M-BCR breakpoint location does not predict survival in Philadelphia chromosome-positive chronic myeloid leukemia.

BACKGROUND. Some reports in the recent literature have shown that the site of molecular rearrangement within the M-BCR area may have a prognostic value in Ph1 + CML patients. A number of studies have, however, failed to demonstrate this finding. Here we report the molecular rearrangements of 107 patients and their clinical follow-up. METHODS. Localization of the breakpoints was determined according to conventional criteria, after digestion with 2 to 4 restriction endonucleases and hybridization with 1 or 2 molecular probes. RESULTS. Sixty two patients were rearranged in the 5' area and 45 in the 3' area: there was no difference between the survival curves of the two groups, at least not after 3 years of follow-up. CONCLUSIONS. The site of the breakpoint within the M-BCR has no prognostic significance in our series.     

Clonal Ph-negative hematopoiesis with trisomy 8 in chronic myeloid leukemia during imatinib therapy

We report a case of chronic myeloid leukemia (CML) with clonal Philadelphia negative hematopoiesis arising after treatment with imatinib. A 45-year-old man was diagnosed as having Ph-positive CML. Therapy with MCNU and hydroxyurea was carried out for 12 days, and was then replaced by imatinib. A major cytogenetic response was obtained after 13 months of therapy with imatinib, but at the same time a new Ph-negative clone with trisomy 8 appeared in the bone marrow. Blood cell counts were still within normal limits. This observation indicates that patients on imatinib should be followed with bone marrow morphologic finding and routine cytogenetic testing, even after induction of a good response.     

Autografting with Ph-negative progenitors in patients at diagnosis of chronic myeloid leukemia induces a prolonged prevalence of Ph-negative hemopoiesis

OBJECTIVE: In many patients with chronic myeloid leukemia (CML), a residual population of primitive normal (Ph-negative) progenitors persists despite the marked expansion of the leukemic (Ph-positive) clone. These cells may be found in the blood of patients studied soon after diagnosis or during the period of endogenous hematopoietic recovery that follows myeloreductive therapy. Based on those observations, we have developed a clinical protocol that allows collection of Ph-negative peripheral blood progenitor cells (PBPC) with transplantable hematopoietic regenerative potential. The aim of this study is to examine changes that occur in the percentage of Ph-negative- and Ph-positive-committed progenitor cells and to determine the relationship between changes and clinical outcome. MATERIALS AND METHODS: We followed 15 patients with CML, mobilized and autografted soon after diagnosis with 85%-100% Ph-negative PBPC for a median time of 28 months (range 18-50) after transplant. At 6 months, 1 year, 2 years, and last follow-up, cytogenetic analyses were performed on fresh bone marrow cells and on colony-forming cells (CFC). RESULTS: Autologous transplantation induces a reduction in the proportion of Ph-positive CFC, from 70%-100% to 0%-25% in the majority of patients (78%). After autografting, 8 of 15 patients achieved a long-lasting cytogenetic remission (median, 24 months; range, 21-43) with a Ph-positivity ranging between 0% and 20% at the level of mature mononuclear cells and colony-forming cells (CFC). In some patients, the majority of CFC remained Ph-negative, whereas the majority of the mature cells were Ph-positive. Other patients (5/15) developed cytogenetic relapse (100% Ph-positive), although they were in hematological remission. We found that detection of Ph-positive long-term-culture initiating cells (LTC-IC) in the marrow at diagnosis was the only factor significantly associated with recurrence of the disease (p < 0.01); on the other hand, the number of Ph-negative LTC-IC infused showed a significant correlation with a better outcome (p < 0.03). CONCLUSION: We have shown that a prolonged period of complete or almost complete Ph-negative hemopoiesis can be achieved in patients with CML who undergo autografting with Ph-negative progenitors. Longer follow-up study will be needed to assess whether these changes are associated with improved survival.     

ABL1 methylation is a distinct molecular event associated with clonal evolution of chronic myeloid leukemia.

Methylation of the proximal promoter of the ABL1 oncogene is a common epigenetic alteration associated with clinical progression of chronic myeloid leukemia (CML). In this study we queried whether both the Ph'-associated and normal ABL1 alleles undergo methylation; what may be the proportion of hematopoietic progenitors bearing methylated ABL1 promoters in chronic versus acute phase disease; whether methylation affects the promoter uniformly or in patches with discrete clinical relevance; and, finally, whether methylation of ABL1 reflects a generalized process or is gene-specific. To address these issues, we adapted the techniques of methylation-specific PCR and bisulfite-sequencing to study the regulatory regions of ABL1 and other genes with a role in DNA repair or genotoxic stress response. In cell lines established from CML blast crisis, which only carry a single ABL1 allele nested within the BCR-ABL fusion gene, ABL1 promoters were universally methylated. By contrast, in clinical samples from patients at advanced stages of disease, both methylated and unmethylated promoter alleles were detectable. To distinguish between allele-specific methylation and a mixed cell population pattern, we studied the methylation status of ABL1 in colonies derived from single hematopoietic progenitors. Our results showed that both methylated and unmethylated promoter alleles coexisted in the same colony. Furthermore, ABL1 methylation was noted in the vast majority of colonies from blast crisis, but not chronic-phase CML. Both cell lines and clinical samples from acute-phase CML showed nearly uniform hypermethylation along the promoter region. Finally, we showed that ABL1 methylation does not reflect a generalized process and may be unique among DNA repair/genotoxic stress response genes. Our data suggest that specific methylation of the Ph'-associated ABL1 allele accompanies clonal evolution in CML.     

Management of chronic myeloid leukemia: targets for molecular therapy

Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by malignant expansion of bone marrow stem cells. Currently, the only unequivocally curative treatment for CML is allogeneic stem cell transplant. Unfortunately, a large proportion of CML patients are ineligible for such treatment and alternative forms of therapy must be employed. Conventional chemotherapy makes use of compounds, such as hydroxyurea, that are cytotoxic for actively dividing cells. Although effective, these agents are not selective for the leukemic clone and this is the cause of undesirable side effects. Moreover, as the disease progresses patients frequently become refractory to chemotherapy. In recent years, knowledge of the molecular pathology of CML has allowed the development of drugs that selectively target the malignant cells: imatinib mesylate is the most prominent example. These agents are selective because they target molecular determinants that are exclusively deregulated in the leukemic cells. In this review we consider some of the novel developments in this field. Particular emphasis is given to chemical agents that target the Bcr-Abl oncoprotein. The latter affords an excellent opportunity for therapy since CML, in contrast to many other human malignancies, is likely caused by the activity of a single oncoprotein. Furthermore, it is believed that Bcr-Abl continues to play a central role throughout the course of the disease.     

Molecular analysis of interferon-induced suppression of Philadelphia chromosome in patients with chronic myeloid leukemia

Treatment with recombinant human interferon alpha-A (Roferon-A) is associated with stable suppression of the population of cells that display the Philadelphia (Ph1) chromosome in some patients with chronic myelogenous leukemia (CML) as defined by cytogenetic analysis. Southern blot analyses employing a 3' breakpoint cluster region (bcr) probe (Pr- 1) were performed to confirm a complete suppression of the Ph1+ chromosome-positive clone of cells at the DNA level. The complete disappearance of rearranged restriction fragments of the bcr gene, which were a characteristic of the disease prior to Roferon-A therapy, was accompanied by the restoration of normal bone marrow and achievement of durable ongoing complete remission for 9 and 6 months, respectively, in two patients with Philadelphia-positive (Ph1+) CML. Molecular analysis is a valuable probe for monitoring the clinical course of disease in patients with Ph1+ CML.     

Collection of Ph-negative progenitor cells from interferon responsive patients with chronic myeloid leukemia: effect of granulocyte-colony-stimulating factor mobilization

BACKGROUND AND OBJECTIVE: The observation that patients with chronic myeloid leukemia (CML) may relapse following stem cell transplantation because of Philadelphia positive cells contaminating the graft have led to a variety of strategies to reduce this contamination. This study investigate the feasibility of collective, Ph-re cells from patients with CML in chronic phase. DESIGN AND METHODS: A total of 18 patients with chronic myeloid leukemia in chronic phase who had responded to varying degrees to treatment with interferon-a (IFN) were subjected to mobilization with granulocyte colony-stimulating factors and peripheral blood progenitor cell collection. Nine patients were in complete cytogenetic remission (CCR) and nine were partial responders. IFN was stopped 2 to 4 weeks before the procedure. G-CSF was given by subcutaneous injection once daily at a dose of 10 microg/kg. RESULTS: Five patients underwent one collection procedure only, 10 underwent two procedures and 3 patients had three collections. The median number of nucleated cells (NC) per patient collected was 10.2 x 10(8)/kg (4.4-19.7) and the median number of CD34(+) cells was 2.5 x 10(6)/kg (0.4-9.4). Analyzable cytogenetic data were available for 26/34 (76%) leukapheresis procedures. The median percentage of Ph- negative metaphases for patients in CCR was 100% (73-100). Patients not in CCR had a higher level of Ph-positive cells in their collections (median 23%, range 0-79%, p=0.01). Of the nine patients in CCR, 8 had at least one apheresis from which progenitor cells were 100% Ph-negative; conversely, patients not in CCR had detectable Ph-positive cells in every collection. Four patients have undergone autologous stem cell transplantation. INTERPRETATION AND CONCLUSIONS: It was possible to collect sufficient Ph negative progenitor cells from patients in CCR but collections from other patients contained significant numbers of Ph-positive cells.     

Further evidence for the molecular heterogeneity of chronic myeloid leukemia

Summary Cytogenetic and molecular techniques were performed on samples obtained from 29 patients with chronic myelocytic leukemia (CML); 27 were in the chronic phase and two were in blast crisis. A further five cases were also analyzed, two with atypical CML (aCML), one with chronic neutrophilic leukemia (CNL), and two with juvenile CML (JCML). Most of the cases with typical CML were Philadelphia chromosome (Ph) positive and had a rearrangement within the major breakpoint cluster region (M-bcr). One of these cases was shown to be Ph positive but showed no rearrangement within the M-bcr. Two cases with clinical features typical of CML were Ph negative. One of these showed a rearrangement within the M-bcr, but no rearrangement was demonstrated in the other. Both patients in blast crisis were Ph positive and M-bcr positive. One showed a second Ph. Patients with aCML were Ph negative and had no M-bcr rearrangement. A polymorphism within the M-bcr was found withBglII in one case. No Ph chromosome or M-bcr rearrangement was found in CNL or JCML. These data support the molecular heterogeneity reported in CML.