Monitoring of remission status by fluorescence in situ hybridisation in chronic myeloid leukaemia patients treated with interferon-alpha

Interferon-alpha (IFN-alpha) can be considered as treatment of choice for patients with chronic myeloid leukaemia (CML) in chronic phase. With this treatment major cytogenetic responses can be achieved in 30% to 50% of patients. Regular monitoring of cytogenetic response is essential for the therapeutic management of these patients. As conventional cytogenetics is not always successful, especially under IFN-alpha treatment, molecular cytogenetic methods have been established for the examination of interphase nuclei for the presence of the BCR-ABL fusion gene, the molecular counterpart of the Philadelphia chromosome. To demonstrate the value of these new methods we have analysed interphase nuclei from sequentially cultured bone marrow cells from 14 CML patients who were treated with IFN-alpha and whose bone marrow was investigated regularly during therapy. Dual-colour FISH with a breakpoint spanning BCR-YAC and a flanking cosmid from the ABL region was applied. When compared with conventional cytogenetics the results achieved by FISH were favourable. The most evident advantage of FISH analysis is that in case of failure of conventional cytogenetics a reliable determination of the remission status can be done. Together with other recent studies our results illustrate the advantages and limitations of the interphase FISH method for monitoring CML patients.     

BCR-ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 x 10(9)/l. P210BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p210BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.     

The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells

The Philadelphia chromosome found in virtually all cases of chronic myeloid leukemia (CML) and in about one third of the cases of adult acute lymphoblastic leukemia is formed by a reciprocal translocation between chromosomes 9 and 22 that results in the fusion of BCR and ABL genetic sequences. This BCR-ABL hybrid gene codes for a fusion protein with deregulated tyrosine kinase activity that can apparently cause malignant transformation. CGP57148B, a 2-phenylaminopyrimidine derivative, has been shown to selectively inhibit the tyrosine kinase of ABL and BCR-ABL. We report here that this compound selectively suppresses the growth of colony-forming unit-granulocyte/macrophage (CFU-GM) and burst-forming unit-erythroid derived from CML over a 2-logarithmic dose range with a maximal differential effect at 1.0 micromol/L. However, almost all CML colonies that grow in the presence of 1.0 micromol/L CGP57148B are BCR-ABL-positive, which may reflect the fact that residual normal clonogenic myeloid precursors are infrequent in most patients with CML. We also studied the effects of CGP57148B on hematopoietic cell lines. Proliferation was suppressed in most of the BCR-ABL-positive lines; all five BCR-ABL-negative lines were unaffected. We conclude that this new agent may have significant therapeutic applications.     

Retroviral vector-mediated transfer of the interferon-alpha gene in chronic myeloid leukemia cells

The transfer and expression of cytokine genes into tumor cells is reportedly a valuable approach to improve the antitumor activity of cytokines in various models. Interferon (IFN)-alpha may induce hematological remission in chronic myeloid leukemia (CML) patients, but only a small proportion of patients achieve a sustained, complete cytogenetic remission. We have investigated the possibility of transducing CML cells with the retroviral vector LIalpha2SN, which encodes the IFN-alpha2 gene. We first optimized the transduction efficiency using the CML-derived K562 cell line. A transduction efficiency of 50% and 85% after three and six infections, respectively, was obtained in K562 cells. We then expressed IFN-alpha2 in CML cells by transducing the latter with LIalpha2SN viral particles. The IFN-alpha secretion after three and six infections was 5,400 and 18,000 U/24 hours/10(6) cells for unselected K562 cells and 7,000 and 290 U/24 hours/10(6) cells for CML CD34+ cells at days 4 and 5. Moreover, the major histocompatibility complex class I antigens were overexpressed after infection with LIalpha2SN in both K562 and CML CD34+ cells. The proliferation (in liquid culture) and the cloning efficiency of these CML cells were significantly decreased after LIalpha2SN treatment. By contrast, the proliferation of cord blood CD34+ cells was not affected by transduction with LIalpha2SN. These results demonstrate the transduction efficiency of CML cells and suggest the possibility of CML cell immunotherapy with retroviral gene transfer of different cytokines such as IFN-alpha.     

Prevalence of antibodies to Rickettsia conorii Ricketsia africae, Rickettsia typhi and Coxiella burnetii in Mauritania

A 24-year-old male developed cytogenetic relapse of chronic myeloid leukemia (CML) four years after allogeneic BMT. After a year of treatment with IFN-alpha, he achieved a partial cytogenetic response. Treatment with donor leukocyte infusions (DLI) was given (total dose 1 x 10(8) T lymphocytes/kg). Two months later, he developed acute GVHD (skin and liver), that improved with CsA and methylprednisolone and resulted in cytogenetic remission with complete donor chimerism. One month later he developed rhinocerebral mucormycosis and was successfully treated with surgical debridement and liposomal amphotericin B (total dose 12 g). This is the first case of mucormycosis described after DLI.     

Inadequate measurement of urinary chloride

Fifteen years ago Talpaz and colleagues were the first to determine that natural interferon alpha (INF-alpha) induces hematologic remission in chronic phase patients with chronic myeloid leukemia (CML). Further research revealed that this agent, contrarily to conventional chemotherapy with busulfan or hydroxyurea, eliminates leukemic hematopoietic cells having Philadelphia chromosome (Ph1-positive) in about 20% of patients, leading to the phase of cytogenetic remission. Comparison of the efficiency of IFN-alpha with conventional chemotherapy was carried out in several randomized clinical trials. It was found, that IFN-alpha delays the occurrence of blastic phase of CML and prolongs patients life span. It does not create, however, a likely chance of full recovery. The results of the randomized trails, carried out in France, showed that the combination of IFN-alpha and cytarabine as compared with INF-alpha alone, increases the rate of major cytogenetic response and prolongs survival in the chronic phase of CML. IFN-alpha efficiency in acceleration and blastic transformation phases of CML has not been proven so far, although this drug may be of certain value in combination with hydroxyurea or other cytostatic agents. At present, it is more often considered that IFN-alpha should be a first line therapy in newly diagnosed CML in its chronic phase, if due to absence of appropriate donors or advanced age, allogenic bone marrow transplantation cannot be performed.     

Expression of the Evi-1 gene in haemopoietic cells of children with juvenile myelomonocytic leukaemia and normal donors

Activation of the Evi-1 gene was first described to be associated with the transformation of murine myeloid leukaemias and has previously been detected in cases of human acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML) in blast crises and in myelodysplastic syndromes. In this study we determined the frequency and the level of Evi-1 expression in juvenile myelomonocytic leukaemia (JMML) and in normal haemopoiesis. Using RT-PCR and Southern blot hybridization mRNA of Evi-1 could be detected in bone marrow (BM) and peripheral blood (PB) mononuclear cells (MNC) of normal donors. In JMML 12/20 patients examined expressed elevated levels of Evi-1 compared to normal controls. In these samples over-expression of the gene was correlated with a higher percentage of blasts (P = 0.02). Expression levels in BFU-E and CFU-GM derived colonies from BM of JMML patients were lower than those in the corresponding MNC samples. Analysis of CD34+ and CD34- cells demonstrated that Evi-1 is primarily expressed in the CD34+ cell population of both JMML and normal donors. These findings suggest that Evi-1 expression is linked to the early stages of haemopoiesis. Studies on the regulation of Evi-1 expression in CD34+ cells will elucidate its function in progenitor cells and clarify its possible role in the pathogenesis of JMML.     

Partial-pKD1 plasmids provide enhanced structural stability for heterologous protein production in Kluyveromyces lactis

We describe a patient with leukocytosis with all the stages of neutrophilic series, peripheral dominant myeloblast proliferation, marked dysplasia of myeloid and erythroid series, and extramedullary hematopoiesis of the lymph nodes. A cytogenetic study of the bone marrow cells showed normal karyotype, and molecular analysis of the leukemic cells showed negative for BCR-ABL by RT-PCR. After chemotherapy, the patient went into complete remission with a normal blood and bone marrow profile with no dysplasia. On relapse, the hematological findings showed a typical bone marrow dominant acute myeloid leukemia, with the leukemic cells having a chromosomal abnormality. The patient exhibited the combined features of myeloproliferative disorder, myelodysplastic syndrome, peripheral dominant myeloblast proliferation (so-called peripheral leukemia) and typical acute myeloid leukemia throughout the clinical course. This is thought to be a rare overlapping disease involving these distinct hematological conditions that do not usually occur in the same patient.     

Cytokines and malignant hemopathies: leukemias and bone marrow graft

Cytokines are now part of the modern armentarium utilized against malignant blood diseases. The essentially lymphocytic haematopoietic growth factors (G-CSF, GM-CSF, IL-3) reduce the infectious morbidity associated with the deep and prolonged neuropoenia induced by the myelo-ablative conditioning for autologous or allogeneic bone marrow transplantations, and widening their indications is tempting. The reluctance expressed about their use in chemotherapy of acute myeloid leukaemia is abating now that controlled studies have shown that they preserve the complete response rate and shorten the duration of leucopoenia. Moreover, they modify the leukaemia biological response, which makes it possible to increase the cytotoxicity of certain drugs and constitutes a new approach to drug-resistant leukaemias. Immuno-modulating cytokines (interferon alpha, interferon gamma, interleukin-2) act through mechanisms that are still ill-defined: antitumoral activity, modification of biological responses, immunoactivation. Nevertheless, interferon alpha has revolutionized the treatment of hairy cell leukaemia and myeloid leukaemia, with a 70% remission rate. The scarcity of complete responses (10% of hairy cell leukaemias) or cytogenetic responses (20% of chronic myeloid leukaemias) justifies a combined treatment (chemotherapy+immunotherapy?) to improve these patients' cure rate. The anti-leukaemic activity of interleukin-2, observed in patients with refractory relapses, produces 33% of responses, including 10% of complete responses, and it is tempting to test the impact of this immunotherapy on the control of residual leukaemia as adjuvant of complete remission using randomized trials.     

Expression of Fas antigen in acute myeloid leukaemia is associated with therapeutic response to chemotherapy

Flow cytometric immunofluorescent analysis was used to assess Fas antigen (CD95) expression in blasts obtained from the bone marrow of 30 patients with acute myeloid leukaemia. The percentage of positive cells in each sample was highly variable. Fas antigen expression did not correlate with age, FAB subtype, white blood cell counts, or CD34 expression. Low expression of Fas was associated with a low complete remission rate after induction chemotherapy (62.5% in cases with < 20% positive cells v 92.9% in cases with > or = 20% positive cells, P < 0.01). The main cause for not achieving remission was resistant disease. Our results suggest that the quantitation of Fas expression can be predictive of treatment outcome in acute myeloid leukaemia.     

Effect of notoginsenoside R1 on the synthesis of components of the fibrinolytic system in cultured smooth muscle cells of human pulmonary artery

Manipulation of autologous bone marrow cells (BM) for transplantation in chronic myeloid leukemia (CML) to enrich for normal cells is a novel approach that may improve survival for patients not suitable for allogeneic transplantation. Limitations of this technique include the reported low frequency of normal stem cells in CML and the difficulties in obtaining sufficient BM for manipulation. To address these problems we compared the apheresis product with the diagnostic bone marrow at diagnosis as a source of primitive BCR/ABL-negative progenitors. We analyzed the CD34+ HLA-DR- and CD34+CD38(-) populations in five CML patients to evaluate the frequency of BCR-ABL-negative progenitors and pre-progenitors in these populations. Progenitor analysis was performed by RT-PCR of individual hemopoietic colonies from a standard CFU-GM assay. Analysis of pre-progenitors involved RT-PCR of secondary colonies derived from a stroma-free pre-CFU assay. Our results show variable levels of BCR-ABL-negative progenitors in the 34+DR- population but very low levels of BCR-ABL-negative progenitors in the 34+38- population in blood. Analysis of pre-progenitors from the 34+DR- fraction of peripheral blood (PB) and BM showed 80-100% and 85-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. Analysis of pre-progenitors from the 34+38- fraction of PB and BM showed 23-100% and 42-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. In summary, pre-progenitors from the 34+DR- and 34+38- populations are predominantly BCR-ABL negative in both marrow and blood at diagnosis. Apheresis product collected at diagnosis is a more abundant sources of BCR-ABL-negative pre-progenitors than BM. Thus, apheresis product could potentially be utilized as a source of BCR-ABL-negative stem cells in CML.     

Skin hyperreactivity of Beh?et's patients (pathergy reaction) is also positive in interferon alpha-treated chronic myeloid leukaemia patients, indicating similarly altered neutrophil functions in both disorders

Typical manifestations of Behcet's disease (BD) and a positive pathergy reaction were observed in a few patients with chronic myeloid leukaemia (CML) on interferon alpha (IFN-alpha) therapy and the significance of this observation was assessed in a prospective study. The skin pathergy test was applied to 15 patients with CML prior to IFN-alpha therapy, 29 patients with CML following IFN-alpha therapy and 30 patients with BD. Twenty-five patients with inflammatory arthropathies (IA), 20 patients with recurrent oral ulcers (ROU), 23 patients treated with IFN-alpha for various disorders and 20 normal individuals were also studied as control groups. The pathergy reaction was positive in nearly a quarter of IFN-alpha-treated CML cases (24%) as well as one-half of the patients with BD (50%). All CML patients prior to IFN-alpha treatment and all patients using IFN-alpha for other diseases were negative for the pathergy reaction. These results may indicate a similarly altered neutrophil function in both BD and IFN-alpha-treated CML patients.     

Granulocyte colony-stimulating factor given in addition to interferon-alpha to mobilize peripheral blood stem cells for autologous transplantation in chronic myeloid leukaemia

In order to potentially mobilize and harvest the Ph cells observed in most patients with chronic myeloid leukaemia (CML) during interferon-alpha (IF-alpha) therapy, G-CSF (filgrastim), 5 microg/kg/d, was administered subcutaneously together with IF-alpha to 30 CML patients in haematological remission but with various degrees of cytogenetic remission, after IF-alpha therapy. Peripheral blood stem cells (PBSC) were harvested using standard aphereses from day 5 of G-CSF Patients underwent one to four (median three) aphereses. Median total yields/kg were 7.6 (range 3.8-25) x 10(8) MNC, 3.4 (0-140) x 10(6) CD34+ cells, and 17 (1.1-107) x 10(4) CFU-GM. No patient had a significant increase in the percentage of Ph+ cells in the bone marrow under G-CSF therapy. The percentage of Ph+ cells in apheresis products tended to decrease between the first and the last apheresis (P = 0.05). 14 patients who were not responsive to IF-alpha were transplanted after conditioning with busulphan 16 mg/kg and melphalan 140 mg/m2. Median time to neutrophils > 0.5 x 10(9)/l was 20 d (16-114 d) and to platelets > 50 x 10(9)/l 18 d (12-149 d). Nine patients had a major cytogenetic response post graft, which correlated with the amount of Ph+ cells reinfused with the graft (P = 0.02). We conclude that this procedure is feasible, allowing the harvest of enough PBSC, some of them Ph- in patients who responded to IF-alpha, to allow autologous transplantation.     

Suppression of cytogenetic clonal evolution with interferon alfa therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia

PURPOSE: To investigate whether cytogenetic clonal evolution can be suppressed with interferon alfa (IFN-alpha) therapy in patients with chronic myelogenous leukemia (CML). PATIENTS AND METHODS: Ninety patients with CML and cytogenetic clonal evolution who received IFN-alpha-based regimens were analyzed. Clonal evolution was defined as the presence of karyotypic abnormalities in addition to the Philadelphia (Ph) chromosome. Patients were evaluated for the suppression of cytogenetic clonal evolution after therapy, the cytogenetic response, and survival. RESULTS: The median age of the population was 39 years (range, 15 to 70 years), median time from diagnosis to clonal evolution 14 months (range, 0 to 145 months), and median percentage of abnormal metaphases 18% (range, 4% to 100%). Fifty six patients (62%) achieved some suppression of cytogenetic clonal evolution; in 41 patients (46%), the suppression was complete. The overall median survival was 51 months, with 43% alive at 5 years. Patients who achieved a complete suppression of cytogenetic clonal evolution had a median survival of 66 months, with 51% alive at 5 years. Characteristics associated with a better response include a lower percentage of abnormal metaphases, time to cytogenetic clonal evolution of 24 months or less, and absence of other features of accelerated disease. A prognostic classification for cytogenetic clonal evolution defined three groups with complete response (CR) rates of 85%, 34%, and 0% (P < .0001) and median survival times of 58, 31, and 30 months, respectively (P=.02). CONCLUSION: Patients with cytogenetic clonal evolution can respond to IFN-alpha therapy, and this response is associated with longer survival. A previously described prognostic model separates patients into subsets with different probabilities of response to IFN-alpha and survival.