MRP1 protein expression in leukemic stem cells as a negative prognostic marker in acute myeloid leukemia patients.

BACKGROUND: It is well established that expression of multi-drug resistance (MDR) proteins (MDR1, BCRP, MDR3, MRP1, and LRP) in leukemic blasts correlates with acute myeloid leukemia (AML) patients' clinical response. Assuming that leukemic stem cells (LSC) are resistant to chemotherapy and responsible for relapse, it might be clinically relevant to evaluate the expression level of MDR proteins in LSC and relate it to the clinical outcome. METHODS: Bone marrow samples from 26 patients with de novo AML were labeled with antibodies to distinguish CD34+CD38-CD123+ LSC population and with antibodies against MDR1, BCRP, MDR3, MRP1, or LRP proteins. Multicolor flow cytometry was applied to evaluate the expression of MDR proteins in blasts and LSC. RESULTS: Nine of 26 patients with AML attained CR (30%). High negative correlation was found between MDR1 and LRP expression in blasts and the patient's remission. MDR proteins were expressed more frequently in LSC than in leukemic blasts. High negative correlation was also observed between remission achievement and MRP1 expression in LSC. CONCLUSIONS: Our data present for the very first time the high negative correlation between MRP1 protein expression in LSC and AML patients' remission. It does strongly suggest that MRP1 expression in LSC is an adverse prognostic marker in patients with de novo AML.

CEBPA copy number variations in normal karyotype acute myeloid leukemia: Possible role of breakpoint-associated microhomology and chromatin status in CEBPA mutagenesis.

Copy number variations (CNV) in CEBPA locus represent heterogeneous group of mutations accompanying acute myeloid leukemia (AML). The aim of this study was to characterize different CEBPA mutation categories in regard to biological data like age, cytology, CD7, and molecular markers, and identify possible factors affecting their etiology. We report here the incidence of 12.6% of CEBPA mutants in the population of 262 normal karyotype AML (NK-AML) patients. We confirmed that double mutant AMLs presented uniform biological features when compared to single CEBPA mutations and accompanied mostly younger patients. We hypothesized that pathogenesis of distinct CEBPA mutation categories might be influenced by different factors. The detailed sequence analysis revealed frequent breakpoint-associated microhomologies of 2 to 12bp. The analysis of distribution of microhomology motifs along CEBPA gene showed that longer stretches of microhomology at the mutational junctions were relatively rare by chance which suggests their functional role in the CEBPA mutagenesis. Additionally, accurate quantification of CEBPA transcript levels showed that double CEBPA mutations correlated with high-level CEBPA expression, whereas single N-terminal CEBPA mutations were associated with low-level CEBPA expression. This might suggest that high-level CEBPA expression and/or accessibility of CEBPA locus contribute to B-ZIP in-frame duplications.

The early reduction of leukemic blasts in bone marrow on day 6 of induction treatment is predictive for complete remission rate and survival in adult acute myeloid leukemia; the results of multicenter, prospective Polish Adult Leukemia Group study.

The aim of this study was to prospectively evaluate the impact of early bone marrow response on complete remission (CR) rate and long-term outcome in adults with acute myeloid leukemia. Bone marrow cytology was assessed on day 6 of induction treatment in 164 patients, revealing the presence of ≥5% blasts in 61 cases. In this subgroup the CR rate was significantly lower compared to the remaining patients (P < 0.00001) resulting in decrease of the overall survival (P = 0.002). Persistence of ≥5% blasts in bone marrow on day 6 of induction is an easily available surrogate marker to be used for treatment decisions.

31P MRS analysis of the phospholipid composition of the peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) of patients with acute leukemia (AL).

The aim of this study was to evaluate the phospholipid concentration in acute leukemia (AL) blast cells from peripheral blood (PBMC) and bone marrow (BMMC). In vitro 31P Nuclear Magnetic Resonance Spectroscopy (31P MRS) was used. The integral intensities of the resonant peaks and the phospholipid concentrations in PBMC and BMMC were analyzed. Differences in the phospholipid concentrations in cells from myeloblastic or lymphoblastic lines were also evaluated. This investigation was carried out on phospholipid extracts from PBMC and BMMC from 15 healthy volunteers and 77 patients with AL (samples taken at the moment of diagnosis). A significant decrease in sphingomyelin (SM) and phosphtidylserine (PS) was observed in the PBMC of patients with AL relative to the results for the healthy volunteers. For ALL, we found a significant decrease in the concentration of phosphatidylcholine plasmalogen (CPLAS), SM, PI+PE (phosphatidylinositol + phosphatidylethanolamine) and PS in comparison with the results for healthy volunteers and patients with AML. Experiments with BMMC cells revealed a significant decrease in the concentration of CPLAS, SM, PI+PE, and PS in ALL relative to AML. Additionally, a significant decrease in phosphatidylcholine (PC) concentration was observed in ALL compared to AML. If the phospholipid extracts were taken simultaneously from the same patient, there were no significant differences in the integral intensities and phospholipid concentrations between PBMC and BMMC.

Status of minimal residual disease after induction predicts outcome in both standard and high-risk Ph-negative adult acute lymphoblastic leukaemia. The Polish Adult Leukemia Group ALL 4-2002 MRD Study.

The treatment of adults with Philadelphia-negative acute lymphoblastic leukaemia (ALL) depends on the presence of risk factors including age, white blood cell count, immunophenotype and time to complete remission. In recent years, status of minimal residual disease (MRD) has been postulated as an additional risk criterion. This study prospectively evaluated the significance of MRD. Patients were treated with a uniform Polish Adult Leukemia Group (PALG) 4-2002 protocol. MRD status was assessed after induction and consolidation by multiparametric flow cytometry. Out of 132 patients included (age, 17-60 years), 116 patients were suitable for analysis. MRD level >/=0.1% of bone marrow cells after induction was found to be a strong and independent predictor for relapse in the whole study population (P < 0.0001), as well as in the standard risk (SR, P = 0.0003) and high-risk (P = 0.008) groups. The impact of MRD after consolidation on outcome was not significant. The combination of MRD status with conventional risk stratification system identified a subgroup of patients allocated to the SR group with MRD <0.1% after induction who had a very low risk of relapse of 9% at 3 years as opposed to 71% in the remaining subjects (P = 0.001). We conclude that MRD evaluation after induction should be considered with conventional risk criteria for treatment decisions in adult ALL.

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.

Side-chain modified vitamin D analogs induce rapid accumulation of VDR in the cell nuclei proportionately to their differentiation-inducing potential.

1,25-Dihydroxyvitamin D(3) (1,25D) regulates gene transcription through a nuclear vitamin D receptor (VDR) which acts as a ligand-regulated transcription factor. Some structural vitamin D analogs (VDAs) are selective in their biological actions, because they retain cell-differentiating potential, while their calcemic activity is reduced. In this article we have shown that in untreated HL60 cells the expression level of VDR is low, in spite of constant presence of VDR mRNA. Furthermore we have shown that one of the most rapid effects of either 1,25D or VDAs is nuclear accumulation of VDR, which is proportional to the differentiation-inducing potential of given analog. We observed this effect not only in HL60 cells, but also in blast cells isolated from patients with acute myeloid leukemias. After longer incubation time of the cells with various VDAs, the expression levels of VDR have become unrelated to the final differentiation effect.

Platelet-activating factor changes in phospholipid extracts from the plasma, peripheral blood mononuclear cells and bone marrow mononuclear cells of patients with acute leukemia--a 31P MRS in vitro study.

The aim of this investigation was to evaluate the changes in PAF concentrations in the plasma, PBMC and BMMC of patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The plasma was from 23 healthy volunteers (HV) and 44 patients with AL (16 ALL, 28 AML). The PBMC were from 15 HV and 55 patients with AL (18 ALL, 37 AML), and the BMMC from 40 patients with AL (11 ALL, 29 AML). Methanol-chloroform phospholipid extraction from 60 x 10(6) cells (PBMC or BMMC) was performed according to a modified version of Folch's method. (31)P MRS data was obtained on an AMX 300 Bruker spectrometer (7.05 T). The PAF concentration in the plasma of the patients with ALL or AML was lower than that for the healthy volunteers. The PAF concentration in the plasma of the patients with ALL did not differ significantly from that of the patients with AML. In the case of both the PBMC and BMMC, the PAF concentration was significantly diminished in patients with ALL relative to the concentration for those with AML and for the healthy volunteers. No differences were observed in the PAF concentrations for the AML patients and the healthy volunteers.

Constitutional mutations of the CHEK2 gene are a risk factor for MDS, but not for de novo AML.

CHEK2 plays a key role in cellular response to DNA damage, and also in regulation of mitosis and maintenance of chromosomal stability. In patients newly diagnosed with myelodysplastic syndrome (MDS, n = 107) or acute myeloid leukemia (AML, n = 117) congenital CHEK2 mutations (c.444 + 1G > A, c.1100delC, del5395, p.I157 T) were tested by PCR and sequencing analysis. The karyotype of bone marrow cells of each patient was assessed at disease diagnosis using classical cytogenetic methods and fluorescence in situ hybridization. The CHEK2 mutations were strongly associated with the risk of MDS (p < 0.0001) but not with the risk of de novo AML (p = 0.798). In CHEK2-positive MDS patients, two times higher frequency of aberrant karyotypes than in CHEK2-negative patients was found (71% vs. 37%, p = 0.015). In CHEK2-positive patients with cytogenetic abnormalities, subtypes of MDS: refractory anemia with excess blasts-1 or 2, associated with unfavorable disease prognosis, were diagnosed two times more often than in CHEK2-negative cases with aberrations (78% vs. 44%). In conclusion, the congenital CHEK2 inactivation is strongly associated with the risk of MDS and with a poorer prognosis of the disease. However, the chromosomal instability in AML is not correlated with the hereditary dysfunction of CHEK2.

Activation of intracellular signaling pathways is necessary for an increase in VDR expression and its nuclear translocation.

1,25-Dihydroxyvitamin D(3) (1,25D) regulates gene transcription through the nuclear vitamin D receptor (VDR) and initiates rapid cellular responses via an unknown mechanism. Here we report that 1,25D induces a rapid increase in synthesis of VDR protein and its transport to the nucleus. These results are similarly obtained in myeloid leukemia cell lines, and in blast cells from blood of patients diagnosed with acute myeloid leukemia, subtypes M2 and M4. Our results suggest that stability of unliganded VDR is LY294002- and PD98059-dependent, and that ligation of VDR leads to its increased translation and nuclear translocation. The receptor localized in the cell nucleus is not exported back to the cytosol by exportin 1. We also show that the cytosolic portion of VDR in leukemia cells is localized in the vicinity of the plasma membrane, close to the F-actin cytoskeleton.

The soluble CTLA-4 receptor: a new marker in autoimmune diseases.

A soluble form of cytotoxic T lymphocyte-associated antigen-4 (sCTLA-4) was recently found and shown to possess B7 binding activity. sCTLA-4 is generated by alternatively spliced mRNA. The mRNA encoding sCTLA-4 consists of 3 exons: exon 1 encodes a leader peptide, exon 2 the ligand binding domain, and exon 4 the cytoplasmic tail, but it lacks the transmembrane domain encoded by exon 3. The altered transcript is detected in resting CD4 and CD8 T cells and its expression is inhibited after 24--48 h of activation and returns to the prestimulation level after 72--120 h of activation. Low levels of sCTLA-4 have been detected in normal human serum and increased serum levels have been observed in several autoimmune diseases (e.g. Graves' disease, myasthenia gravis, systemic lupus erythematosus, and systemic sclerosis). The biological significance of increased sCTLA-4 serum level has not been clarified. On one hand, sCTLA-4 may bind B7 expressed on antigen-presenting cells and is thus able to interfere with the B7:CD28-mediated costimulation of T cell responses. On the other hand, sCTLA-4 may also be capable of interfering with B7:CTLA-4 interactions, thereby blocking the negative signal imparted via the full-length form of CTLA-4. This double-edged nature of B7 blocking by sCTLA-4 may result in different outcomes of the clinical course of disease.

Monocytic differentiation induced by side-chain modified analogs of vitamin D in ex vivo cells from patients with acute myeloid leukemia.

The differentiation-inducing potential of side-chain modified analogs of vitamins D, compared to the reference compound, 1,25-dihydroxyvitamin D3, was studied in blast cells from patients with acute myeloid leukemia and in cell lines. Analogs PRI-1906 and PRI-1907 showed increased cell-differentiation activities, PRI-1907 even at a very low concentration. Our study revealed a high variability of individual patients' blasts in their susceptibility to vitamin D analogs. The blasts of the patients with normal karyotype and with mutated NPM1 reacted to analogs with stronger differentiation than the blasts of the remaining patients, while the blasts with mutated FLT3 receptor reacted with weaker differentiation than the remaining blasts.

Variations in suppressor molecule ctla-4 gene are related to susceptibility to multiple myeloma in a polish population.

Various phenotype and functional T-cell abnormalities are observed in multiple myeloma (MM) patients. The aim of this study was to investigate the association between polymorphisms in the gene encoding cytotoxic T-lymphocyte antigen-4 (CTLA-4), a negative regulator of the T-lymphocyte immune response and susceptibility to multiple myeloma in a Polish population. Two hundred MM patients and 380 healthy subjects were genotyped for the following polymorphisms: CTLA-4c.49A>G, CTLA-4g.319C>T, CTLA-4g.*642AT(8_33), CT60 (CTLA-4g.*6230G>A), Jo31 (CTLA-4g.*10223G>T). Our study is the largest and most comprehensive evaluation to date of the association between genetic polymorphisms in the CTLA-4 molecule and multiple myeloma. It was found that CTLA-4c.49A>G[G], CT60[G], and Jo31[G] alleles were more frequently observed in MM patients than in controls (0.50 vs. 0.44, p = 0.03, 0.65 vs. 0.58, p = 0.04, and 0.63 vs. 0.57, p = 0.03, respectively). Moreover, the haplotype CTLA-4c.49A>G[G], CTLA-4g.319C>T[C], CTLA-4g.*642AT(8_33) [8], CT60[G], Jo31[G] including all susceptibility alleles increases the risk of MM about fourfold (OR: 3.79, 95%CI: 2.08-6.89, p = 0.00001). These findings indicate that genetic variations in the CTLA-4 gene play role in susceptibility to multiple myeloma and warrant further investigation through replication studies.

Cladribine, but not fludarabine, added to daunorubicin and cytarabine during induction prolongs survival of patients with acute myeloid leukemia: a multicenter, randomized phase III study.

PURPOSE: The goal of this study was to evaluate whether the addition of a purine analog, cladribine or fludarabine, to the standard induction regimen affects the outcome of adult patients with acute myeloid leukemia (AML). PATIENTS AND METHODS: A cohort of 652 untreated AML patients with median age 47 years (range, 17 to 60 years) were randomly assigned to receive one of three induction regimens: DA (daunorubicin plus cytarabine), DAC (DA plus cladribine), or DAF (DA plus fludarabine). Postremission treatment was the same for all arms. RESULTS: Complete remission rate in the DAC arm was higher compared with the DA arm (67.5% v 56%; P = .01) as a consequence of reduced incidence of resistant disease (21% v 34%; P = .004). There was no significant difference in early outcome between the DAF and DA arms. The probability of overall survival was improved for the DAC arm (45% ± 4% at 3 years) compared with the DA arm (33% ± 4%; P = .02), and leukemia-free survival was comparable. Long-term outcome did not differ significantly for the comparison of the DAF and DA arms. A survival advantage of the DAC arm over the DA arm was observed among patients age 50 years or older (P = .005), those with initial leukocyte count above 50 × 10(9)/L (P = .03), and those with unfavorable karyotype (P = .03). DAF revealed a significant advantage over DA in patients with adverse karyotype (P = .02). CONCLUSION: The addition of cladribine to the standard induction regimen is associated with increased rate of complete remission and improved survival of adult patients with AML.

ICOS gene polymorphisms in B-cell chronic lymphocytic leukemia in the Polish population.

There is strong evidence that altered immunological function entails an increased risk of B-cell chronic lymphocytic leukemia (B-CLL). The main mechanism of an anti-tumor response depends on T-cell activation. Unlike the constitutively expressed CD28, inducible costimulatory molecule (ICOS) is expressed on the T-cell surface after activation. ICOS enhances all the basic T-cell responses to a foreign antigen, namely proliferation, secretion of lymphokines, the upregulation of molecules that mediate cell-cell interaction, and effective help for antibody secretion by B cells. ICOS is essential for both efficient interaction between T and B cells and normal antibody responses to T cell-dependent antigens. It does not upregulate the production of interleukin-2, but superinduces the synthesis of interleukin-10. Our previous results indicated the ICOS gene has a role as a susceptibility locus to B-CLL. Therefore an extended study was undertaken to evaluate the association between four ICOS polymorphisms (which were recently described as functional ones) and susceptibility to B-CLL in the Polish population. A case-control study of 296 individuals, including 146 B-CLL patients, was conducted on four polymorphisms in the ICOS gene. Genotyping of the polymorphisms ICOS ISV1+173T>C (rs10932029), ICOSc.1624C>T (rs10932037), ICOSc.2373G>C (rs4675379), and ICOSc.602A>C (rs10183087) was carried out using allelic discrimination methods with the TaqMan SNP Genotyping Assay. There were no statistically significant differences in the allele, genotype, or haplotype distributions between B-CLL patients and healthy controls for any of the investigated polymorphic markers in the ICOS gene. However, we noted that patients carrying genotype ICOS ISV1+173T>C [TT], ICOSc.602A>C [AA], ICOSc.1624C>T [CC], and ICOSc.2373G>C [GG] have a decreased frequency of progression to a higher Rai stage during 60-month follow-up (21.35% vs. 40.8%, p = 0.013) compared to other individuals. This indicates that the investigated polymorphisms do not modulate the risk of B-CLL in the Polish population, but are associated with disease dynamics, in particular with the time to Rai stage progression.

Different susceptibilities to 1,25-dihydroxyvitamin D3-induced differentiation of AML cells carrying various mutations.

This study was designed to compare the differentiation-inducing potential of 1,25-dihydroxyvitamin D(3) (1,25D) with some analogs (VDAs) in a panel of acute myeloid leukemia (AML) cell lines and in blast cells isolated from patients with AML. Of the cell lines studied, HL60 proved to be the most sensitive to each of the differentiation-inducing agents when compared to THP-1, NB-4 and U-937 cell lines. Three of the VDAs tested (PRI-1906, PRI-2191 and PRI-2201) were similarly effective as 1,25D in all the cell lines tested. However, blast cells from AML showed a varying sensitivity towards 1,25D. For example, blast cells isolated from patients in which the whole or part of chromosome 7 was deleted were extremely sensitive to 1,25D and its analogs. In contrast, 1,25D failed to increase the expression of differentiation markers in blast cells isolated from patients carrying activating mutations in Flt3 gene. Since, the expression of vitamin D receptor (VDR) in cells with Flt3 mutations was increased to the same extent as in other AML cells this suggests that failure of these cells to differentiate lies downstream of the receptor. That blast cells with different cytogenetic abnormalities have dissimilar responses to 1,25D and its analogs, may have implications in the use of 1,25D as a 'differentiation therapy' for myeloid leukemias. The analog PRI-2191 (tacalcitol) was found to be the most potent in inducing patient's cells differentiation.

Infectious complications in patients with acute myeloid leukemia treated according to the protocol with daunorubicin and cytarabine with or without addition of cladribine. A multicenter study by the Polish Adult Leukemia Group (PALG).

OBJECTIVES: The addition of cladribine to the standard regimen consisting of daunorubicin and cytarabine has been reported to increase the efficacy of induction therapy in acute myeloid leukemia (AML). The goal of this study was to determine the effect of this modification on the incidence and spectrum of infectious complications. METHODS: Case report forms of 309 patients with newly diagnosed AML who had been enrolled in the prospective, randomized 'DAC-7 vs. DA-7' trial were reviewed. The frequency, etiology, localization, severity, and outcome of infections were compared for patients receiving only daunorubicin and cytarabine (DA-7) and those additionally treated with cladribine (DAC-7). RESULTS: A total of 443 febrile episodes were reported with no significant difference between the treatment groups. A trend towards a higher frequency of bacteremias was observed among DA-7 patients compared to those in the DAC-7 group (31% vs. 21%; p=0.08). The treatment arms did not differ in terms of the distribution of the isolated Gram-positive, Gram-negative, fungal, and viral organisms. However, when bacteremias were considered, Gram-positive blood cultures tended to be more frequent in the DA-7 compared to the DAC-7 group (16% vs. 8.5%; p=0.07). This difference reached statistical significance when major blood bacteremias were analyzed separately (13% vs. 5%; p=0.02). Complete recovery from infections was observed in the majority of patients across both treatment arms and no significant difference was noted regarding infection-related mortality. CONCLUSIONS: The addition of cladribine to standard induction chemotherapy has no impact on the incidence and spectrum of infectious complications in newly diagnosed AML patients.