Cytokine and Chemokine Profile in Patients with Multiple Myeloma Treated with Bortezomib.

The aim of the study was to determine the levels of selected cytokines and chemokines in the serum of multiple myeloma (MM) patients treated with bortezomib-based regimens. A total of 71 MM patients were examined: 41 with primary refractory disease (17) or early relapse (28), and 30 who were bortezomib sensitive with no progression for at least six months. Patients who demonstrated CR or PR after bortezomib-based therapies longer than six months after treatment discontinuation were designated bortezomib sensitive. Serum cytokine levels were assayed with Bio-Rad Bio-Plex Pro Human Cytokine 27-Plex Assay on the MAGPIX Multiplex Reader and the Bio-Plex® 200 System (Bio-Rad). Higher levels of MIP-1α and lower levels of MIP-1ß and IL-9 were associated with better responses to bortezomib-based treatment, and higher levels of IL-1ra and IL-8 were associated with bone involvement. MCP-1 was elevated in patients with hemoglobin < 10 g/dl compared to those without anemia. The levels of IL-8, MIP-1α, and TNF-α were significantly higher in patients with renal insufficiency. Only MIP-1α was elevated in patients with hypercalcemia compared to patients with normal calcium levels. In conclusion, distinct cytokines are involved in the pathogenesis of MM and may play a prominent role in the prediction of treatment response. However, a single measurement of serum cytokines should be interpreted with caution and further studies are needed.

The distribution and potential prognostic value of SMAD protein expression in chronic lymphocytic leukemia.

The SMAD proteins are responsible for transducing signals from activated transforming growth factor-beta. This is the first study assessing the expression of SMAD-1/8, SMAD-2/3, SMAD-4, and SMAD-7 in chronic lymphocytic leukemia cells with regard to their clinical significance and potential prognostic value. Overexpression of SMAD-1/8 was observed in 160 chronic lymphocytic leukemia patients compared to 42 healthy volunteers (p = 0.023) and was associated with a more progressive course of the disease (p = 0.016). Moreover, the high expression of SMAD-1/8 correlated with other, well-established prognostic factors, including clinical stage (p = 0.010) and lymphocyte doubling time (p = 0.021). The expression of SMAD-4 was lower in chronic lymphocytic leukemia patients compared with the control group (p = 0.003). Importantly, lower SMAD-4 levels correlated with longer progression-free survival (p = 0.009), progressive course of the disease (p = 0.002), advanced clinical stage (p = 0.0004), elevated beta-2-microglobulin and lactate dehydrogenase levels (p < 0.05), shorter lymphocyte doubling time (p = 0.009), and CD38 antigen expression (p = 0.039). In addition, lower SMAD-4 expression correlated with lower apoptotic index (p = 0.0007) and lower expression of receptors for vascular endothelial growth factors VEGFR-1 and VEGFR-2. A significant association was found between the low expression of inhibitory protein SMAD-7 and both zeta-chain-associated protein kinase 70-negative cells (p = 0.04) and lower apoptotic index (p = 0.004). No differences were observed in SMAD-2/3 expression. In conclusion, our results demonstrate a significant correlation between greater SMAD-1/8 and lower SMAD-4 expression in chronic lymphocytic leukemia cells, as well as more progressive outcome and poor prognosis. These data provide supporting evidence that the expression of SMAD proteins plays an important role in disease development and may be considered as a novel, biologic prognostic factor in this disease.

Addition of cladribine to the standard induction treatment improves outcomes in a subset of elderly acute myeloid leukemia patients. Results of a randomized Polish Adult Leukemia Group (PALG) phase II trial.

Intensive induction chemotherapy using anthracycline and cytarabine backbone is considered the most effective upfront therapy in physically fit older patients with acute myeloid leukemia (AML). However, outcomes of the standard induction in elderly AML are inferior to those observed in younger patients, and they are still unsatisfactory. As addition of cladribine to the standard induction therapy is known to improve outcome in younger AML patients. The present randomized phase II study compares efficacy and toxicity of the DAC (daunorubicin plus cytarabine plus cladribine) regimen with the standard DA (daunorubicin plus cytarabine) regimen in the newly diagnosed AML patients over 60 years of age. A total of 171 patients were enrolled in the study (DA, 86; DAC, 85). A trend toward higher complete remission (CR) was observed in the DAC arm compared to the DA arm (44% vs. 34%; P = .19), which did not lead to improved median overall survival, which in the case of the DAC group was 8.6 months compared to in 9.1 months in the DA group (P = .64). However, DAC appeared to be superior in the group of patients aged 60-65 (CR rate: DAC 51% vs. DA 29%; P = .02). What is more, a subgroup of patients, with good and intermediate karyotypes, benefited from addition of cladribine also in terms of overall survival (P = .02). No differences in hematological and nonhematological toxicity between the DA and DAC regimens were observed.

Prognostic value of thymidine kinase activity in patients with chronic lymphocytic leukemia.

Thymidine kinase (TK) activity is a marker of biological activity that allows the indolent and aggressive forms of chronic lymphocytic leukemia (CLL) to be distinguished. The aims of the study were to determine the relationship between TK activity and clinical status and prognosis, as well as to compare its activity with that of other prognostic and predictive factors. TK activity was measured in patient sera at the time of diagnosis using the DiviTum method, with the mean value being 439 Du/L. A correlation was found between TK activity and risk of disease progression (p=0.045). The optimal discriminative value of TK activity in the prediction of CLL progression was found to be 600 Du/L. TK activity significantly differed between the patients who achieved complete remission and those who only partially responded to therapy. In 93% of patients without any response to treatment and 18 out of 20 patients with progressive disease, TK activity over 600 Du/L was noted. In addition, all of the 10 patients with 17p13 deletion displayed TK activity of over 600 Du/L (p=0.0004). High TK activity also correlated with elevated levels of LDH (p=0.001) and ß2­microglobulin (p=0.03) in the study group. The results of the study indicated the importance of TK activity as a prognostic factor in patients with CLL.

Clonal evolution in CLL patients as detected by FISH versus chromosome banding analysis, and its clinical significance.

The acquisition of new aberrations during the course of chronic lymphocytic leukemia (CLL) named clonal evolution (CE) is usually detected by one of the two methods: chromosome banding analysis (CBA) and interphase fluorescence in situ hybridization (I-FISH). The purpose of this study was to compare the usefulness of FISH and CBA for detecting CE and to evaluate its influence on clinical outcome. FISH and CBA were performed at two time points: baseline and follow-up. Thirty-eight previously untreated patients with CLL were included in this study. CBA and I-FISH revealed CE in 15 (39.5%) and 10 (26.3%) patients, respectively. High-risk CE was detected in six cases by CBA and in five cases by I-FISH. In four cases with CE-dependent 17p abnormalities detected by CBA, metaphase FISH was needed for the confirmation of 17p13.1 deletion. Time from first-line to second-line treatment (TTST) and overall survival (OS) did not differ between patients with and without CE, irrespective of the CE-detecting method used. However, shorter OS (P = 0.043) and TTST (P = 0.006) were observed for the patients with potentially relevant CE (rCE) detected by CBA, in which acquired aberrations were present in at least 20% of undivided cells and/or changed baseline karyotype to abnormal or complex and were not resulting from 13q deletion. Our results suggest that some, but not all, CE-dependent aberrations detected by CBA influence clinical outcome. Moreover, I-FISH, which was aimed at detecting aberrations of prognostic significance, was found to be more precise than CBA in their detection, especially TP53 deletion.

Neutrophil to lymphocyte ratio (NLR) impact on the progression-free survival and overall survival of multiple myeloma patients treated with high-dose chemotherapy and autologous stem cell transplantation.

High-dose chemotherapy with autologous stem-cell transplantation (ASCT) remains the standard of care in multiple myeloma (MM) patients. This retrospective study aimed to assess the impact of neutrophil-to-lymphocyte ratio (NLR) and other complete blood count (CBC)-based predictors on PFS and OS of transplant-eligible MM patients. The CBC-based biomarkers were evaluated in a single-center cohort of 176 MM patients at three time points: at the diagnosis, the time of ASCT, and +100 d after ASCT. Univariable and multivariable Cox's regression analyses and Kaplan-Meier estimate were used in statistical analysis. NLR at ASCT (HR 1.15, 95% CI: 1.05-1.26) and hemoglobin at ASCT (HR 0.80, 95% CI: 0.68-0.94) were independent factors influencing PFS. In the model for OS, the only statistically significant factors were NLR at ASCT (HR 1.15, 95% CI: 1.04-1.27), bortezomib administration prior to ASCT (HR 0.52, 95% CI: 0.33-0.83) and age at diagnosis (HR 1.03, 95% CI: 1.00-1.06). NLR at ASCT is an independent predictive factor in MM patients undergoing ASCT.

The role of NPM1 alternative splicing in patients with chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with heterogeneous clinical course. Recent studies revealed a link between NOTCH1 mutation and the overexpression of MYC and MYC-related genes involved in ribosome biogenesis and protein biosynthesis, such as nucleophosmin-1 (NPM1), in CLL cells. In the present study, we aim to evaluate the impact of the NOTCH1 mutation on the MYC and MYC induced NPM1 expression in CLL cells via quantification of their transcripts. METHODS: Using qRT-PCR, we analyzed the levels of MYC and three main NPM1 splice variants in 214 samples collected from CLL patients. We assessed the impact of each splice variant on CLL prognostic markers, including the IGHV, TP53, NOTCH1, SF3B1, and MYD88 mutational status, cytogenetic aberrations, and laboratory features. RESULTS: Significantly higher levels of NPM1.R1 transcripts in patients with unmutated compared to mutated IGHV status were found. The median time to first treatment (TTFT) in patients with a high level of NPM1.R1 was significantly shorter compared to the group with low NPM1.R1 levels (1.5 vs 33 months, p = 0.0002). Moreover, in Multivariate Cox Proportional Hazard Regression Model NPM1.R1 splice variant provided an independent prognostic value for TTFT. CONCLUSION: In conclusion, our study indicates the prognostic significance of the level of NPM1.R1 expression and suggests the importance of splicing alterations in the pathogenesis of CLL.

The Predominant Prognostic Significance of NOTCH1 Mutation Defined by Emulsion PCR in Chronic Lymphocytic Leukemia.

PURPOSE: NOTCH1 mut represents a new prognostic marker in chronic lymphocytic leukaemia (CLL). The low sensitivity of the current methods may increase the risk of false-negative results, particularly in patients with low NOTCH1 mut allelic burden. This study compared two methods of the NOTCH1 mut assessment including droplet digital PCR (ddPCR) and amplification-refractory mutation system PCR (ARMS-PCR) untreated CLL patients. PATIENTS AND METHODS: This study included 319 untreated CLL patients. Two PCR-based methods; ddPCR and ARMS-PCR were performed to assess the mutational status of NOTCH1. The Mann-Whitney, Fisher's exact test, Kruskal-Wallis, Kaplan-Meier, Log rank tests and multivariate Cox proportional hazard regression model were used to analyze collected data. RESULTS: We proved that ddPCR increased the detectability of the NOTCH1 mut compared to ARMS-PCR in CLL (18.55% vs 6%). We showed a shorter time to first treatment (TTFT) in the NOTCH1 mut group of patients compared to the NOTCH1 wt defined by ddPCR (1.5 vs 33 months, p=0.01). The TTFT survival curves analysis in subgroups divided according to the mutational status of IGHV and NOTCH1 assessed by ddPCR discriminated group with the best prognosis: IGHV mut NOTCH1 wt. Multivariate analysis revealed that the mutational status of IGHV represented an independent prognostic factor for TTFT, while NOTCH1 mut determined by ddPCR constituted as a dependent prognostic factor for TTFT. CONCLUSION: The selection of the precise method of NOTCH1 mut detection as ddPCR might significantly improve prognostic stratification of CLL patient. Assessment of IGHV might be relevant to more accurate discrimination of prognostic groups of CLL patients, especially in harboring NOTCH1 mut irrespective of the quantity of allelic burden.

Early induction intensification with cladribine, cytarabine, and mitoxantrone (CLAM) in AML patients treated with the DAC induction regimen: a prospective, non-randomized, phase II study of the Polish Adult Leukemia Group (PALG).

We present the results of a prospective, non-randomized phase 2 trial in which 253 AML patients (pts) under 60 years old received DAC (Daunorubicin + AraC + Cladribine) as first induction followed by CLAM (Cladribine + AraC + Mitoxantrone) as early second induction on day 16 based on bone marrow (BM) blasts on day 14 (D14). The CR/CRi rate after a single course of DAC was 83% for pts with D14 BM blasts less than 10%. Forty-six pts had >10% BM blasts on D14, of whom 35 received CLAM with rates of CR/CRi 60% and early death (ED) 23%. The remaining 11 pts were not fit to receive CLAM, with rates of CR/CRi 28%, PR 18%, and ED 18%. Median OS was 7.2 versus 7.5 months, respectively. The overall CR/CRi rate was 77% after the first induction, with final CR/CRi rate 80% after DAC reinduction for pts who achieved PR with initial DAC course. CLAM used as early second induction might improve CR/CRi rates for younger AML pts with poor early response to DAC induction, but may be associated with higher mortality.

CD38 gene polymorphisms contribute to genetic susceptibility to B-cell chronic lymphocytic leukemia: evidence from two case-control studies in Polish Caucasians.

Given the recent findings on the importance of CD38 signaling in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL), we hypothesized that single nucleotide polymorphisms (SNP) in the CD38 gene may be related to B-CLL risk. We evaluated two potentially functional CD38 SNPs, intronic rs6449182 (184C>G) and missense rs1800561 (418C>T, Arg140Trp) in two hospital-based case-control studies (study A and validation study B). Genotyping was done using PCR-based assays in a total of 460 Polish Caucasian patients with B-CLL and 503 age-matched and gender-matched controls. We found that frequencies of both variant alleles (rs6449182 G and rs1800561 T) were significantly higher in B-CLL. In study A, logistic regression analysis revealed an association between B-CLL and genotypes: rs6449182 CG [odds ratio (OR), 3.57; 95% confidence interval (95% CI), 2.4-5.3], rs6449182 GG (OR, 5.2; 95% CI, 2.36-11.5), and rs1800561 CT (OR, 6.72; 95% CI, 1.5-30.1), although no homozygous rs1800561 TT genotype was detected in either study. These results were confirmed in study B, which showed an association between B-CLL and genotypes rs6449182 CG (OR, 4.00; 95% CI, 2.7-6.0), rs6449182 GG (OR, 12.84; 95% CI, 4.3-38.7), and rs1800561 CT (OR, 10.12; 95% CI, 1.3-81.6), and in the combined analysis of both studies. We also observed that rs6449182 G carriers had more advanced clinical stage (P=0.002) and tended to be younger at diagnosis (P=0.056). Furthermore, we found higher CD38 transcript levels and higher proportions of CD38-positive cells in carriers of rs6449182 G and rs1800561 T alleles (P<0.05 for all comparisons). In conclusion, our data show that CD38 SNPs may affect CD38 expression and contribute to the increased risk of B-CLL carcinogenesis.

Cladribine combined with high doses of arabinoside cytosine, mitoxantrone, and G-CSF (CLAG-M) is a highly effective salvage regimen in patients with refractory and relapsed acute myeloid leukemia of the poor risk: a final report of the Polish Adult Leukemia Group.

OBJECTIVES: Patients with primary refractory AML and with early relapses have unfavorable prognoses and require innovative therapeutic approaches. Purine analogs fludarabine (FA) and cladribine (2-CdA) increase cytotoxic effect of Ara-C in leukemic blasts and inhibit DNA repair mechanisms; therefore its association with Ara-C and mitoxantrone (MIT) results in a synergistic effect. In the current report, we present the final results of multi-center phase II study evaluating the efficacy and toxicity of CLAG-M salvage regimen in poor risk refractory/relapsed AML patients. METHODS: The induction chemotherapy consisted of 2-CdA 5 mg/m2, Ara-C 2 g/m2, MIT 10 mg/m2, and granulocyte-colony stimulating factor. In the case of PR, a second CLAG-M was administered. Patients in CR received consolidation courses based on high doses of Ara-C and MIT with or without 2-CdA. RESULTS: One hundred and eighteen patients from 11 centers were registered; 78 primary resistant and 40 relapsed. Sixty-six patients (58%) achieved CR after one or two courses of CLAG-M, 49 (35%) were refractory, and 8 (7%) died early. WBC >10 g/L and age >34 yr were factors associated with increased risk of treatment failure. Hematological toxicity was the most prominent toxicity of this regimen. The probability of OS at 4 yr was 14% (95% CI 4-23%). OS was influenced by age, WBC >10 g/L and poor karyotype in both univariate and multivariate analyses. The probability of 4 yr DFS was 30% for all 66 patients in CR (95% CI 11-49%). Poor karyotype was the only factor associated with decreased probability of DFS. CONCLUSIONS: We conclude that CLAG-M is a well-tolerated and highly effective salvage regimen in poor risk refractory/relapsed AML.

Concomitance of monosomal karyotype with at least 5 chromosomal abnormalities is associated with dismal treatment outcome of AML patients with complex karyotype - retrospective analysis of Polish Adult Leukemia Group (PALG).

Monosomal karyotype (MK) and complex karyotype (CK) are poor prognostic factors in acute myeloid leukemia (AML). A comprehensive analysis of cytogenetic and clinical factors influencing an outcome of AML-CK+ was performed. The impact of cladribine containing induction on treatment results was also evaluated. We analyzed 125 patients with AML-CK+ treated within PALG protocols. MK was found in 75 (60%) individuals. The overall complete remission (CR) rate of 66 intensively treated patients was 62% vs. 28% in CK+ MK- and CK+ MK+ group (p = .01). No difference in CR rate was observed between DA and DAC arms. The overall survival (OS) in intensively treated patients was negatively influenced by MK, karyotype complexity (≥5 abnormalities), and WBC >20 G/L in multivariate analysis. The addition of cladribine to DA regimen improved OS only in MK- but not in MK+ group. In conclusion, concomitance of MK with ≥5 chromosomal abnormalities is associated with dismal treatment outcome in AMK-CK+.

Relationship between in vitro drug sensitivity and clinical response of patients to treatment in chronic lymphocytic leukemia.

To improve the efficacy of therapeutic options in chronic lymphocytic leukemia (CLL) an in vitro system to determine the response of mononuclear blood cells from blood of patients was elaborated. The study combines four approaches, i.e., cell viability, apoptosis rate, differential scanning calorimetry (DSC), and immunoblotting to develop personalized therapy protocols based on the cell sensitivity to drug exposure of individual CLL patients. The complementary analyses were performed on 28 peripheral blood samples from previously untreated CLL patients before therapy. The induction and progress of apoptosis in CLL cells exposed in vitro to purine analogs combined with mafosfamide, i.e., cladribine + mafosfamide (CM) and fludarabine + mafosfamide (FM) were assessed using the above approaches. The changes in thermal profiles (decrease/loss of transition at 95±5˚C) coincided with an accumulation of apoptotic cells, a decrease in the number of viable cells, and differences in the expression of the apoptosis­related protein PARP­1. No significant changes were observed in the thermal profiles of nuclei isolated from CLL cells resistant to the treatment. The complementary assays revealed a strong relationship between both the in vitro sensitivity of leukemia cells to drugs and the clinical response of the patients, determined usually after the sixth course of treatment (after ~6 months of therapy). As a summary of studies followed by complementary tests, our findings demonstrate the value of in vitro exposure of CLL cell samples to drugs intended to treat CLL patients, before their administration in order to recommend the most suitable and effective therapy for individual patients.

Jagged-1: a new promising factor associated with favorable prognosis in patients with acute myeloid leukemia.

In this study Jagged-1 and Dll-1 surface expression as well as Notch-1 receptor intracellular domain (Notch-1-IC) expression were assessed by multi-color flow cytometry in leukemic blasts obtained from 88 patients with acute myeloid leukemia (AML). CD34+peripheral blood stem cells (PBSCs) were used as a control. The median expression of Jagged-1 and Dll-1 was significantly higher in AML blasts than in PBSCs (p=0.001 and p=0.002, respectively). Higher expression of Notch-1-IC was detected in patients with poor-risk karyotype as compared to good- and intermediate-risk groups (p=0.035). In our study, poor-risk cytogenetics and low (

The influence of low molecular weight heparin on the intravascular activation of the coagulation system in patients with acute leukemia during induction chemotherapy--report of a prospective randomized study.

UNLABELLED: Intravascular activation of the coagulation system can already be observed in the majority of patients with acute leukemia (AL) at the time of diagnosis. This activation can be further enhanced by chemotherapy. The study comprised of 46 patients with AL, randomly divided into two groups. Twenty-three patients received prophylactic doses of nadroparin (Fraxiparine). Thrombin-antithrombin complexes (TAT), prothrombin fragment (F1 + 2), D-dimer (DD), plasmin-antiplasmin complexes (PAP) and antithrombin III (AT III) activity were determined in all patients. The tests were performed before treatment, and on the 3rd and 8th days of chemotherapy. The TAT, F1 + 2, DD and PAP concentrations were found to be elevated in 83% of patients already at the time of diagnosis. No significant difference between either groups test results was noted when either tested before treatment or on the third day of therapy. DIC (disseminated intravascular coagulation) syndrome appeared in two patients receiving heparin prophylaxis and in three patients to whom this treatment was not administered. The concentrations of activation markers on the eighth day of chemotherapy were lower than at the beginning of treatment in most of the patients receiving nadroparin. At this time there were no laboratory signs of DIC in any of the patients receiving heparin prophylaxis. IN CONCLUSION: although the application of prophylactic doses of nadroparin does not prevent DIC syndrome during first days of chemotherapy, it may limit the intravascular activation of the coagulation system during later chemotherapy.

Chronic myelomonocytic leukemia coexisting with B-cell chronic lymphocytic leukemia.

Coexistence of B-cell chronic lymphocytic leukemia (B-CLL) and chronic myelomonocytic leukemia (CMML) is an unusal event, and to our knowledge, only four such cases have been reported in the literature. We report a 68-year-old white woman in whom these two diseases were diagnosed concomitantly. The diagnosis was made on the basis of peripheral blood count, morphology and immunophenotyping, and bone marrow cytology and histology. Interphase FISH analysis detected a 13q14.3 deletion in lymphocytes nuclei and no such abnormality in monocytes nuclei. The PCR analysis of IgH gene rearrangement in the bone marrow, as well as the peripheral blood lymphocytes, showed two different monoclonal IgH configurations as the result of biallelic clonal rearrangement of IgH genes suggesting an origin of lymphocytes from B-cell progenitors. The patient was originally treated with prednisone 1 mg/kg/day because of progressive significant thrombocytopenia, without improvement. Subsequently, she received one course of cladribine (2-CdA). Significant reduction of lymphocytes in the peripheral blood was observed. However, rapid increase of monocytes was seen shortly after the 2-CdA treatment. Subsequently, she received hydroxyurea (1.5 g/day) without hematological improvement. The patient died in January 2003, three months after diagnosis because of progression of both leukemias and associated pneumonia. Possible etiopathogenic relationship between both disorders is discussed.

Hodgkin's type of Richter's syndrome in familial chronic lymphocytic leukemia treated with cladribine and cyclophosphamide.

Second malignancies are frequent complications in patients with chronic lymphocytic leukemia (CLL). Hodgkin's disease (HD) has been observed in approximately 0.5% of the patients with CLL and is known as Hodgkin's type Richter's syndrome (H-RS). We present a 64-year-old male patient with a familial history of CLL who developed H-RS in abdominal lymph nodes 6 years after CLL diagnosis and 18 months after treatment with cladribine (2-CdA) and cyclophosphamide. HD was diagnosed by fine needle aspiration. The disease was refractory to treatment with two courses of CHOP and three courses of ABVD chemotherapy. In the current literature we found case reports of only 6 patients with H-RS who were treated with fludarabine (FA) before transformation, and, to our knowledge the presented patient is the first to develop H-RS after treatment with 2-CdA combined with cyclophosphamide. He is also the first published patient with familial CLL in whom this complication developed.

Coexistence of chronic lymphocytic leukemia and essential thrombocythemia.

The association of chronic lymphocytic leukemia (CLL) with essential thrombocythemia (ET) is an extremely rare event and until now 3 patients with such coexistence have been reported in the literature. We report a 77-year-old white woman in whom these two disorders were diagnosed concomitantly on the basis of peripheral blood count and cytology, bone marrow cytology and histology, immunophenotyping, as well as exclusion criteria. The diagnosis of ET was also supported by spontaneous in-vitro erythroid colony growth and by evaluation of thrombopoietin (TPO) serum level. Interphase FISH analysis allowed to detect 13q14.3 deletion in 98% of lymphocytes nuclei. In contrast this aberration was not observed in the megakaryocytes. The results of PCR analysis of IgG gene rearrangement showed distinct bands characteristic for monoclonal lymphoid population in bone marrow, peripheral blood and inguinal lymph node. The patient was started on hydroxyurea 1 g/day and normalization of the platelet count was achieved. Possible etiopathogenic relationships between both disorders and differential diagnosis of ET and reactive thrombocytosis (RT) are discussed.

In vivo and ex vivo responses of CLL cells to purine analogs combined with alkylating agent.

BACKGROUND: The heterogeneity of chronic lymphocytic leukemia (CLL) is thought to be due to differences in the expression of factors that regulate apoptosis and cell cycle, giving rise to diverse apoptotic disturbances and tumor properties. Therefore, the primary goal in CLL treatment is to overcome resistance to apoptosis and efficiently trigger this process in leukemic cells. METHODS: Mononuclear cells were obtained from the blood of CLL patients by Histopaque-1077 sedimentation. CLL cell samples from the blood of drug treated patients, (cladribine or fludarabine with cyclophosphamide; CC or FC), as well as the cell samples of untreated patients exposed to the used drug combinations (CM, FM) or mafosfamide alone for 48 h were fractionated into nuclear and cytoplasmic fractions or were lysed. DNA fragmentation was evaluated by agarose electrophoresis and also cytometrically as sub-G1 population. The expression of apoptosis related proteins and H1.2 histone translocation were evaluated in lysates and nuclear and cytoplasmic fractions, respectively with appropriate antibodies. RESULTS: Cladribine (C) and fludarabine (F) combined with cyclophosphamide/mafosfamide in vivo, as well as ex vivo trigger apoptosis in CLL cells. These drug combinations (CC; FC/CM; FM) induce leukemic cell apoptosis confirmed by DNA fragmentation, sub-G1 cell number, down-regulation of anti-apoptotic proteins (Mcl-1, Bcl-2), and H1.2 histone translocation in comparison with appropriate control cells, however, to a different degree. CONCLUSIONS: The kinetics and rate of drug-induced apoptosis in leukemic cells under ex vivo experiments differ between patients, mirroring the differences noticed during in vivo treatment. Individual model cell samples indicate comparable susceptibility to the used drug combinations under in vivo and ex vivo conditions.