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.

Richter syndrome first manifesting as cutaneous B-cell lymphoma clonally distinct from primary B-cell chronic lymphocytic leukaemia.

Richter syndrome (RS) is a transformation to high-grade non-Hodgkin lymphoma in patients with chronic lymphocytic leukaemia (CLL). RS may develop in lymph nodes or rarely extranodally. Skin localization of RS has been described in only a few cases. We present a 77-year-old woman who developed isolated diffuse large B-cell lymphoma (LBCL) in the skin of the nose without any other symptoms of RS. The LBCL in the skin was clonally distinct from the original bone marrow CLL cells. Moreover, LBCL cells were positive for LMP-1 segment of Epstein-Barr virus and overexpressed p53 protein. The patient was successfully treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) and adjuvant local radiotherapy.

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.

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.

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 in combination with mitoxantrone and cyclophosphamide(CMC) in the treatment of heavily pre-treated patients with advanced indolent lymphoid malignancies.

The aim of our study was to determine the effectiveness and toxicity of combined chemotherapy consisting of cladribine (2-chloro-deoxyadenosine, 2-CdA), mitoxantrone and cyclophosphamide (CMC regimen) in the treatment of refractory or relapsed indolent lymphoproliferative disorders. The treatment course consisted of 2-CdA given at a dose of 0.12 mg/kg/24 h in a 2-h intravenous infusion for 5 (CMC5) or 3 (CMC3) consecutive days, mitoxantrone 10 mg/m2 on day 1 and cyclophosphamide 650 mg/m2/iv on day 1. Thirty-three patients (19 with B-CLL and 14 with LG-NHL) entered the study and all of them were eligible. Twenty patients (60.6%) were recurrent after prior therapy and 13 (39.4%) had refractory disease. All patients received 5 or more cycles of chemotherapy before CMC treatment. Twenty-one patients were treated with CMC5 regimen and 12 with CMC3 regimen. The overall response rate, including CR and PR, was 48.6% (95% CI 32-66). There were no differences in the frequency of responses between the CMC3 and CMC5 treated groups (p>0.05). One patient with B-CLL and three patients with lymphocytic lymphoma achieved CR (12.1%). Among 12 patients (36.4%) who achieved PR there were 6 CLL patients, and 6 lymphoma patients. The major toxicity was myelosuppression. Severe neutropenia was seen in 11/33 (33.3%) patients, more frequently in patients who received CMC5 than in the patients who received CMC3, both in the CLL (50.0% and 28.5%, respectively) and in the LG-NHL group (22.2% and 0%, respectively). The rate of thrombocytopenia was similar in both groups. Infections and fever of unknown origin complicated the treatment with CMC5 more often than with CMC3: five episodes were seen in 3 patients treated with CMC3 when compared to 15 episodes in 12 patients treated with CMC5. In conclusion, the CMC programme is an active combined regimen in heavily pre-treated CLL and LG-NHL patients. However, its toxicity is significant and we suggest a shortening of 2-CdA infusion from 5 to 3 d in further studies. Whether a combination of 2-CdA with cyclophosphamide and mitoxantrone would result in improved outcome as compared to 2-CdA alone, is being investigated in a prospective, randomised trial.

Toxic epidermal necrolysis in a patient with severe aplastic anemia treated with cyclosporin A and G-CSF.

Toxic epidermal necrolysis (TEN) and severe aplastic anemia (SAA) are rare nonpredictable side effects of several drugs. To our knowledge there are no reports on the coexistence of these two disorders. This study presents a 23 year-old man with TEN diagnosed 6 days after treatment with aminophenazone, paracetamol and phenoxymethylpenicillin. The resolution of the skin disorder was observed after few days of treatment with G-CSF and cyclosporin A. In contrast, SAA only partially responded to treatment with these agents.

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.

The susceptibility of Philadelphia chromosome positive cells to FAS-mediated apoptosis is not linked to the tyrosine kinase activity of BCR-ABL.

We investigated whether inhibition of the BCR-ABL tyrosine kinase by the CGP57418B compound would render chronic myeloid leukaemia (CML) cells susceptible to Fas (CD95, Apo-1)-mediated cell death. Only two (AR230 and SD1) out of 10 BCR-ABL positive cell lines were found to express the CD95 protein. No change in Fas expression was observed in any of the 10 cell lines after 48 h exposure to CGP57418B. AR230 cells were resistant and SD1 cells were partially resistant to Fas-mediated apoptosis induced by ligation of the Fas receptor to an anti-Fas IgM antibody. Pre-incubation with 1 microM CGP57418B did not change the susceptibility of these cell lines to Fas-mediated cell death. Similar results were observed in experiments with CD34+ cells from CML patients and from normal individuals. The data suggest that, in contrast to some cytotoxic drugs, the CGP57148B tyrosine kinase inhibitor utilizes a pathway other than the CD95 system in order to induce apoptosis in CML cells.

The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease.

The number of genetic lesions necessary to generate leukemia in humans is unknown, but it is possible that certain specific abnormalities, eg, fusion genes, known to be associated with acute and chronic leukemia are produced relatively frequently in human cells but require other events to occur before the leukemia becomes manifest. We investigated this possibility by studying peripheral blood leukocytes from normal individuals and various hematopoietic cell lines for the presence and expression of the p210 and the p190 types of the BCR-ABL gene associated with chronic myeloid leukemia (CML) and acute lymphoblastic leukemia. We used two-step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in which batches of 10(8) cells per sample were tested in 40 replicate reactions. We estimate that this assay is 1.5 logs more sensitive than the two-step RT-PCR assays that we use routinely to assess minimal residual disease. BCR-ABL fusion gene transcripts of various configurations were found in circulating leukocytes from 12 of the 16 healthy adults analyzed. Transcripts with an e1a2 junction (p190 BCR-ABL) were present in 11 and p210-type transcripts with b2a2 and/or b3a2 junctions were detected in 4 individuals. The same RT-PCR assays in non-CML cell lines showed the presence of classical or aberrant p210-type mRNA in 3 of 7 lines and of p190-type transcripts in all 7 lines of hematopoietic origin (HL60, KG1, U937, Kasumi, Jurkat, JVM13, and JVM25), whereas the NIH3T3 murine fibroblast line was reproducibly negative for these fusion genes. These findings confirm and extend previous reports on the detection of leukemia-associated genes in normal leukocytes and suggest that certain fusion genes are generated relatively frequently in hematopoietic cells, but only infrequently do the cells acquire the additional changes necessary to produce leukemia in humans. Although there is only a small probability that such innocent BCR-ABL-carrying leukocytes are detected by conventional RT-PCR assays, they may be the source of some sporadically positive tests in leukemia patients in long-term remission.

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.

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.

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.

[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.

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.

The influence of 2- chlorodeoxyadenosine in combination with tumour necrosis factor-alpha or its mutein on murine leukaemias L1210 and P388.

We investigated the influence of 2-chlorodeoxyadenosine (2-CdA) in combination with tumour necrosis factor-alpha (TNFalpha) or its mutein VI, which differs from the native molecule by N-terminal amino acid composition, on the survival time of mice inoculated with leukaemia L1210 or P388. Groups of mice with leukaemia L1210 and P388 receiving 2-CdA combined with TNFalpha had shorter survival times than animals treated with these agents separately. In contrast, the administration of 2-CdA in conjunction with mutein VI, prolonged the survival of mice inoculated with these leukaemias as compared with animals receiving these agents separately. The results of the present study emphasize the importance of the biological activity of the TNFalpha molecule N-terminus.

[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.