Ph-positive Chronic Myelogenous Leukemia Research Articles

Therapeutic Choices in Patients with Ph-Positive Chronic Myelogenous Leukemia In Mexico in the Era of Tyrosine Kinase Inhibitors: Stem Cell Transplantation or Tyrosine Kinase Inhibitors? Fifteen Years Later.

Background: Chronic myelogenous leukemia is a neoplastic proliferation of the granulocytic series. In Mexico, chronic myelogenous leukemia accounts for approximately 10% of all leukemias. Tyrosine-kinase inhibitors are considered front-line therapy in high-income countries, whereas allogeneic hematopoietic stem cell transplantation is a recognized therapeutic approach, mainly in low- and middle-income countries. Objective: To analyze the overall survival of persons with chronic myelogenous leukemia who have received tyrosine-kinase inhibitors or allogeneic hematopoietic stem cell transplantation in a medical center, since 1994, and briefly discuss the current indications of these treatments in the tyrosine-kinase inhibitors era. Methods: We retrospectively analyzed all patients with a diagnosis of chronic myelogenous leukemia treated in a medical center between 1994 and 2023; subsets of individuals who received an allogeneic hematopoietic stem cell transplantation or tyrosine-kinase inhibitors therapy as first-line treatment were analyzed. Results: 60 persons with chronic myelogenous leukemia were treated with allogeneic hematopoietic stem cell transplantation or tyrosine-kinase inhibitors: 35 received an allogeneic hematopoietic stem cell transplantation, whereas 25 were given tyrosine-kinase inhibitors. All patients who underwent an allogeneic hematopoietic stem cell transplantation engrafted successfully, and the procedure was completed on an outpatient basis in most cases (29/35). The median survival in allogeneic hematopoietic stem cell transplantation was 78.3 months (CI 95%: 0-205.6) and in persons given tyrosine-kinase inhibitors the median was not reached. Conclusion: Tyrosine-kinase inhibitors were significantly superior to allogeneic hematopoietic stem cell transplantation in prolonging the overall survival of persons with chronic myelogenous leukemia in our single institution experience. (Rev Invest Clin. 2024;76(2):91-6).

Exome Sequencing Reveals The Genetic Landscape Of Rare Atypical e6a2 CML

BackgroundOver 90% of Ph-positive chronic myelogenous leukemia (“typical CML”) patients have breakpoints in the M-bcr, which typically result in b2a2 (e13a2) and/or b3a2 (e14a2) fusion mRNAs, both of which are translated into the p210 BCR-ABL protein. CML patients with the p190 BCR-ABL (m-bcr) or p230 BCR-ABL (μ-bcr) fusion genes have been reported. Atypical BCR breakpoints outside these cluster regions are extremely rare. For instance, only 8 cases have been described of e6a2 fusion CML. Very little is known about the clinical or biological characteristics of this subtype of CML, including the role of collaborating gene mutations in the development of disease. In this study, we defined the gene mutations that occurred in a rare e6a2 CML case and compared the observed gene mutations to those in “typical” chronic phase (CP)-CML cases. To our knowledge, this is the first comparison of the genetic mutations occurring in typical CML and in this rare atypical form of CML. MethodologyWe identified the index e6a2 CML patient, and eight additional typical CML patients for whom we had bone marrow aspirate, peripheral blood and paired normal tissue. We performed whole-exome sequencing for all of these samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 3 GB of sequencing data using high throughput sequencing on the Illumina platform. ResultsWe identified 15 candidate cancer genes that were somatically mutated in our e6a2 CML patient. Commonly implicated biological processes comprising these genes included transcription (STAT5A, TET2, GTF2F1), cellular differentiation (TP73), and signal transduction (GPR116). Interestingly, the majority of these mutations also occurred in typical CML, albeit at lower frequency. Thus, genes mutated common to our atypical case and typical CMLs included STAT5A, TET2, GTF2F1, ABL1 and CYP2A6. Thus, while atypical e6a2 BCR-ABL fusion CML cases are extremely rare, they appear to share many aspects of the biology with typical CMLs. ConclusionThis study represents an in-depth analysis of a rare e6a2 CML in combination with one of the first analyses of gene mutations that occur in typical CML. Our data provide a significant first step to identifying genes that play a role in the pathogenesis along with BCR-ABL that perhaps contribute to drug resistance, and ultimately impact overall survival. Disclosures:No relevant conflicts of interest to declare.

Characterization of a Monoclonal Antibody Specific for Novel Bcr/Abl Out-of-Frame Fusion Proteins

The new tumor-specific antigens Bcr/Abl-OOF, identified in Philadelphia chromosome (Ph)-positive leukemia cells, are derived from an alternative splicing event involving BCR exons 1, 13, or 14 and ABL exons 4 and 5. The COOH-terminus of these transcription products contain an amino acid portion derived from an out-of-frame (OOF) reading of the ABL gene; these variants are expressed in Ph-positive chronic myelogenous leukemia (CML) and acute lymphocytic leukemia patients. Previously, we confirmed the presence of out-of-frame peptide-specific T cells in the peripheral blood of CML patients with the ability to lyse primary autologous CML cells. We also demonstrated that the out-of-frame Abl portion was immunogenic in HLA-A2.1 transgenic mice. Here we describe the production and characterization of monoclonal antibody 1D8G8, a new tool for localization and functional studies of the tumor antigen Bcr/Abl-OOF. This antibody recognizes the out-of-frame protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence, and binds to a specific epitope of this antigen presented in association with HLA-A2.1 molecules at the surface of these cells, as demonstrated by flow cytometry. Thus this MAb could be useful to better understand how this new protein presents in Ph-positive cells beside the canonical Bcr/Abl fusion proteins.

Dysplastic basophils in the accelerated phase of chronic myelogenous leukemia

Dysplastic basophils can occur in the myelodysplastic syndromes but they are not diagnostically very useful as they are usually infrequent and the tendency of normal basophils to degranulate can make them difficult to recognize. They are much more readily recognized in the accelerated phase of chronic myelogenous leukemia (CML). The images shown are from the blood film of a 64-year-old woman with a 33-year history of Ph-positive CML. At the time of the film, she was about to commence bosutinib following relapse after a third autologous stem cell transplant. Splenectomy had been performed the year after presentation and during the course of her long illness she had also received busulfan, hydroxycarbamide, imatinib, and nilotinib. Her white cell count was 80.3 × 109/l and 87% of cells were basophils. She had become anemic and thrombocytopenic (hemoglobin 107 g/l and platelet count 17 × 109/l, respectively). The left hand image shows a dysplastic basophil (left) in comparison with a cytologically normal neutrophil (right). The cell in the center is a basophil precursor in which typical large basophilic granules can be recognized. The right hand image shows two dysplastic basophils. The dysplastic features, which were shown in the majority of basophils in this patient, included cytoplasmic vacuolation, nuclear hypersegmentation and granules that were sparse, abnormally small and had abnormal staining characteristics (often pink rather than purple on a Romanowsky stain). At presentation of CML, the basophils are usually cytologically normal. The appearance of increased numbers of basophils and dysplastic basophils are common features of accelerated phase disease.

Chromosome abnormalities additional to the Philadelphia chromosome at the diagnosis of chronic myelogenous leukemia: pathogenetic and prognostic implications

Additional chromosome abnormalities (ACAs) occur in less than 10% of cases at diagnosis of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). In some cases, on the basis of the persistence of the ACAs in Ph-negative cells after response to imatinib, a secondary origin of the Ph chromosome has been demonstrated. In this study, the possible prognostic value of this phenomenon was evaluated. Thirty-six Ph-positive CML patients were included in the study. In six patients, ACAs persisted after the disappearance of the Ph. A complete cytogenetic response (CCR) was obtained in five of these six patients, and five of six also had a high Sokal score. In all the other cases, ACAs disappeared together (in cases of response to therapy with imatinib) or persisted with the Ph (in cases of no response to imatinib). In the former cases, the primary origin of the Ph was demonstrated. CCR was obtained in 22 cases (17 with low to intermediate Sokal scores), while no response was observed in 8 patients (5 with a high Sokal score). Sokal score seems to maintain its prognostic value for patients in whom the Ph occurs as a primary event, but not in those in whom it occurs as a secondary one.

Emergence of chronic myelogenous leukemia during treatment for essential thrombocythemia

A 72-year-old male patient was initially diagnosed with essential thrombocythemia (ET), a Philadelphia chromosome-negative (Ph1(-)) chronic myeloproliferative disorder (CMPD), and was treated with hydroxyurea (HU). After 9 years of diagnosis of ET, his peripheral leukocytes gradually increased, while his platelet count showed a decrease. Bone marrow analysis disclosed Ph-positive chronic myelogenous leukemia (CML) in the chronic phase. Administration of imatinib mesylate (IM), a Bcr-Abl tyrosine kinase inhibitor (TKI), induced complete hematologic response in a month, but was discontinued after 4 months because of Grade 3 pleural effusion (PE). The treatment was switched to nilotinib which successfully induced a complete cytogenetic response (CCyR) after 5 months of TKI therapy and resolved the PE. Despite CCyR, however, ET recurred. Since then, the patient has been treated for 8 months with a combination of nilotinib and HU which has successfully controlled both CML and ET. This report includes a review of the characteristics of 15 reported cases with co-occurrence of CML and Bcr-Abl-negative CMPDs, including ours. Although rare, care needs to be taken since, despite the often similar clinical features of the two diseases, they require completely different treatments.

Influence of complex variant chromosomal translocations in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

Cytogenetic variants of the Philadelphia (Ph) chromosome can be observed in 5–8% of patients diagnosed with Chronic Myelogenous Leukemia (CML), and usually involve at least one chromosome other than 9 and 22. Despite the genetically heterogeneous nature of these alterations, available data indicate that CML patients displaying complex variant translocations (CVTs) do not exhibit a less favorable outcome as compared to individuals presenting conventional Ph-positive CML. Patients and methods. We report our experience with 10 CML patients carrying CVTs among 153 newly diagnosed cases followed at our Institution. Results and discussion. Unlike previously published reports, in our series only two CML patients exhibiting CVTs achieved an optimal response to tyrosine kinase inhibitors (TKI) treatment. The remaining eight patients obtained either a suboptimal response or failed drug therapy. Our data suggest that the presence of CVTs at diagnosis might confer an unfavorable clinical outcome, as these genetic alterations might be markers of genomic instability and indicate a higher likelihood of disease progression.

Loss of Heterozygosity (LOH) for HumACTBP2 (SE33) Is Detected in Philadelphia (Ph)-Positive and Ph-Negative Hematopoietic Cells of Some Patients with Ph-Positive CML.

Introduction: In the majority of patients with Philadelphia (Ph)-positive chronic myelogenous leukemia (CML), the non-leukemic hematopoiesis is considered polyclonal. Occasionally, clonal cytogenetic abnormalities have been reported in Ph-negative cells from patients in cytogenetic remission after IFN-alpha or imatinib. In the present project we determined clonality of flow-sorted hematopoietic cells from untreated and treated patients with Ph-positive CML in first chronic phase by analyzing the two alleles of short tandem repeats (STR) of given loci. The loss of one of the two alleles of STR identifies loss of heterogzygosity (LOH) and clonal expansion. These results were compared to conventional cytogenetics and correlated with response to therapy.Patients and methods: A total of 25 consecutive patients (13 m/12 f, median age 58 y) treated at the University of Leipzig from April to June 2005 were included. Conventional cytogenetics were analyzed by R-banding. FISH using the LSI BCR/ABL ES probe in unsorted as well as in flow-sorted monocytes (CD14+), granulocytes (CD14-/CD33+), T- lymphocytes (CD3+), B-lymphocytes (CD19+), and CD34+-cells was performed. For LOH analysis, DNA was extracted from flow-sorted cells. 8 autosomal loci were analysed: D3S1358 (3p), vWA (12p12-pter), D8S1179 (8q), D21S11 (21q11.2-q21), D18S51 (18q21.3), TH01 (11p15.5), FGA (4q28), SE33 (6q15). PCR products were detected by capillary electrophoresis. Loss of, or a strong decrease in the signal intensity of one allele was considered as evidence for LOH. An allelic imbalance factor (AIF) was calculated by the quotient of the peak ratios from constitutional and flow-sorted cell DNA (N1:N2/T1:T2). An AIF > 1.8 was considered as LOH. Buccal swabs were used as controls.Results: Median time from diagnosis to analysis was 60 months. Of the 25 patients, twenty (80%) received Imatinib for a median of 26 months, three (12%) were newly diagnosed and two (8%) were treated with IFN-alpha. Both patients with IFN-alpha as well as 8 (40%) patients treated with Imatinib had sustained complete cytogenetic remission (CCR). Furthermore, 8 (40%) responded to Imatinib with hematological remission (HR) only, 3 (15%) patients with major CR (MCR) and one patient (5%) with minor CR. Conventional cytogenetics revealed additional abnormalities [t(7;17), del (20q), trisomy 8, and -Y] in Ph- negative cells in 4 (16%) patients. No LOH was detected in these patients. Ph-positive B-cells were detected in 4 (16%) with HR. Interestingly, the median quotient of Ph-positive CD34+ and Ph-positive unsorted cells was 0.65. LOH for SE33 in CD34+ cells, monocytes and granulocytes was detected in 6 (24%) patients [3 in CCR (2 patients treated with IFN-alpha and 1 with imatinib)], 1 patient in MCR under imatinib, and 2 patients with newly diagnosed CML. LOH was not detected in buccal swabs.Conclusions: We confirm the occurrence of clonal cytogenetic abnormalities in Ph-negative cells. LOH did not parallel abnormal cytogenetics and LOH for SE33 only was detected in patients with Ph-negative and Ph-positive cytogenetics. The detection of LOH which could be a sign of inactivation of tumor suppressor genes in treated and untreated patients with CML argues against a treatment-induced event and raises speculations on the role of this finding in the pathogenesis of at least some cases of CML.

Transgenic Mouse Models of BCR/ABL-Positive Chronic Myelogenous Leukemia: A Review

There are three methods for making chronic myelogenous leukemia (CML) mouse models: xenotransplantation of primary Ph-positive CML cells into immunodeficient mice, BCR/ABL-expressing transgenic mice, and BCR/ABL retroviral bone marrow transduction and transplantation. These animal models have provided us with important new insights into the molecular pathophysiology of CML and answered directly many questions regarding this disease. To date, The model mice using the BCR/ABL retroviral bone marrow transduction and transplantation method has provided the most valuable knowledge compared with the other two methods. After a long history of failure, however, recent studies have reported successful establishment of making the CMLphenotype in transgenic mice. The transgenic CML mouse model now appears to be more accurate, informative and advantageous model for studying CML. This article reviews the history of BCR/ABL-expressing CML mouse models and the recent findings in the transgenic mice with the CML-phenotype. Keywords: chronic myelogenous, cml-phenotype, bcr, abl

Gene Expression Profiling of Philadelphia Chromosome(Ph) Negative CD34+ Hematopoietic Stem and Progenitor Cells of Patients with Ph Positive CML in Complete Molecular Remission during Therapy with Imatinib.

Treatment of chronic myelogenous leukemia (CML) with Imatinib has shown its efficacy and superiority to conventional therapies. Beside inhibition of BCR-ABL Imatinib also has inhibitory effects on other tyrosine kinases such as wild-type ABL, c-KIT and platelet-derived growth factor receptors (PDGF-R) alpha and beta which play a role in differentiation and proliferation of hematopoietic stem cells and are involved in DNA repair mechanisms. Inhibition of these genes might therefore result in functional disturbance of hematopoiesis or even in secondary karyotypic abnormalities. Therefore, we compared gene expression profiles of immunomagnetically isolated CD34+ bone marrow (BM) cells from six healthy volunteers with Ph negative CD34+ cells from BM of eight patients (6 males, 2 females; median age: 52 years, range: 25–68 years) with Ph positive CML in chronic phase who reached major molecular remission during imatinib therapy (400 mg/day; 28 months (range: 11–39 months)). Cytogenetics, FISH analysis and quantitative real-time RT-PCR for BCR-ABL transcripts showed that all patients were in complete cytogenetic and in major molecular remission ( ≥ 3 log reduction of the BCR-ABL/G6PDH ratio in comparison to pretreatment value). CD34+ cells (median: 5 x 105; range 2 x 105–1.8 x 106) were isolated immunomagnetically from bone marrow with a purity >98%. Total RNA (median: 550 ng; range: 130 – 990 ng) from isolated CD34+ cells was used to generate biotin-labelled cRNA (median: 7.5 μg; range: 2.6–12.3 μg). Labeled cRNA was hybridized to Affymetrix HG-Focus GeneChips covering 8793 genes representing a broad spectrum of different functional groups. For normalization and data analysis we used the statistical scripting language “R”. Raw data were normalized using a method of variance stabilizing transformations (VSN). To compare the normalized data from CD34+ cells of imatinib-treated patients and healthy volunteers we used the Significance Analysis of Microarrays (SAM) algorithm v1.21. We found that CD34+ cells of patients after reaching major molecular remission during first-line treatment with 400 mg imatinib per day showed no significantly differentially expressed genes in comparison to CD34+ cells of healthy volunteers in vivo. There was no evidence for transcriptional changes of genes involved in DNA repair or oncogenesis supporting the view that secondary chromosomal aberrations in Ph negative hematopoiesis observed during imatinib therapy might not be induced by the tyrosine kinase inhibitor itself. Our data suggest that imatinib as first-line therapy might not result in a major functional disturbance of Ph negative hematopoiesis.

Long-term results of imatinib mesylate therapy in philadelphia chromosome (Ph) positive chronic phase chronic myelogenous leukemia (CML) post interferon-a (IFN) failure M.D. Anderson experience in 261 patients

6622 Background: The initial results of imatinib in Ph-positive CML in chronic phase post IFN failure were positive with a median follow up times of 17 months. The major cytogenetic (CG) response rate (Ph 5% (p<0.05) or prior hematologic resistance to IFN (p=0.01). Achieving any CG response at 3 months was associated with a 4-year survival rate of 95% (versus 78% for others; p=0.0001); the 4-year FFP rates were 94% versus 64%, respectively (p<0.0001). Achieving a major CG response at 6 months was associated with a 4-year survival rate of 95% (versus 81% for others; p=0.001); the 4-yr FFP rates were 94% versus 68% (p<0.0001). Among 167 patients achieving CG CR, 152 pts had quantitative PCR (QPCR) studies: 82 pts (49%; 31% total) had a major molecular response (QPCR= BCR-ABL/ABL <0.05%) and 38 pts (15%) had a complete molecular response (BCR-ABL undetectable). The 3-yr cumulative major molecular response rate was 55%, and complete molecular response rate 22%. Compared with a historical group of 131 similar patients in CML chronic phase post IFN failure treated with non-imatinib therapies, imatinib was associated with better 4-year survival rates (88%, versus 35% p<0.0001); this survival advantage for imatinib was confirmed by multivariate analysis (p<0.0001). Conclusions: The results with imatinib in CML chronic phase post IFN failure are improving and durable. Major and complete responses are now noted in a substantial proportion of patients with longer follow-up time. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis; Supergen

Long-term results of imatinib mesylate therapy in philadelphia chromosome (Ph) positive chronic phase chronic myelogenous leukemia (CML) post interferon-a (IFN) failure M.D. Anderson experience in 261 patients

6622 Background: The initial results of imatinib in Ph-positive CML in chronic phase post IFN failure were positive with a median follow up times of 17 months. The major cytogenetic (CG) response rate (Ph 5% (p<0.05) or prior hematologic resistance to IFN (p=0.01). Achieving any CG response at 3 months was associated with a 4-year survival rate of 95% (versus 78% for others; p=0.0001); the 4-year FFP rates were 94% versus 64%, respectively (p<0.0001). Achieving a major CG response at 6 months was associated with a 4-year survival rate of 95% (versus 81% for others; p=0.001); the 4-yr FFP rates were 94% versus 68% (p<0.0001). Among 167 patients achieving CG CR, 152 pts had quantitative PCR (QPCR) studies: 82 pts (49%; 31% total) had a major molecular response (QPCR= BCR-ABL/ABL <0.05%) and 38 pts (15%) had a complete molecular response (BCR-ABL undetectable). The 3-yr cumulative major molecular response rate was 55%, and complete molecular response rate 22%. Compared with a historical group of 131 similar patients in CML chronic phase post IFN failure treated with non-imatinib therapies, imatinib was associated with better 4-year survival rates (88%, versus 35% p<0.0001); this survival advantage for imatinib was confirmed by multivariate analysis (p<0.0001). Conclusions: The results with imatinib in CML chronic phase post IFN failure are improving and durable. Major and complete responses are now noted in a substantial proportion of patients with longer follow-up time. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis; Supergen

Effects of age on prognosis with imatinib mesylate therapy for patients with Philadelphia chromosome-positive chronic myelogenous leukemia.

Older age is a consistent poor prognostic factor in patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). Whether this is related to an intrinsic worse disease biology or to inadequate drug delivery or excessive treatment-associated toxicity is unknown. The availability of imatinib mesylate, a selective, Bcr-Abl-targeted therapy that is administered orally with minimal side effects, may clarify whether older age would remain an adverse factor (thus, implying a different age-related CML biology). Seven hundred forty-seven patients in different phases of Ph-positive CML who were treated with imatinib from 1999 until the time of last follow-up were evaluated. Among them, 187 patients had newly diagnosed, early chronic phase CML; 351 patients had chronic phase CML after interferon alpha (IFN) failure; 133 patients had accelerated phase CML; and 76 patients had blastic phase CML. The imatinib daily dose varied from 400 mg to 800 mg orally, according to the protocol design. Patients were categorized into a group of older patients (age 60 years or older) or younger patients (age younger than 60 years). Their characteristics, responses to therapy, and survival were compared by univariate and multivariate analyses. One hundred eighty-seven patients had newly diagnosed CML, and 49 patients (26%) were in the older age group. Older patients had similar cytogenetic response rates and survival compared with younger patients. Among 351 patients with late chronic phase CML after IFN failure, 120 patients (34%) were in the older age group. Although the older patients had a lower incidence of achievement of complete cytogenetic response (Ph, 0%) by univariate analysis (56% vs. 44%; P = 0.05), age was not found to be an independent poor prognostic factor in the multivariate analysis. Similarly, older age was not an adverse poor prognostic factor for survival. Forty-two of 133 patients (32%) with accelerated phase CML were older. The incidence of any cytogenetic response was lower in older patients (53% vs. 33%; P = 0.04), but age was not significant in the multivariate analysis. Older patients also had a trend toward worse survival (P = 0.09) that was not significant in the multivariate analysis. Twenty-eight of 76 patients (37%) evaluated in blastic phase were older. Older age was not a significant prognostic factor either for achieving response or for survival. With imatinib therapy, older age appears to have lost much of its prognostic relevance. This suggests that the previous poor prognosis observed with older age was related to treatment-associated factors (e.g., toxicity with allogeneic transplantation or with IFN therapy) rather than to an intrinsic, different disease biology of CML in older patients.

Anagrelide and imatinib mesylate combination therapy in patients with chronic myeloproliferative disorders.

The tyrosine kinase inhibitor imatinib mesylate inhibits the function of the Bcr-Abl oncoprotein associated with Philadelphia-positive chronic myelogenous leukemia (CML). Anagrelide suppresses megakaryocyte proliferation and differentiation. The objectives of this study were to investigate the feasibility and safety of imatinib mesylate and anagrelide combination therapy in patients with Ph-positive CML or chronic myeloproliferative disorders (MPD) with persistent thrombocythemia. This study was a retrospective review of all available records of patients with chronic MPD presenting to the M.D. Anderson Cancer Center between October 1998 and May 2002, treated with imatinib mesylate combined with anagrelide. Of 22 patients identified, 18 had Ph-positive CML (chronic phase, 16 patients; accelerated phase, 2 patients), 1 had agnogenic myeloid metaplasia (AMM), 2 had essential thrombocythemia (ET) and 1 had MPD transformed into refractory anemia with excess blasts (RAEB). The median age was 57 years (range 26-82 years). The median dose of imatinib mesylate administered was 400 mg (range 300-800 mg) and the median dose of anagrelide was 1.5 mg (range 0.5-4.0 mg). Imatinib mesylate and anagrelide combination therapy was feasible and tolerable. Of the 18 patients with Ph-positive CML, 15 in chronic phase and 1 in accelerated phase achieved a complete hematologic response (CHR), and 9 of the 18 achieved cytogenetic response (complete in 8 patients). No responses were noted in patients with AMM, ET or MPD transformed into RAEB. The combination of imatinib mesylate and anagrelide was safe and was associated with an 89% CHR rate in patients with CML in chronic phase and persistent thrombocythemia.

Prediction of initial cytogenetic response for subsequent major and complete cytogenetic response to imatinib mesylate therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia.

Obtaining a major (Philadelphia chromosome [Ph] of < 35%) or a complete cytogenetic response (Ph of 0%) has been associated with excellent long-term survival. Cytogenetic response may continue to improve with therapy. Because early allogeneic stem cell transplantation (SCT) may be associated with a better outcome, early parameters predicting for subsequent cytogenetic responses would optimize the treatment decision-making. The current study was performed to analyze whether early cytogenetic responses may be predictive of later major or complete cytogenetic responses to imatinib mesylate therapy in patients with Ph-positive chronic myelogenous leukemia (CML). Two hundred sixty-one patients with Ph-positive, chronic-phase CML after failure with interferon therapy who were treated with imatinib mesylate therapy were analyzed. A persistence of 100% Ph-positive cells after > or = 6 months of imatinib mesylate therapy was associated with a major cytogenetic response rate of 9-13% and a complete cytogenetic response rate of 0-4%. However, a minor cytogenetic response after 3-12 months of therapy still was associated with high rates of major (68-83%) or complete (35-54%) cytogenetic response rates. Patients with Ph-positive, chronic-phase CML who have persistent 100% Ph-positive disease after > or = 6 months of imatinib mesylate therapy may be offered allogeneic SCT or considered for alternative investigational therapies.

Involvement of protein phosphatase 2A in interferon-α-2b-induced apoptosis in K562 human chronic myelogenous leukaemia cells

Interferon-alpha (IFN-α)-2b is known to have antiproliferative effects on hematological malignant cells, especially chronic myelogenous leukaemia (CML). However, it can induce cytogenetical remissions in a very small percentage of the patients. Also during interferon therapy, resistance can emerge in the CML clones. K562 is an in vitro model cell line transformed from a Ph positive CML patient. It can be induced to differentiate to granulocytic and/or monocytic lineages with certain molecules. IFN-α-2b generally exerts its effects on CML cells by Janus family kinases (Jak/Stat) pathway, mostly through tyrosine kinase system. However, there is almost no data on the relevance of serine/threonine (Ser/Thr) protein phosphatase (PP) system in the interferon induced signal transduction pathways. In this study, we investigated serine/threonine protein phosphatases in the IFN-α-2b induced K562 cytotoxicity. Trypan blue dye exclusion test and MTT assay were utilised for determining cytotoxicity. IC 50 of IFN-α-2b on K562 cells was found to be 600 IU/ml. However, no differentiation was determined by analysis of cell surface antigen expressions. Serine/threonine protein phosphatase inhibitors calyculin A (Cal A) and okadaic acid (OKA) augmented the IFN-α-2b induced cytotoxicity. Apoptosis assay by the mono-oligonucleosome detection and acridine orange/propidium iodide dye revealed marked apoptosis underlying cytotoxicity. Phosphatase enzyme assay revealed a gradual increase in protein phosphatase 2A (PP2A) activity during interferon induced cytotoxicity. Conversely, immunoblots showed no change in the expression of PP2A catalytic and regulatory subunits. In conclusion, PP2A plays a role in IFN-α-2b induced apoptosis of K562 cells and should be investigated as a new window furthermore.

Philadelphia-positive chronic myelogenous leukemia with a 5q− abnormality in a patient following interferon-alpha therapy

In this report, we describe a rare 5q−/CML association in a patient with Ph-positive chronic myelogenous leukemia (CML) who achieved complete cytogenetic response on interferon-alpha (IFN-α) treatment, but who developed a new clone in the blastic crisis. The patient was treated with interferon-alpha beginning in 1996 and a serial chromosome and molecular study was performed over the clinical course of the disease. The patient remained in complete hematologic and cytogenetic remission until November 1998, when a reverse transcriptase PCR study performed on the bone marrow and peripheral blood cells was negative for chimeric BCR/ABL mRNA. The treatment was discontinued until April 1999, when the patient developed acute transformation of the disease. In June 1999, cytogenetic examination showed the development of a new clone, consisting of the deletion of the long arm of chromosome 5 in addition to the standard Ph translocation. The unusual association of a Ph with an abnormality usually observed in a secondary myeloproliferative disease raises the question of whether the new finding is treatment-induced or part of the disease process and casually related to the acute transformation.

BCR rearrangement-negative chronic myelogenous leukemia revisited.

To document the characteristics of patients with major breakpoint cluster region (M-bcr) rearrangement-negative chronic myelogenous leukemia (CML). The hematopathologist, who was blinded to patients' molecular status, reviewed the referral bone marrows and peripheral-blood smears from 26 patients with Philadelphia (Ph) translocation-negative CML who lacked Bcr rearrangement (and other evidence of a Bcr-Abl anomaly) and 14 patients (controls) with chronic-phase Ph-positive CML. Clinical data was ascertained by chart review. Among the 26 M-bcr rearrangement-negative CML patients, three pathologic subtypes emerged: (1) patients indistinguishable from classic CML (n = 9), (2) patients with atypical CML (n = 8), and (3) patients with chronic neutrophilic leukemia (n = 9). Among the 14 patients with Ph-positive CML who were included in the blinded review, 13 were classified as classic CML, and one was classified as atypical CML. The only statistically significant difference between M-bcr rearrangement-negative subgroups was in the proportion of patients having karyotypic abnormalities, an observation common only in patients with atypical CML (P = 0.008). However, the small number of patients in each subgroup limited our ability to differentiate between them. Interferon alfa induced complete hematologic remission in five of 14 patients; four of these remissions lasted more than 5 years. Only one of 26 patients developed blast crisis. The median survival of the 26 patients was 37 months. Patients with M-bcr rearrangement-negative CML fall into three morphologic subgroups. Disease evolution does not generally involve blastic transformation. Instead, patients show progressive organomegaly, leukocytosis, anemia, and thrombocytosis. Some patients in each subgroup can respond to interferon alfa.

Donor lymphocyte infusion followed by interferon-α plus low dose cyclosporine A for modulation of donor CD3 cells activity with monitoring of minimal residual disease and cellular chimerism in a patient with first hematologic relapse of chronic myelogenous leukemia after allogeneic bone marrow transplantation

A 15-year-old girl with Ph-positive chronic myelogenous leukemia in first chronic phase received bone marrow from her human leukocyte antigen matched brother. Twenty three months after bone marrow transplantation hematological relapse occured which was treated with two infusions of donor lymphocytes (DLI) (0.5×10 8 CD3/kg b.w./infusion). To enforce the graft-versus-leukemia effect (GvL), the first DLI was followed by administration of interferon-α (INF-α) 6×10 6 U/day for 30 days, whereas, after the second infusion INF-α was given at the same dose until hematological remission was achieved (80 doses). Minimal residual disease (MRD) was detected by conventional cytogenetics (Ph chromosome), fluorescence in situ hybridization (FISH) cytogenetics (BCR/ABL translocation) and reverse transcriptase-polymerase chain reaction (RT-PCR) Ecotropic virus integration site-1 (EVI-1 gene expression), whereas cellular chimerism was monitored by assessment of microsatellite markers PCR and Y-chromosomal DNA content FISH. When hematological remission was achieved the pancytopenia was observed and the cytogenetic and molecular investigations revealed only partial remission and mixed chimerism, however, with predominance of donor origin hematopoiesis. To diminish the myelosupressive effect of donor CD3 cells without switching-off the GvL effect, a low dose of cyclosporine A was given. Further observation revealed significant improvement of hematopoiesis with parallel gradual decline of MRD and increase of donor hematopoiesis up to complete chimerism. Graft-versus-host disease was not observed at any stage of the treatment.

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.