived mutants containing different bcr domains fused to abl were constructed. All 4 mutants, Delta(1-63) bcr/abl, (1-63) bcr/abl, Tyr177Phe bcr/abl, and (1-210) bcr/abl exhibited elevated tyrosine kinase activity and conferred factor-independent growth in cell lines. In contrast, differences in the transforming potential of the 4 mutants occurred in our mouse model, in which all mice receiving P210 bcr/abl-expressing bone marrow cells exclusively develop a myeloproliferative disease (MPD) resembling human CML. Of the 4 mutants assayed, only 1-210 bcr/abl, containing both the coiled-coil domain and Tyr177, induced MPD. Unlike full-length P210, this mutant also caused a simultaneous B-cell acute lymphocytic leukemia (ALL). The other 3 mutants, (1-63) bcr/abl, Tyr177Phe bcr/abl, and Delta(1-63) bcr/abl, failed to induce an MPD but instead caused T-cell ALL. These results show that both the bcr coiled-coil domain and Tyr177 are required for MPD induction by bcr/abl and provide the basis for investigating downstream signaling pathways that lead to CML.     

Identification of molecular variants of p210bcr-abl in chronic myelogenous leukemia

The aberrant abl protein product of a chronic myelogenous leukemia (CML) blast crisis cell line (K562) and of five Philadelphia chromosome- positive CML patients in blast crisis were analyzed by an immune complex kinase assay using two antipeptide sera generated against the hydrophilic domain of v-abl and a region within the third exon of the breakpoint cluster region (bcr) respectively. Both the anti-abl and anti-bcr sera detected a 210 kd band in extracts derived from K562 cells and from two CML patients with myeloid blast crisis. p210 was detected by the anti-abl but not the anti-bcr sera in three CML patients with myeloid (one patient) and lymphoid (two patients) blast crisis, indicating the absence of bcr exon 3 in this protein. Southern blot analysis on DNA derived from one of the patients in the latter group was consistent with the break on chromosome 22 occurring 5' to bcr exon 3. Our observations demonstrate that the Philadelphia translocation results in the generation of a chimeric bcr-abl protein with at least two molecular variants, both of which are enzymatically active as protein kinases.     

Mutations of the N-ras gene in juvenile chronic myelogenous leukemia

Juvenile chronic myelogenous leukemia (JCML), a myeloproliferative disorder of childhood, is distinct from adult-type chronic myelogenous leukemia (CML) and bears resemblance to chronic myelomonocytic leukemia (CMMoL). Since mutations in the N-ras gene have been found at high frequencies in CMMoL, but only rarely in CML, we analyzed mutations activating the N-ras gene in 20 patients with JCML. We used the strategy for analysis of gene mutations based on in vitro DNA amplification by polymerase chain reaction (PCR) followed by single- strand conformation polymorphism (SSCP) analysis and/or direct sequence analysis. Nucleotide sequence analysis showed single nucleotide substitutions involving codons 12, 13, or 61 in six of 20 patients (30%). Four of six patients with mutations were in chronic phase and the other two in blast crisis, indicating no apparent correlation with disease stage. Most of the patients with mutations were in the older age group with poor prognosis, although one patient in the younger age group also harbored the mutation. These data suggest that N-ras gene mutations may be involved in the pathogenesis and/or prognosis of JCML and provide further evidence that JCML is an entity distinct from CML.     

No rearrangement of the breakpoint cluster region in two juvenile chronic myeloid leukemia

We performed cytogenetic studies and breakpoint cluster region (bcr) rearrangement analysis in two cases of juvenile chronic myeloid leukemia (JCML) which is special type of chronic myeloid leukemia (CML). Case 1 (8-month-old male) and case 2 (3-month-old female) showed clinical and hematologic manifestations similar to CML. Each of case 1 and 2 had normal karyotype and no bcr rearrangement. These findings suggest that JCML is a different heterogeneous disorder from that of adult CML.     

应用RT-nest-PCR法检测慢性髓细胞白血病非亲缘异基因骨髓移植后微小残留病变

目的：应用筑巢式RT—PCR（RT—nest-PCR）法检测慢性髓细胞性白血病（CML）患者非亲缘异基因骨髓移植（URD）后微小残留病变（MRD），并探讨它与复发的相关性。方法：分别采用RT-nest—PCR法和常规细胞遗传学方法检测bcr／abl融合基因和Ph染色体。结果：20例CML行URD患者术前Ph染色体和bcr／abl融合基因检测均为阳性，移植术后30dPh染色体皆转为阴性，bcr／abl融合基因完全转阴时间为1～3个月，中位时间2个月；在随防的18例CML患者中，有16例患者bcr／abl融合基因完全转阴后没再转为阳性。在1例复发的CML患者中可见到血型、Ph染色体和bcr／abl融合基因动态变化，另1例CML患者在移植成功后的21个月和36个月检测到bcr／abl融合基因，经随访未见临床和血液学复发征象。结论：检测bcr／abl融合基因是目前观察CML患者非亲缘异基因骨髓移植后微小残留病变最敏感的方法之一，非亲缘异基因骨髓移植能最大限度地消除CML患者体内残留病变，患者能长期无病生存。