Selective induction of leukemia-associated fusion genes by high-dose ionizing radiation.

There is strong clinical and epidemiological evidence that ionizing radiation can cause leukemia by inducing DNA damage. This crucial initiation event is believed to be the result of random DNA breakage and misrepair, whereas the subsequent steps, promotion and progression, must rely on mechanisms of selective pressure to provide the expanding leukemic population with its proliferative/renewal advantage. To investigate the susceptibility of human cells to external agents at the genetic recombination stage of leukemogenesis, we subjected two hematopoietic cell lines, KG1 and HL60, to high doses of gamma-irradiation. The irradiation induced the formation of fusion genes characteristic of leukemia in both cell lines, but at a much higher frequency in KG1 than in HL60. In KG1 cells, the AML1-ETO hybrid gene [associated with the t(8;21) translocation of acute myeloid leukemia] occurred significantly more often than the BCR-ABL [associated with t(9;22) chronic myeloid leukemia] or the DEK-CAN [associated with t(6;9) acute myeloid leukemia] fusion genes. These findings support the notion that ionizing radiation can directly generate leukemia-specific fusion genes but emphasize the differing susceptibility of different cell populations and the differing frequency with which the various fusion genes are formed. The selectivity observed at the primary level of gene fusion formation may explain at least in part the differential risk for development of some but not other forms of leukemia after high-dose radiation exposure.

Second malignancies and Richter's syndrome in patients with chronic lymphocytic leukaemia treated with cladribine.

The increased frequency of second malignancies in chronic lymphocytic leukaemia (CLL) is well known. Moreover, antineoplastic therapy additionally increases the risk of secondary cancers. In this study, we analysed whether treatment with cladribine (2-chlorodeoxyadenosine, 2-CdA) during the course of CLL had an impact on the subsequent occurrence of either secondary solid tumours or Richter's syndrome. There were 1487 eligible patients, 251 treated with 2-CdA alone, 913 treated with alkylating agents (AA)-based regimens alone and 323 treated with both 2-CdA and AA. Median time from the start of CLL treatment to the diagnosis of secondary malignancy was 1.9 years (0.5-5.1 years) for the 2-CdA group, 1.8 years (0.3-7.9 years) for the AA group and 3.9 years (0.3-8.4 years) for the 2-CdA+AA group. A total of 68 malignancies were reported in 65 patients. Ten events were non-melanotic skin cancers and were excluded from the analysis, leaving 58 events in 58 patients. In the group of patients treated with 2-CdA alone, there were 15 (6.0%) cases, in the group of patients treated with AA alone there were 26 (2.8%) cases, and in the group treated with 2-CdA+AA there were 17 (5.3%) cases of secondary malignancies. The differences between the frequency of secondary malignancies in the 2-CdA and 2-CdA+AA versus AA alone groups were not significant (P=0.05 and P=0.06, respectively). Only lung cancers occurred significantly more frequently in the 2-CdA (2.8%) and 2-CdA+AA (2.2%) treated groups compared with the AA patients (0.3%) (P<0.001 and P<0.01, respectively). In conclusion, 2-CdA in CLL patients does not seem to increase the risk of secondary malignancies except for lung cancers. However, further studies are necessary to establish the real risk of lung cancer in CLL patients treated with 2-CdA.

The interaction of 2-chlorodeoxyadenosine (2-CDA) and interferon alpha (IFN-alpha) on normal and myeloid leukemia hematopoiesis in vitro.

The influence of 2-CDA and interferon alpha (IFN-alpha) on the CFU-GM cells from ten normal individuals as well as on the CFU-GM from ten patients with chronic myeloid leukemia (CML) and on clonogenic leukemic blasts (CFU-L) from ten patients with acute myeloid leukemia (AML) in semi-solid cultures in vitro was investigated. The agents were added to the cultures alone and in combinations at concentrations of 20, 40 and 80 nM/l of 2-CDA and 10(2), 10(3) and 10(4) U/ml of IFN-alpha. A dose-dependent growth inhibition of CFU-GM as well as of CFU-L was observed. The inhibitory effect of both drugs used alone was significantly greater on the CFU-GM cells from patients with CML than on cells from normal donors. However, 2-CDA and IFN-alpha used together showed greater additive effect on the CFU-L than on the CFU-GM from normal donors and from CML patients.

Combination regimen of 2-chlorodeoxyadenosine (cladribine), mitoxantrone and dexamethasone (CMD) in the treatment of refractory and recurrent low grade non-Hodgkin's lymphoma.

The aim of our phase II study was to determine the effectiveness of combined chemotherapy consisting of 2-hour intravenous infusion of 2-CdA, mitoxantrone and dexamethasone (CMD) regimen in the treatment of heavily previously treated patients with refractory or relapsed low grade non-Hodgkin's lymphoma (LGNHL). All of the 14 patients had clinical stage IV disease, most of them had B symptoms and elevated LDH levels. All cases were refractory to standard chemotherapy or had recurrent relapses having received at least 5 courses of prior chemotherapy. All patients received at least one cycle of CMD (range, 14). A total of 35 courses of CMD were given to the entire group. Complete response (CR) was obtained only in one patient (7.1%) and partial response (PR) in 3 (21.4%) with an overall response rate of 28.5%. The major toxicity was myelosuppression and 35% of the patients had infection. One patient died of sepsis. These results suggest that the addition of other drugs to 2-CdA in heavily treated patients with refractory or relapsing disease may not be more advantageous when composed to giving 2-CdA alone.

Fulminant 2-chlorodeoxyadenosine-related peripheral neuropathy in a patient with paraneoplastic neurological syndrome associated with lymphoma.

2-chlorodeoxyadenosine (2-CdA) has been demonstrated to be a neurotoxic agent when used at significantly greater doses than currently recommended for clinical use. In this report we describe a case of a 37-years-old man lymphoplasmacytoid malignant lymphoma and pre-existing paraneoplastic neurological syndrome who died of an apparent rapidly progressive sensorimotor peripheral neuropathy after completing treatment with two courses of low-doses of 2-CdA.

Activity of 2-chlorodeoxyadenosine (Cladribine) in 2-hour intravenous infusion in 94 previously treated patients with low grade non-Hodgkin's lymphoma.

The purpose of our study was to determine the efficacy of 2-chlorodeoxyadenosine (2-CdA) administered in 2-hour intravenous infusions in previously treated patients with low grade non-Hodgkin's lymphoma (LGNHL). We treated 94 LGNHL patients with 2-CdA at a dosage of 0.12 mg/kg/24h in 2-hour intravenous infusion for 5 consecutive days. The treatment consisted of from 1 to 7 courses (median 3), repeated usually at monthly intervals. All patients were refractory to or relapsed after standard chemotherapy. Of these 94 patients 78 (83%) had clinical stage IV of the disease. Complete response (CR) was obtained in 12 (12.8%) and partial response (PR) in 36 (38.3%) giving an overall response rate of 51.1%. In 12 (12.8%) grade 4 thrombocytopenia with haemorrhagic diathesis was noted, grade 4 neutropenia was observed in 12 (12.8%) and infections complicated the course of treatment in 38 (40.4%) patients. 2-CdA treatment was the cause of death of 3 patients. The results of our study show that 2-CdA given in 2-hour infusions is an effective agent in advanced, heavily pretreated patients with LGNHL.

Treatment of drug-induced agranulocytosis with colony stimulating factors (G-CSF or GM-CSF).

The application of granulocyte-macrophage and granulocyte colony stimulation factors (GM-CSF and G-CSF) has been progressively increased in the treatment of patients with agranulocytosis. The aim of our study was to compare the time of neutrophil recovery in patients with severe agranulocytosis treated with G-CSF or GM-CSF and the historical control group. We have studied 6 patients with agranulocytosis treated with stimulating factors and 7 patients in historical control group. Most of the patients have been exposed to thiamazole or non-steroid antiinflammatory drugs. Our results demonstrate that patients receiving colony stimulating factor have a significantly shorter period of recovery (the mean time 8.7 +/- 1.98 days) than the historical control group (the mean time 11.0 +/- 2.24 days). We observed also a shorter time of antibiotico-therapy and hospitalization in the group of patients treated with colony stimulating factor.

Influence of 2-chloro-2'-deoxyadenosine alone and in combination with cyclophosphamide or methotrexate on murine leukemia L1210.

The influence of 2-CdA administrated alone and in combination with CY or MTX on the survival time of mice bearing L1210 leukemia was investigated. 2-CdA treated mice lived longer than control animals. Survival times of mice receiving combined therapy of 2-CdA and CY were significantly prolonged as compared with mice treated with these agents separately. Survival times of mice treated with 2-CdA and MTX were not significantly prolonged, compared with animals receiving 2-CdA alone.

The influence of recombinant human tumor necrosis factor (rh-TNF alpha) on granulocyte-macrophage progenitor cells (CFU-GM) and clonogenic leukaemic blasts (CFU-L) in cultures in vitro.

We investigated the influence of recombinant human tumor necrosis factor alfa (rh-TNF alpha) on the clonal growth of CFU-GM from 14 normal individuals and clonogenic blasts (CFU-L) from 16 patients with acute myeloid leukaemia (AML) in semi-solid cultures in vitro. Recombinant human TNF was produced by the Center of Molecular and Macromolecular Studies of the Polish Academy of Sciences (Lódz, Poland) as a lyophylized powder. This factor was added to the culture medium at the concentrations of 10, 100, 1000 U/ml. A dose-dependent growth inhibition of CFU-GM and AML CFU-L was observed. The inhibitory effect of rh-TNF alpha was significantly greater on CFU-L than CFU-GM.

Cladribine combined with mitoxantrone in the treatment of blastic phase of chronic myeloid leukemia.

A phase II clinical study was performed to evaluate the effectiveness and toxicity of cladribine (2-CdA) combined with mitoxantrone (CM regimen) in the treatment of chronic myeloid leukemia in blastic phase (CML BP). A total of 12 adult patients with CML BP were included in this study. 2-CdA was given at a dose 0.12 mg/kg in 2-hour iv infusion on days 1-5 and mitoxantrone 10 mg/m2 i.v. day 1. The cycles were repeated at 4 week intervals in most cases. Complete remission (CR) was defined as the presence of < 5% of blasts in a normo- or hypercellular bone marrow in addition to normal peripheral blood counts and with normal physical examination. A partial response (PR) required normal peripheral blood counts but 5 to 25% marrow blasts. Toxicity was assessed according to WHO criteria. The patients received 21 courses of CM (median 2, range 1-3). Of 12 patients only 2 (17%), achieved PR. Responses were observed in patients with myeloid BP, after 3 and 2 courses, respectively. Myelosuppression was the main toxicity. Four patients (33.3%) had grade 3 or 4 neutropenia and 3 (25%) had grade 3 or 4 thrombocytopenia. Infections occurred in 4 patients (33.3%) and 2 of them died of sepsis shortly after CM treatment. This preliminary results in a small group of patients suggest that CM programme has limited value in pre-treated patients with CML BP. However, this regimen may be used as palliation in the end stage of disease.

Cladribine decreases the level of angiogenic factors in patients with chronic lymphocytic leukemia.

We investigated the serum concentration of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGFbeta1), using an enzyme linked immunosorbent assay (ELISA) in a group of 18 chronic lymphocytic leukemia (CLL) patients, before and after a successful treatment with cladribine (2-chlorodeoxyadenosine, 2-CdA) and 16 healthy volunteers. The serum level of bFGF was found to be significantly lower in the control group (median 0.15 pg/ml, range 0.0-15.7 pg/ml), when compared to the untreated CLL patients (median 41.4 pg/ml, range 2.1-292.6 pg/ml) (p=0.0002). After a successful 2-CdA treatment we observed a significantly lower level of this cytokine (median 10.55 pg/ml, range 0.4-140.4 pg/ml) (p=0.0019) in the same patients. However, the level of bFGF in this group was still higher than in the control group (p=0.003). The levels of TGFbeta1 were higher in the group of untreated CLL patients (median 31.36 ng/ml, range 14.36-75.71 ng/ml) than in the control group (median 28.35 ng/ml, range 10.85-70.10 ng/ml) (p=0.029). After the 2-CdA treatment serum concentration of this cytokine decreased significantly (median 20.34 ng/ml, range 3.02-43.85 ng/ml) (p=0.031) with similar levels present to that of the healthy control group (p=0.3). In conclusion, we have shown that the serum concentration of bFGF and TGFbeta1 in CLL patients were significantly reduced after 2-CdA chemotherapy that resulted in remission. The level of these factors might correlate with the activity of the disease.

[Clinical pharmacology of 2-chlorodeoxyadenosine (Cladribine)]. / Farmakologia kliniczna 2-chlorodeoksyadenozyny (Kladrybiny).

2-chlorodeoxyadenosine (2-CdA) is a simple nucleoside derived from deoxyadenosine by substituting chloride for hydrogen in 2' position of the purine ring, which renders it resistant to degradation by adenosine deaminase. Many studies have revealed high efficiency of 2-CdA in the treatment of lymphoid malignancies. Hairy cell leukemia is notably responsive to 2-CdA, with 85% of patients entering complete remissions after treatment of this drug.

[Clinical pharmacology of hydroxyurea]. / Farmakologia kliniczna hydroksymocznika.

Hydroxyurea, an antineoplastic drug evaluated clinically more than 30 years ago, is still the principal drug in patients with myeloproliferative syndromes. It is suggested now that it should be used as initial therapy for chronic myelogenous leukemia. Recently hydroxyurea is used in the treatment of patients with sickle cell disease. It has been shown to augment production of fetal hemoglobin and decrease the propensity of abnormal hemoglobin to polymerize and from the sickle cells in this way.

The influence of 2-chlorodeoxyadenosine alone and in combination with cyclophosphamide or methotrexate on normal hematopoiesis in mice.

The influence of 2-chlorodeoxyadenosine (2-CDA) administered alone or in combination with cyclophosphamide (CY) or methotrexate (MTX) on normal hematopoiesis in mice was investigated. The peripheral blood counts such as the number of leukocytes, platelets, and erythrocytes, hematocrit values and hemoglobin concentration were measured. The number of mononuclear cells and multipotential stem cells (CFU-S) in bone marrow was also checked. 2-CDA caused both the decrease in white cell count (granulocytes, monocytes, lymphocytes) and the suppression of mononuclear cells and multipotential stem cells in the bone marrow. The addition of CY significantly enhanced the suppression of these parameters. In contrast to CY, MTX did not augment myelotoxic effect of 2-CDA. 2-Chlorodeoxyadenosine administered singly and in combination with CY or MTX had no influence on the number of erythrocytes, hemoglobin concentration, hematocrit values and number of platelets.

Synergistic action of 2-chlorodeoxyadenosine and cyclophosphamide on murine leukemias L1210 and P388.

The influence of 2-chlorodeoxyadenosine (2-CDA) administered alone and in combination with cyclophosphamide (CY) or methotrexate (MTX) on the survival time of mice bearing L1210 or P388 leukemia was investigated. 2-CDA given alone significantly prolonged survival time of mice bearing L1210 leukemia and caused only a slight prolongation of survival time in mice bearing P388 leukemia. Survival times of mice bearing both kinds of leukemia, receiving combined therapy of 2-CDA and MTX were not significantly prolonged as compared with mice treated with these agents separately. The groups of mice with both leukemia L1210 and P388 treated with 2-CDA and CY lived significantly longer as compared with the control group and with mice receiving only single cytostatic.

Emerging therapies in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) therapy has dramatically changed in the last decade due to the introduction of tyrosine kinase inhibitors (TKIs) - imatinib, nilotinib and dasatinib. Despite the significant prolongation of overall survival of CML patients there is still room for improvement. Approximately 20-25% of patients initially treated with imatinib will need alternative therapy, due to drug resistance which is often caused by the appearance of clones expressing mutant forms of BCR-ABL. Second generation TKIs dasatinib and nilotinib have shown promising results in imatinibresistant or intolerant CML patients, but are not active against CML clones with highly resistant T315I mutation. In recent years special attention is placed on small pool of leukemic stem cells which may contribute to the persistence of the leukemia. This article provides a review of preclinical and clinical data concerning the most promising new directions in CML treatment, with special emphasis on new drugs active in T315I mutation and compounds affecting leukemic stem cells.