Ph-positive Chronic Myeloid Leukemia Research Articles

Blast Crisis of Ph-Positive Chronic Myeloid Leukemia with Isochromosome 17q: Report of 12 Cases and Review of the Literature

Isochromosome 17q [i(17q)] is frequently observed in the blast crisis (BC) of chronic myel-ogenous leukemia (CML). It has been suggested that this chromosome abnormality is associated with special hematological characteristics of the BC, but the information on this subject is scarce. The clinical, hematological and cytogenetic features of patients with i(17q) were analyzed in a series of 121 patients with BC of Ph-positive CML. 'helve patients (10%) displayed an i(17q), representing the third commonest cytogenetic abnormality, after trisomy 8 and Ph chromosome duplication. In seven of the 12 patients the BC was preceded by an accelerated phase, and 10 had more than 10% blood basophils at BC diagnosis. The blast cells had a myeloid phenotype in the 12 patients. Five patients exhibited cytogenetic abnormalities in addition to i(17q), with trisomy 8 and duplication of the Ph chromosome being the alterations most frequently observed. Median survival of patients with i(17q) was 22 weeks, which was not significantly different from the survival of patients with myeloid BC in the overall series. These results are similar to the findings in 181 patients with i(17q) from 12 series of the literature, and confirm the special hematologic profile of BC of CML with this cytogenetic abnormality.

Unusual Clinical Course and Acquisition of del(11)(q23) in Second Lymphatic Blastic Phase of a Ph-Positive Chronic Myeloid Leukemia

We describe unusual clinical and cytogenetic findings of a 29-year-old female with a Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML), who showed a mosaic of apparently normal cells and cells bearing the classical t(9;22)(q34;q11) during the first lymphatic blastic phase (BP). The second lymphatic BP developed 10 years later. In addition to the t(9;22), which was detected in all metaphases, a del(11)(q23) was identified as a subclonal change in 4 of 25 metaphases. Fluorescence in situ hybridization (FISH) analysis using a chromosome 11-specific library probe and a probe covering the breakpoint cluster region of the MLL gene revealed hybridization signals of both probes on the normal and the deleted chromosome 11, indicating that the breakpoint on chromosome 11 occurred telomerically to the breakpoint cluster region of the MLL gene. Chemotherapeutic treatment resulted in reconstitution of the chronic phase with persistence of the Ph translocation as the sole chromosomal abnormality.

Clonally unrelated BCR-ABL-negative acute myeloblastic leukemia masquerading as blast crisis after busulphan and interferon therapy for BCR-ABL-positive chronic myeloid leukemia

We report a patient with Philadelphia (Ph)-positive, BCR-ABL rearrangement positive, chronic myeloid leukemia (CML) with a prolonged chronic phase of 24 years who was first prescribed alpha-2 interferon 22 years after initial diagnosis. This therapy was tolerated poorly on account of thrombocytopenia, but an eventual major cytogenetic response was followed soon afterwards by transformation to terminal acute myeloid leukemia (AML). Cytogenetic studies indicated that the transformed myeloblasts were karyotypically normal and Ph negative. Although polymerase chain reaction (PCR) analysis of total leukemic mRNA remained BCR-ABL positive, other molecular studies, including Southern blotting and fluorescent in situ hybridization (FISH) analyses, showed that myeloblasts were BCR-ABL rearrangement negative. PCR-based clonality studies using an X-chromosome-linked restriction fragment polymorphism within the phosphoglycerate kinase gene (PGK1) further showed that the Ph-negative blast cells had a different clonal origin from the Ph-positive clone of chronic phase. We suggest that cases of underlying Ph-negative leukemic transformation in Ph-positive CML warrant further study and should be considered for trial of intensive remission induction therapy as appropriate for acute leukemia.

No parental origin bias for the rearranged chromosomes in myeloid leukemias associated with t(9;22), t(8;21) and t(15;17)

We investigated parental origin of rearranged chromosomes 9 and 22 (9q+ and 22q−) in five patients with Ph-positive chronic myeloid leukemia (CML) using the C-banding and silver-staining methods of nucleolus organizer regions, respectively; of rearranged chromosome 21 (21q+) in seven patients with t(8;21)-positive acute myeloid leukemia (AML); and of rearranged chromosome 15 (15q+) in six patients with t(15;17)-positive AML. It was found that these rearranged chromosomes can be of either paternal or maternal origin. Although the number of patients examined was small, these results indicate that the genes rearranged as a result of these chromosome translocations ( ABL, BCR, AML-1 and PML) are not genomically imprinted.

Simple competitive two-step RT-PCR assay to monitor minimal residual disease in CML patients after bone marrow transplantation.

Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assessing the amount of transcripts of the BCR/ABL gene, the molecular marker of chronic myeloid leukemia (CML), is the only method sensitive enough for monitoring of minimal residual disease (MRD) in CML patients after bone marrow transplantation (BMT). In this study we present a simple modification of competitive Q-RT-PCR using natural competitors from cell lines K562 and BV173. The competitors were used in the form of unpurified RNA in cell lysates which ensured their high stability. Mixing competitors and samples before RNA extraction eliminated problems with quantification of cDNA or RNA entering the competitive reaction and with checking for the RNA quality and reverse transcription (RT) efficiency. The bulk of the malignant clone was expressed as the number of leukemic cells in 10(6) leukocytes when the overproduction of the BCR/ABL mRNA in the cell lines we used as competitors was taken into account. It was found to be 82-fold and 14-fold in K562 and BV173, respectively, in comparison with 100% Ph-positive CML standard. The assay reliability was verified by comparison of results with the mathematical model of competitive PCR. The assay is highly reproducible and sensitive (10(-5)). Its accuracy was proved to be excellent in a wide range of malignant cell concentrations. The method is demonstrated on three CML patients suffering from MRD after BMT. In conclusion, this method fulfills all criteria of competitive Q-RT-PCR. Because of its simplicity it is suitable for clinical laboratories and due to the high stability of the lysates used it may serve in the standardization of results between different laboratories.

Absence of influence of prior treatment with interferon on the outcome of allogeneic bone marrow transplantation for chronic myeloid leukemia.

Timing of transplantation in the chronic phase of chronic myeloid leukemia (CML) and previous treatment with interferon remains controversial. We have tried to discover what influence pretreatment with interferon alpha (IFN-A) has on the results of allogeneic bone marrow transplantation for CML patients treated in a single institution. Fifty-one consecutive patients with chronic phase Ph-positive CML who received an allogeneic bone marrow transplantation from a HLA-identical familial donor were evaluated. Thirty had been treated with IFN-A (IFN+ group) prior to BMT and twenty-one had not (IFN- group). Both groups were homogeneous for clinical characteristics such as age, sex, previous chemotherapy, disease status, and time from diagnosis to transplant. No difference was found in neutrophil and platelet count recovery between the IFN+ and IFN- group. The incidence of acute and chronic GVHD, VOD and severe mucositis was not significantly different. Relapse and both overall survival and DFS were similar for both groups. No adverse effects of prior IFN exposure on the outcome of HLA-identical sibling donor BMT for chronic phase CML patients were found in this study.

Chronic myeloid leukemia with thrombocythemic onset may be associated with different BCR/ABL variant transcripts

Ph-positive chronic myeloid leukemia (CML) mimicking essential thrombocythemia (ET) at onset seems to be a distinct clinical entity. Whether this rare clinical form of CML is associated with single, specific variants of BCRIABL transcripts is a matter of debate. Among 82 consecutive patients with Ph-positive CML, we identified 3 patients in which the disease mimicked ET at presentation, because of marked thrombocytosis and moderate leukocytosis, with few immature myeloid cells in peripheral blood and blood basophilia in 2 of them. Molecular analysis with the reverse transcriptase-polymerase chain reaction technique showed the presence of b2a2, b3a2, and b3a2-b2a2 transcript variants in the three patients, respectively. The results of our study together with a review of literature data suggest that different BCRIABL transcript variants may occur in CML mimicking ET, without an apparently significant prevalence of one type.

Value of fluorescence in situ hybridization for detecting the bcr/abl gene fusion in interphase cells of routine bone marrow specimens.

Fluorescence in situ hybridization (FISH) is a new technique that allows demonstrating of the bcr/abl gene fusion in bone marrow cells of patients with Philadelphia translocation (Ph)-positive chronic myeloid leukemia (CML). In this study, bone marrow samples of 150 patients were investigated routinely by interphase FISH, cytogenetics, and bone marrow histopathology. In 20 patients with reactive hyperplasia of the granulopoiesis and normal karyotypes, FISH revealed nonspecific bcr/abl fusion signals at a mean frequency of 2.7% of the cells examined. The cutoff level for specific fusion signals was set at three times the standard deviation (9.0%). None of the 29 cytogenetically Ph-negative patients with myeloproliferative disease other than CML had fusion signals exceeding 9%. The mean frequency of specific fusion signals in nontreated patients with CML (n = 59) was 92.7%, and 49.3% in patients with CML who received therapy (n = 42). For diagnosing Ph-positive CML, interphase FISH has been faster, more reliable, and more sensitive than cytogenetics, which was successful in 54 of 59 patients investigated at first diagnosis but only in 27 of 42 patients receiving therapy, and it failed to detect Ph-positive cells in three patients with CML. However, small percentages of less than 9.0% of cells with bcr/abl fusion signals were below the threshold of interphase FISH, thereby limiting its use for detecting minimal residual disease.

Location of theBCR-ABL fusion gene on the 9q34 band in two cases of Ph-positive chronic myeloid leukemia

Two new variant Philadelphia (Ph) chromosomes with an aberrant location of the BCR-ABL fusion gene on 9q34 of the derivative 9 are reported. One presented cytogenetically as a standard t(9;22)(q34;q11), whereas the other was classified as an ins(9;22)(q34;q11.1q11.2) using the combined interpretation of cytogenic, FISH, and molecular data. The mechanisms of the two rearrangements are presented. It is suggested that the insertion has occurred in a single event in the patient with ins(9;22). In the patient with t(9;22), both a translocation and an insertion, occurring either sequentially or simultaneously, can account for the location of the BCR-ABL fusion gene on the derivative 9. A possible poor prognostic impact of this aberrant location of the BCR-ABL is also suggested by the clinical data reported in such patients.

Histopathological changes in bone marrow biopsies from patients with chronic myeloid leukaemia after treatment with recombinant alpha-interferon.

Recombinant alpha-interferon (r-IFN) is an effective therapy for chronic myeloid leukaemia (CML), inducing haematological and major cytogenetic response in 70% and 30% of patients, respectively. In this study we have evaluated the significance of bone marrow (BM) histology on the subsequent response to r-IFN therapy, as well as the morphological changes induced by r-IFN within BM. 73 BM biopsies were studied from 21 patients with Ph1-positive CML in chronic phase at diagnosis and at different times during r-IFN treatment. At diagnosis the probability of achieving a major or complete cytogenetic response was significantly higher in patients with a total marrow cellularity lower than 90% (P = 0.02). During therapy with r-IFN, significant BM changes included disappearance of the CML pattern (P = 0.0002), reduction of M:E ratio (P = 0.0009) and total cellularity (P = 0.0027), and increase in number of terminal megakaryocytes (P = 0.0009) and of fatty tissue regeneration (P = 0.037); only after long-term therapy (mean 20 months), did reticulin fibrosis increase significantly (P = 0.032). The overall BM morphology in response to treatment displayed different pictures, ranging from persistence of CML (25 biopsies out of 51), to reversion to normal histology (14 out of 51). Persistence of diffuse morphological abnormalities was associated with lack of cytogenetic responsiveness (P = 0.025).

The relationship between telomere length and therapy-associated cytogenetic responses in patients with chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal disease with specific cytogenetic changes involving the Philadelphia (Ph) translocation. The authors examined the relationship between telomere length (terminal restriction fragment [TRF]) and therapy-associated cytogenetic responses in CML patients. The authors examined the telomere length and telomerase activity in 44 patients with Ph-positive CML in the chronic phase. TRF was determined by Southern blot analysis using the (TTAGGG)4 probe and telomerase activity was assessed by the telomeric repeat amplification protocol and fluorescent-labeled primers. At the time of CML diagnosis, 19 patients had TRFs within the age-matched normal range (mean +/- 2 x standard deviation [SD]) and the remaining 25 patients had TRFs shorter than the age-matched normal range (< mean +/- 2 x SD). Hematologic findings, including leukocyte count, hemoglobin level, platelet count, and percentage of bone marrow blasts at the time of diagnosis did not significantly differ between patients with normal and shortened TRFs; however, those with shortened TRFs had high levels of telomerase activity (P = 0.045). In a group of patients treated with alpha-interferon (n = 32), those with normal TRFs had a significantly lower frequency of blast crises (P = 0.0328), a significantly higher incidence of cytogenetic responses (P = 0.0185), and a favorable prognosis (P < 0.01) compared with those with shortened TRFs. These findings suggest that normal TRFs in a small number of CML patients at the time of diagnosis may have a significant amount of normal stem cells remaining. The authors suggest that normal TRFs at the time of diagnosis indicate a subset of CML patients who may respond favorably to alpha-interferon therapy.

Frequency of pseudo-Gaucher cells in diagnostic bone marrow biopsies from patients with Ph-positive chronic myeloid leukaemia.

Pseudo-Gaucher cells (PGC) are a characteristic finding in Ph-positive CML, and prolongation of survival was observed when PGC were detected within the bone marrow. However, the conspicuous variation in the reported frequencies indicates the necessity for analysis of their natural occurrence in the bone marrow from untreated CML patients. A total of 833 diagnostic bone marrow biopsies from patients with Ph-positive CML were examined for PGC by 7 observers. Proof of PGC was based on systematic examination of Giemsa-stained slides with and without polarization at high magnification. Birefringence within the cytoplasm turned out to be highly specific for PGC. The risk of overlooking PGC was at least 80% when the number of these storing histiocytes was 70 per slide or less, and at least 50% when the total amount per slide was < or = 250. This high risk of failure explained the disagreement among the authors. An intensive investigation by at least two observers is mandatory if results are to be evaluated in research. Under the conditions used in this study, the natural frequency of PGC within the bone marrow from untreated patients with a Ph-positive CML is much higher than assumed to date, amounting to about 70%. On the basis of these findings, the prognostic importance of PGC in CML must be evaluated critically.

Clinical and biological aspects of philadelphia-Negative/BCR-Negative chronic myeloid leukemia

The Philadelphia (Ph) chromosome was the first chromosomal abnormality associated with a specific leukemia, chronic myeloid leukemia (CML). This chromosome arises from the t(9;22)(q34;q11) translocation which results in the juxtaposition of the bcr gene and the abl proto-oncogene. This BCR/ABL fusion gene encodes for a hybrid protein with the capacity of oncogenic transformation of hematopoietic cells. Nonetheless, very few myeloproliferative disorders (about 10%) included under the generic term of CML have no Ph chromosome. Half of these Ph-negative CML have the BCR/ABL fusion gene (BCR-positive) and are considered equivalent to Ph-positive CML. In contrast, the patients without detectable BCR/ABL fusion (BCR-negative) fulfil the criteria for atypical CML (aCML) of the French-American-British (FAB) classification, despite considerable variability at the individual level. Due to the very small number of patients with precise cytological descriptions already published, cooperative studies focused on aCML are warranted to draw definitive conclusions and to provide some pointers on physiopathology

Unusual expression of mRNA typical of Philadelphia positive acute lymphoblastic leukemia detected in chronic myeloid leukemia

The Philadelphia chromosome (Ph) is found in both chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The Ph translocation, t(9;22)(q34;q11), can disrupt the BCR gene on chromosome 22 in one to two areas called the major (Mbcr1) and minor (mbcr1) breakpoint cluster regions. In CML the breakpoint has been mapped almost exclusively to Mbcr1, whereas in Ph positive ALL both Mbcr1 and the upstream mbcr1 breakpoints have been described. In this communication we describe an unusual patient with typical chronic phase Ph positive CML and evidence of the uncharacteristic mbcr1 breakpoint, predicting expression of the ALL-type p190 fusion protein. Fluorescence in situ hybridization demonstrated BCR gene rearrangement, the reverse transcription polymerase chain reaction detected the BCR-ABL fusion mRNA characteristic of the mbcr1 breakpoint, and failed to detect BCR-ABL mRNA characteristic of the Mbcr1 breakpoint. Southern blot analysis revealed no rearrangement in Mbcr1, and direct sequencing of the PCR product confirmed it to be the ALL-type mbcr1 fusion mRNA with the first exon of the BCR gene fused to ABL exon a2. This case differs from the previously reported cases of “p190” CML in that the patient presented without abnormal hematopoietic features other than those found in typical CML and provides further evidence that the p190 mRNA is not sufficient to cause an acute rather than chronic leukemia. © 1996 Wiley-Liss, Inc.

Lack of correlation between ABL-BCR expression and response to interferon-alpha in chronic myeloid leukaemia.

Interferon-alpha (IFN-alpha) is useful in the treatment of Philadelphia (Ph)-positive chronic myeloid leukaemia (CML). There is, however, a marked heterogeneity among CML patients in relation to their response to IFN-alpha treatment, the reasons for which are unknown. Since the reciprocal ABL-BCR gene is transcriptionally active in only a proportion of CML patients, it has been suggested that response to IFN-alpha may correlate with ABL-BCR expression. In the present study we have tested 209 Ph-positive CML patients for expression of ABL-BCR, BCR-ABL and the normal BCR and ABL genes by reverse transcriptase/polymerase chain reaction (RT/PCR). Whereas BCR-ABL, BCR and ABL transcripts were detected in all the patients, ABL-BCR expression was observed in 59% of the cases. A group of 105 patients within this series was treated with IFN-alpha; 33% achieved a complete or major cytogenetic response (< 35% Ph-positive metaphases) and the remaining 67% showed minimal or no response to IFN-alpha. The proportions of patients who were ABL-BCR positive (63%) and ABL-BCR negative (37%) were the same for good responders and poor responders, suggesting that there is no correlation between ABL-BCR expression and cytogenetic response to IFN-alpha in CML.

Does the type of BCR/ABL junction predict the survival of patients with Ph1-positive chronic myeloid leukemia?

The prognostic value of the site of DNA rearrangement within the M-BCR on chromosome 22 or of the type of transcript has been debated in the last years. The majority of the studies do not support the hypothesis of a predictive value of such molecular parameters. Results coming from a multicentric, prospective trial, based on alpha-IFN therapy, seem to indicate a better karyotypic response in 3' rearranged patients. The possibility of evoking a cytotoxic immune response directed towards peptides originating from each of the different BCR/ABL junctions constitute an important challenge for the future.

Karyotypic findings as an independent prognostic marker in chronic myeloid leukaemia blast crisis

Fifty-three patients with Ph positive chronic myeloid leukaemia in blastic phase were studied. Additional abnormalities were found in 29 (55%) patients and were more common in myeloid (64%) than lymphoid (45%) blast crisis. The most frequent were +Ph (32%), +8 (28%), +19 (19%), +20 (9%) and +21 (9%). i(17q) (9%) was associated with thrombocytopenia ( 5 5 ) and basophilia ( 2 5 ). The incidence of additional abnormalities was higher in patients treated with busulphan (70%) than hydroxyurea (44%). No significant differences were noted in the mean values of the clinical and haematological findings recorded at blast crisis between patients with only Ph positive (PP) cells and those with additional abnormalities (AP + AA). Univariate analysis identified karyotypic findings as an independent prognostic marker indicating its significance in assessing the response to therapy and survival after the onset of transformation.

Effects of herbimycin A and its derivatives on growth and differentiation of Ph 1-positive acute lymphoid leukemia cell lines

The molecular basis of the Philadelphia chromosome (Ph 1) is a structurally altered c-abl ( bcr/abl) gene which encodes an abnormally large protein with protein tyrosine kinase activity. Herbimycin A, an inhibitor of tyrosine kinase, preferentially inhibited the growth of Ph 1-positive acute lymphoid leukemia (ALL) cell lines, as well as Ph 1-positive chronic myeloid leukemia (CML) cell lines. Although noncytotoxic concentrations of herbimycin A induced erythroid differentiation of two CML-derived cell lines, K562 and KU812, in a previous study, the differentiation-inducing effect of herbimycin A on Ph 1-positive ALL cell lines was less strong. Herbimycin A enhanced some differentiation-associated properties of one Ph 1-positive ALL cell line, L2, but the effect of herbimycin A on the other Ph 1-positive ALL cell lines was cytotoxic rather than cytostatic (differentiation-inducing). Several derivatives of herbimycin A were synthesized and their effects on the cell proliferation of Ph 1-positive CML and ALL cell lines were examined. The sensitivities of the Ph 1-positive cell lines to herbimycin A derivatives were different from the data on the rat kidney cell line infected with Rous sarcoma virus (v- src) derived from a previous study, suggesting bcr/abl kinase may differ in sensitivity from other tyrosine kinases. Moreover, the sensitivities of the ALL cell lines were not the same as those of the CML cell lines. These results suggest that a specific inhibitor of bcr/abl kinase could be an effective antileukemic agent against Ph 1-positive CML or ALL.

Blastic phase chronic myeloid leukemia with a four-break rearrangement: t(11;9)(9;22)(q23;p22q34;q11)

Chromosome analysis of bone marrow (BM) aspirate from a 36-year-old man with chronic myeloid leukemia (CML) in blastic phase (BP) showed a four-break rearrangement t(11;9)(9;22)(q23;p22q34;q11), which can be considered a t(9;22)(q34;q11) and a secondary t(9;11)(p22;q23). It is not surprising that additional chromosome abnormalities occur in patients with Ph-positive CML in BP, but it is of interest that t(9;11)(p22;q23), characteristic of acute myeloid leukemia French-American-British (FAB) type M5 (ANLL-M5) was observed. The possible meaning of this additional change in BP of CML is discussed.

The cytogenetic scenario of chronic myeloid leukemia.

The Philadelphia chromosome (Ph), i.e., the reciprocal translocation t(9;22)(q34;q11), is found with great specificity in bone marrow cells from patients with chronic myeloid leukemia (CML). Variant Ph-producing translocations, seen in 5-10% of all patients, are all complex and involve the same molecular rearrangement as the regular t(9;22). Patients with classic and variant Ph-producing translocations are clinically and hematologically identical, and as a group differ from Ph-negative CML patients. In all patient groups, the occurrence of additional chromosome changes is an ominous sign indicating that disease progression is imminent. The chromosome changes occurring in excess of the Ph in CML are clearly nonrandom and two pathways of cytogenetic evolution may be distinguished. Major route changes comprise trisomy 8, i(17q), trisomy 19, and an extra Ph; totally, 71% of Ph-positive CML patients have at least one of these four major route changes. Six minor route changes, including five numerical abnormalities (-7, -17, +17, +21, and -Y) but also one structural aberration, t(3;21) (q26;q22), have been identified. At least one of these changes is found in 15% of all Ph-positive CML cases. Altogether, the four major route aberrations and the six minor route changes are present as part of the clonal evolution in 86% of CML with cytogenetic abnormalities in addition to the Ph chromosome.