Diagnostic and prognostic value of the terminal deoxynucleotidyl transferase (TdT) activity in treating the blastic phase (bp) of chronic granulocytic leukaemia (CGL)

The blastic cell phenotypes of 26 cases of CGL in blastic phase were estimated and the patients were treated with different schemes. The following methods of typisation of the blast cells were used: cytochemical stainings (POX, Sudan B, PAS, nonspecific esterase), estimation of TdT activity, and in 11 patients the testing with monoclonal antibodies of VI series. Using these methods 10 patients (38%) with lymphoid form of the blastic phase, 11 (43%) with the myeloid type and 5 patients (19%) with undifferentiated type were diagnosed. In the group of lymphoblastic type a longer survival time and complete remissions were observed. High TdT activity in blastic cells did correspond with favourable response to Vincristin and Prednison. The introduction of TdT assessment into the diagnosis of CGL allows the cells to be classified more precisely, thus helping in defining the prognosis and in the choice of treatment programme.     

Daunomycin, cytosin-arabinoside and VP-16 (DAV) for myeloid blast crisis of CML

Nine patients with myeloid blast crisis of Philadelphia chromosome-positive chronic myelocytic leukemia received 1-3 courses of intensive induction chemotherapy with DAT (daunomycin, cytosin-arabinoside and 6-thioguanin) or DAV (daunomycin, cytosin-arabinoside and VP-16). Eight patients responded with clearing of blasts from peripheral blood giving a response rate of 89%. However, bone marrow aplasia with less than 5% blasts was seen in only 2 patients. These 2 patients subsequently received an allogeneic bone marrow transplant and achieved complete remissions of 3 and 6 month duration. All patients died due to progression of blast crisis. Median survival of the group was 164 days. These results were compared to a historical control group of 31 patients with myeloid blast crisis treated with vincristine and prednisone. Despite a significantly better response rate with DAV or DAT (8 of 9 versus 9 of 31, p = 0.01) survival was not significantly different than that of the control group.     

Blast crisis in chronic myeloid leukemia

Although the worst prognosis among acute forms of leukemia is persistently attributed to the blast crisis of chronic myeloid leukemia, a therapeutic nihilism seems to us to be unreasonable. Even in the developing stage of a blast crisis we consider an aggressive chemotherapy to be justified particularly in younger patients. In this connection, value and necessity of supporting of therapy are especially emphasized. Remissions can be achieved in about one third of patients during the blast crisis of CML. As in other forms of tumour therapy, the initial status, age, and secondary diseases have to be taken into account and a careful, individual therapy planning has to be made. For the future there is the hope that the increasing possibilities of differentiating blasts will allow more systematic therapeutic conclusions to be made. According to the present knowledge the greatest hopes for achieving long-term remissions or even healings can, in our opinion, be expected by bone-marrow transplantation.     

BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia: Not Quite Enough to Cause Resistance to Imatinib Therapy?

Patients with chronic myeloid leukemia (CML) treated with imatinib in early chronic phase tend to have durable remissions, but there is a high rate of relapse in patients with advanced disease. Mutations in the kinase domain of BCR-ABL that impair drug binding have been identified as the major mechanism of resistance. It is not known when exactly these mutations arise, but in some patients retrospective analysis of pretherapeutic samples demonstrated identical mutations, suggesting selection in the presence of drug. In the present study we have used a highly sensitive PCR assay to screen for kinase domain mutations in pretherapeutic samples from CML patients, irrespective of their subsequent response to imatinib. We find that kinase domain mutations are demonstrable in approximately 1/3 of patients with accelerated phase or blast crisis and that the presence of 2 copies of the Philadelphia chromosome is strongly correlated with mutation detection. Unexpectedly, kinase domain mutant clones were not invariably selected in the presence of drug, suggesting that additional mechanisms must contribute to a fully drug resistant leukemia.     

In vitro culture studies of blood and marrow cells in chronic myeloid leukemia at different phases of the disease

The in vitro culture growth of peripheral blood (PB) and bone marrow (BM) cells were studied simultaneously from 100 adult patients with chronic myeloid leukemia at different phases. Sixty-five patients were investigated at initial diagnosis, 30 patients in control phase, and 41 patients in blast phase. In untreated chronic phase, the relative concentrations of granulocyte-macrophage progenitor cells (CFU-GM) in BM were not significantly different from those of normal controls, but there was generally a marked increase in circulating CFU-GM. The 6 Ph1-negative patients did not show different growth characteristics. We were unable to correlate the CFU-GM number to any of the hematologic parameters as well as to the response to busulfan therapy. Pretreated patients with excessive cluster formation did not necessarily indicate impending blast crisis. In hematologic remission, the numbers of CFU-GM in both BM and PB were well within the ranges of normal controls. Culture results in blast phase revealed a spectrum of abnormal growth. In myeloid crisis, 14/29 BM and 12/29 PB samples showed increased colony and cluster formations which were composed predominantly of immature cells with variable degeneration. Marrow cells in lymphoid crisis produced low numbers of both colonies and clusters in 5 out of 8 patients, while blood cells from 8 out of 10 patients formed large amount of colonies of normal morphology. This study indicates that the in vitro CFU-GM assay may have diagnostic utility in differentiating lymphoid crisis from myeloid crisis.     

Immunotherapy of myeloid leukaemia

The treatment of myeloid leukaemia has progressed in recent years with the advent of donor leukocyte infusions (DLI), haemopoietic stem cell transplants (HSCTs) and targeted therapies. However, relapse has a high associated morbidity rate and a method for removing diseased cells in first remission, when a minimal residual disease state is achieved and tumour load is low, has the potential to extend remission times and prevent relapse especially when used in combination with conventional treatments. Acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) are heterogeneous diseases which lack one common molecular target while chronic myeloid leukaemia (CML) patients have experienced prolonged remissions through the use of targeted therapies which remove BCR-ABL⁺ cells effectively in early chronic phase. However, escape mutants have arisen and this therapy has little effectivity in the late chronic phase. Here we review the immune therapies which are close to or in clinical trials for the myeloid leukaemias and describe their potential advantages and disadvantages.     

Detection of an antigen, associated with cells during the blast crisis of chronic myeloleukemia, in a cytotoxic test using xenogenic antibodies]

The immune antimyeloblast serum (AMS) was obtained from horses immunized with white blood cells from patients suffering from chronic myeloid leukemia (CML) at the blast crisis stage; the serum was completely absorbed with normal red blood cells and white blood cells (WBC). The absorbed antiserum remained cytotoxic to blast cells from 20 of 42 patients with CML at the blast crisis stage. AMS failed to react with the WBC from patients with CML in its chronic phase, and from patients with other types of leukemia Morphological studies indicated a possibility of identification of the antigen associated with myeloblasts from the blood of patients with CML blast crisis, by means to AMS.     

Lysis of fresh leukemic blasts by interferon-activated human natural killer cells

In a comprehensive study of 30 leukemia patients, it was found that a measurable fraction of fresh leukemic blasts from 8 of 8 adult patients with chronic myelogenous leukemia (CML) in blast crisis and 10 of 11 pediatric patients with childhood acute lymphocytic leukemia (ALL) were efficiently lysed by human peripheral blood natural killer (NK) cells as measured in 4-hour chromium release assays. The observed lysis of these fresh, noncultured, neoplastic blasts was mediated by a population of interferon-augmentable, FcR-positive, non-adherent large granular lymphoid cells from normal donors, which were also able to kill the 'standard' NK target K562. It was of further interest that all 8 of the patients with blast crisis CML exhibited myeloid type morphology. Furthermore, neoplastic lymphoblasts from 9 of 10 patients with NK-susceptible childhood ALL lacked easily detectable B or T cell markers and were of 'null' cell type. In marked contrast to the lytic susceptibility of fresh leukemic blasts from patients with ALL and CML in blast crisis, fresh neoplastic granulocytes from 5 patients with chronic phase CML (2 of which eventually progressed to myeloid type blast crisis), as well as leukemic blasts from 8 patients with acute myeloid leukemias (AML, AMMoL, and AMoL) were resistant to lysis as mediated by human NK cells from normal donors. The clinical implications of these findings are discussed.     

Prognostic significance of the histomorphometric features of bone marrow trephine biopsies in patients with chronic myeloid leukemia

OBJECTIVE: To study the correlation of histomorphometric data of bone marrow trephine biopsies at the time of initial diagnosis in chronic myeloid leukemia (CML) patients with the patient prognosis. STUDY DESIGN: A total of 40 CML patients were divided equally in group I (developed accelerated phase or blast crisis within 18 months of initial diagnosis of chronic phase of CML) and group II (developed accelerated phase or blast crisis > 30 months after initial diagnosis of chronic phase of CML). The clinical, hematologic and histomorphometric data were compared in the 2 groups of CML patients. RESULTS: The percentage of bone marrow promyelocytes was significantly increased in group I. On morphometry, the number of blasts per square millimeter, the area of reticulin fibers per square millimeter and the percentage area occupied by reticulin fibers were statistically significant. CONCLUSION: There seems to be grounds for hope for predicting prognosis of CML patients at initial diagnosis based on histomorphometric findings. The percentage area of reticulin fibers and the number of blasts per square millimeter are important prognostic predictors in histomorphometry data.     

错配修复基因hMSH2与慢性粒细胞白血病相关性研究

目的:通过对慢性粒细胞白血病(chronic myeloid leukemia,CML)患者骨髓细胞中错配修复基因(mismatch repair,MMR)h MSH2的表达水平及其调控机制的分析,探讨h MSH2与慢性粒细胞白血病疾病进展的联系。方法:用实时定量PCR方法检测10例对照,27例CML患者(包括慢性期9例,进展期8例,急变期10例)骨髓中4个MMR基因(h MSH2、h MSH6、h MLHl、h PMS2)m RNA的表达;用MSP方法检测MMR基因启动子区甲基化水平;用Western blot方法观察MMR蛋白水平在各组之间的差异。结果:与正常对照比较,CML患者的h MSH2的表达明显降低(P0.05),其表达随疾病恶化而下降,依次为急变期加速期慢性期,而h MLHl、h PMS2、h MSH6的表达却未见异常;27例CML患者中出现3例h MSH2启动子区高甲基化。结论:CML患者的h MSH2表达水平比正常人显著降低,且随着疾病恶化其表达水平逐下降,提示h MSH2可能与CML疾病进展相关。     

Tiazofurin: biological effects and clinical uses

Inosine 5'-phosphate dehydrogenase (IMPDH) activity is increased in all cancer cells. It is the rate-limiting enzyme of guanosine triphosphate (GTP) biosynthesis, and therefore, a sensitive target of chemotherapy. Tiazofurin selectively blocks IMPDH activity. Tiazofurin was found to have an antiproliferative effect on tumor cells in vitro and in the murine system. Based on these findings, Phase I trials were started elsewhere in patients with solid tumors, but were discontinued because of toxicity. In leukemic patients, we were able to demonstrate a good correlation between biochemical parameters (i.e., decline in IMPDH activity and GTP concentrations in blast cells) and clinical response. The most consistent responses to therapy were seen in patients with myeloid blast crisis of chronic myeloid leukemia. Severe toxicity was seen in the earlier patients in the study. However, better patient selection, limitation of treatment duration and earlier recognition and treatment of complications have now made it possible to administer tiazofurin without undue toxicity.     

The dimeric Mcm8–9 complex of Xenopus laevis likely has a conserved function for resistance to DNA damage

The success of Imatinib mesylate (STI571, Gleevec) in treating chronic myeloid leukemia (CML) is, to date, the crowning achievement of targeted molecular therapy in cancer. Nearly 90% of newly diagnosed patients treated with Imatinib in the chronic phase of the disease achieve a complete cytogenetic response. However, more than 95% of these patients retain detectable levels of BCR-ABL mRNA and patients discontinuing Imatinib therapy almost invariably relapse, demonstrating that an Imatinib insensitive population of leukemia-initiating cells (LICs) persists in nearly all patients. These findings underscore the need for treatments specifically targeting the leukemia-initiating population of CML cells. While mounting evidence suggests that the LIC in the chronic phase of CML is the BCR-ABL positive hematopoietic stem cell, several recent publications suggest that during CML blast crisis, a granulocyte-macrophage progenitor (GMP) population also acquires LIC properties through activation of the β-catenin pathway. Characterization of these cells and evaluation of their sensitivity to Imatinib is critical to our understanding and treatment of CML blast crisis.     

Aggressive chemotherapy in adult primary myelodysplastic syndromes. A report on 29 cases

Twenty-nine adult patients with primary myelodysplastic syndromes (MDS) and an excess of marrow blasts were treated by aggressive chemotherapy while still in MDS phase (20 cases) or after progression to ANLL (9 cases). Median age was 47.5 (range 18-68). Twenty-eight patients received a combination of Rubidazone and Ara C and 1 received High dose Ara C. Fourteen patients (48%) achieved complete remission (CR), 5 (17%) were treatment failures (F) and 10 (35%) died during therapy induced aplasia (DA). Median disease free survival was 8.5 months. Median survival of the whole population was 6 months from the onset of treatment, and 17 months in patients achieving CR. These results were significantly less favorable than those obtained at our institution in de novo ANLL with the same chemotherapy regimens. No statistically significant prognostic factors of treatment outcome emerged but patients with normal cytogenetic findings seemed to have both a higher CR rate and longer remissions than patients with abnormal karyotypes. Patients under 50 did not have higher CR rates than older patients, although they had longer remissions (with 3 out of 6 CRs exceeding 2 years). Finally, treatment outcome and survival were identical in patients treated in the MDS phase and in those treated after progression to ANLL. Combination chemotherapy is a highly toxic approach in MDS and essentially seems to benefit younger patients with a normal karyotype, in whom some long remissions can be obtained.     

Low dose cytosine arabinoside in refractory anemia with excess of blasts in transformation

Myelodysplastic syndromes (MDS) are heterogeneous diseases. Patients with blast counts of more than 20% of nucleated bone marrow cells have a high risk of short survival. We treated six patients with refractory anemia with excess of blast in transformation (RAEBiT) with low dose cytosine arabinoside (LD Ara-C). We had one partial remission (PR), surviving 16 weeks and two complete remissions (CR), surviving 22 and 55+ months. Myelosuppression was dominant in all patients, but was not as serious as with conventional remission-induction treatments for leukemias. Bone marrow aplasia occurred in all responding patients, but a differentiation effect is possible too. Maintenance therapy with LD Ara-C may be important for the two long-lasting CR.     

Treatment of poor-risk myelodysplastic syndromes and acute myeloid leukemia with a combination of 5-azacytidine and valproic acid

5-azacytidine (AZA) has become standard treatment for patients with higher-risk myelodysplastic syndrome (MDS). Response rate is about 50% and response duration is limited. Histone deactylase (HDAC) inhibitors are attractive partners for epigenetic combination therapy. We treated 24 patients with AZA (100?mg/m(2), 5?days) plus valproate (VPA; continuous dosing, trough serum level 80-110?μg/ml). According to WHO classification, 5 patients had MDS, 2 had MDS/MPD, and 17 had acute myeloid leukemia (AML). Seven patients (29%) had previously received intensive chemotherapy, and five had previous HDAC inhibitor treatment. The overall response rate was 37% in the entire cohort but significantly higher (57%) in previously untreated patients, especially those with MDS (64%). Seven (29%) patients achieved CR (29%) and two PR (8%), respectively. Hematological CR was accompanied by complete cytogenetic remission according to conventional cytogenetics in all evaluable cases. Some patients also showed complete remission according to FISH on bone marrow mononuclear cells and CD34(+) peripheral blood cells, as well as by follow-up of somatic mitochondrial DNA mutations. Four additional patients achieved at least marrow remissions. Factors influencing response were AML (vs. MDS), marrow blast count, pretreatment, transfusion dependency, concomitant medication with hydroxyurea, and valproic acid (VPA) serum level. This trial is the first to assess the combination of AZA plus VPA without additional ATRA. A comparatively good CR rate, relatively short time to response, and the influence of VPA serum levels on response suggest that VPA provided substantial additional benefit. However, the importance of HDAC inhibitors in epigenetic combination therapy can only be proven by randomized trials.     

Recombinant human colony stimulating factor-granulocyte/macrophage and -granulocyte, but not macrophage induce the development of a respiratory burst in primary human myeloid leukemic cells in vitro

Respiratory burst develops in myeloid blast cells if they differentiate functionally along the monocytic or granulocytic lineage. Using the nitroblue tetrazolium (NBT) assay we studied the effects of recombinant human granulocyte/macrophage colony stimulating factor (rhuGM-CSF), rhuG-CSF and rhuM-CSF on development of respiratory burst activity in primary blast cells from patients with myeloid leukemia. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (AML, n = 13) or myeloid-blast crisis (myBC) of chronic myeloid leukemia (CML, n = 5) it was found that the percentage of NBT positive cells was increased by at least 20% as compared to control cultures by rhuGM-CSF in 6/17 cases, by rhuG-CSF in 7/17 cases and by rhuM-CSF in 0/16 cases, representing in 'responders' a mean increase of 267% and 270% in the absolute number of NBT positive cells by rhuGM-CSF and rhuG-CSF, respectively. Morphological examination of cultured cells from 'responders', as compared to controls, showed decreased blast cell content but generally no evidence of terminal differentiation. The demonstration of Auer rods in NBT positive cells indicates that respiratory burst developed in a leukemic clone. These findings may be of physiological, pathophysiological and clinical relevance.     

Prognostic significance of Ki67-negative blast cell clone in the high risk group of children treated for acute myeloid leukaemia

The aim of this study was to demonstrate the value of immunocytochemical staining of Ki67 antigen expression in blast cells of children with acute myeloid leukemia (AML) and to evaluate its correlation with treatment failure. The material included bone marrow specimens obtained during induction treatment from 46 children treated for AML between 1998-2003. Immunocytochemical staining for Ki67 was based on the ABC technique. Expression of Ki67 antigen on day 0 of induction treatment was confirmed in all patients. The percentage of immunopositive blasts ranged from 88.4% to 99.8% (mean 91.8%). On day 15, according to chemotherapy response, patients were divided into two groups: G1-36 children who responded to induction treatment and reached remission (blast level 5%, low risk group) and G2-10 patients who did not meet remission criterion (blast level > 5%) and were assigned to the high risk (HR) group. Out of 10 children assigned to this group, Ki67 expression in blast cells was confirmed in 4 cases. The fraction of immunopositive blasts ranged from 78.4% to 88.6%. In the other 6 cases, blasts were Ki67-negative. In 12-month period after beginning the treatment, 18 cases of treatment failure (including 7 deceases) were observed in both groups. Five deaths, observed in the HR group, concerned the patients characterized by Ki67-negative blasts. The results indicate a possible correlation between the Ki67-immunonegative blast pattern on day 15 of treatment induction and early decease of AML children assigned to HR group.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.     

L-Asparaginase in Treatment of Acute Leukaemia and Lymphosarcoma

L-Asparaginase was used to treat 40 patients with acute leukaemia or lymphosarcoma. Fifteen with acute lymphoblastic leukaemia either untreated or in relapse after previous therapy were given “Squibb,” “Bayer,” or “Porton” L-asparaginase. Five of these patients had complete remission of their disease, and four had good partial remission. Eleven patients with acute myeloid leukaemia were treated for a short period with L-asparaginase alone. None of them went into remission though a pronounced fall in the numbers of circulating white cells was seen. Six patients with lymphosarcoma received L-asparaginase, two of them having good partial remissions.The toxic side-effects of the L-asparaginase from the three sources seemed to vary, and L-asparaginase from Erwinia carotovora appeared to be antigenically different from the enzyme produced by Escherichia coli.The way in which leukaemic cells become resistant to the action of the enzyme requires further investigation. To overcome this resistance asparaginase should be used in combination with other drugs in the treatment of acute leukaemia.