Frieda Kontsioti

A clinicopathological study of B-cell differentiation markers and transcription factors in classical Hodgkin’s lymphoma: a potential prognostic role of MUM1/IRF4

Background and Objectives Although most patients with classical Hodgkin’s lymphoma (CHL) are cured, a significant minority are refractory to treatment. The investigation of biological markers could improve the predictive capacity of clinical staging systems. The aim of our study was to detect B-cell differentiation markers and transcription factors in CHL in order to define subgroups with different histogeneses and prognoses. Design and Methods We evaluated 107 cases of CHL for BCL6, CD79a, MUM1/IRF4 and B-cell transcription factors BOB.1, OCT.2 expression by immunohistochemistry. Statistical analysis was performed using Fisher’s exact test, the Mann-Whitney test, the Kaplan-Meier method and the log rank test. Univariate and multivariate regression analyses were performed to identify variables with a significant effect on survival. Results CD79a was expressed in 5.8%, BCL6 in 14.7%, MUM1/IRF4 in 92.3%, BOB.1 in 53.4% and OCT.2 in 12.6% of cases. There was no significant association between CD79a or BCL6 expression and clinical characteristics. Univariate analysis showed that age of 45 or more, stage III and IV disease and MUM/IRF4 negative status were associated with significantly shorter time to progression (TTP) and overall survival (OS). On multivariate analysis the lack of MUM/IRF4 expression was associated with significantly shorter TTP while age of 45 or more and the presence of extralymphatic sites of disease were associated with significantly shorter OS. Interpretation and Conclusions Our study has confirmed that MUM1/IRF4 is expressed in most cases of CHL and shows that lack of this expression in a minority of cases may be a potential adverse prognostic factor.

Cell cycle and apoptosis regulatory gene expression in the bone marrow of patients with de novo myelodysplastic syndromes (MDS)

Deregulation of cell cycle and apoptosis pathways are known contributors to the pathogenesis of myelodysplastic syndromes (MDS). However, the underlying mechanisms are not fully clarified. The aim of our study was to examine mRNA expression levels of cell cycle and apoptosis regulatory genes, as well as the percentage of apoptotic and S phase cells and to correlate the findings with clinical characteristics and prognosis. Sixty patients with MDS, classified according to FAB (17 RA, five RARS, 19 RAEB, nine RAEBT, ten CMML) and WHO (ten RA, three RARS, seven RCMD, two RCMD-RS, 11 RAEBI, eight RAEBII, ten CMML, and nine AML) were included in the study. We found increased expression of anti-apoptotic bclxL and mcl1 genes and decreased expression of p21 gene in MDS patients. Moreover, we found increased expression of anti-apoptotic mcl1 gene in patients with higher than Intermediate-1 IPSS group. Multivariate analysis confirmed that combined expression of apoptotic caspases 8, 3, 6, 5, 2, 7, and Granzyme B was decreased in MDS patients. Regarding cell cycle regulatory genes expression, we demonstrated increased expression of cyclin D1 in patients with CMML Increased combined expression of cyclins B, C, D1, and D2 was found in patients with cytogenetic abnormalities. The two pathways seem to be interconnected as shown by the positive correlation between CDKs 1, 2, 4, p21 and the level of apoptosis and positive correlation between apoptotic caspase 3 expression and the percentage of S phase cells. In conclusion, our study showed altered expression of genes involved in apoptosis and cell cycle in MDS and increased expression of cyclin D1 in patients with CMML.

Dasatinib inhibits proliferation and induces apoptosis in the KASUMI-1 cell line bearing the t(8;21)(q22;q22) and the N822K c-kit mutation

Activating mutations of the c-kit gene are frequently found in CBF (core binding factor) leukemias. We evaluated the effect of tyrosine kinase inhibitor dasatinib in leukemic cell lines bearing or not c-kit mutations. Our data demonstrate that in the AML Kasumi-1 cell line, bearing the N822K c-kit mutation, dasatinib is a potent suppressor of c-kit and Src kinase activity and inhibits the phosphorylation of their downstream target AKT, possibly through the Src-mediated VEGF/VEGFR receptor type 2 pathway. Dasatinib also effectively blocks proliferation and induces apoptosis through caspase-3 activation in Kasumi-1 cells. These data further encourage the integration of dasatinib in the treatment of CBF AML with c-kit mutations in the context of clinical trials, which are eagerly anticipated.

Expression analysis of proteins involved in the non homologous end joining DNA repair mechanism, in the bone marrow of adult de novo myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are characterized by genetic instability which is associated with abnormal DNA repair mechanisms. The most lethal type of DNA damage are double strand DNA breaks (DSBs), which are mainly repaired by Non Homologous End Joining Mechanism (NHEJ), whose core enzyme components include the Ku70/Ku80 heterodimer, DNA–PKcs, XRCC4 and DNA Ligase IV. The aim of the present study was the analysis of expression of proteins required for NHEJ in bone marrow cells of adult de novo MDS and their association with clinical characteristics and prognosis. Our analysis included 48 cases of MDS; 19 RA, 5 RARS, 19 RAEB, 3 RAEB-T, 1 CMML, 1 transformation to AML according to FAB classification. The expression of the enzymes Ku70, Ku80, XRCC4, DNA-PKcs and Ligase IV was determined by Western Blotting. The mean Ligase IV expression value was significantly lower in MDS patients compared to normal controls (0.53 vs. 0.78, p = 0.03). A negative correlation was found between karyotype risk group and Ligase IV values. (p = 0.05). Moreover, Ku70 expression levels were significantly lower in patients with a good prognosis karyotype (p = 0.04). Furthermore, a negative correlation between Ku70 expression values and Hb levels was observed (p = 0.04). Finally, a positive correlation was observed between enzyme Ku70 expression values and level of blasts (p = 0.04). Our findings suppor-t a potential role of NHEJ enzyme Ligase IV in the pathogenesis of MDS. Larger numbers of cases need to be screened in order to draw definite conclusion.

Apoptosis Induction and Gene Expression Profile Alterations of Cutaneous T-Cell Lymphoma Cells following Their Exposure to Bortezomib and Methotrexate

Mycosis fungoides (MF) and its leukemic variant Sézary syndrome (SS) comprise the majority of CTCL, a heterogenous group of non-Hodgkins lymphomas involving the skin. The CTCL’s resistance to chemotherapy and the lack of full understanding of their pathogenesis request further investigation. With the view of a more targeted therapy, we evaluated in vitro the effectiveness of bortezomib and methotrexate, as well as their combination in CTCL cell lines, regarding apoptosis induction. Our data are of clinical value and indicate that the bortezomib/methotrexate combinational therapy has an inferior impact on the apoptosis of CTCL compared to monotherapy, with bortezomib presenting as the most efficient treatment option for SS and methotrexate for MF. Using PCR arrays technology, we also investigated the alterations in the expression profile of genes related to DNA repair pathways in CTCL cell lines after treatment with bortezomib or methotrexate. We found that both agents, but mostly bortezomib, significantly deregulate a large number of genes in SS and MF cell lines, suggesting another pathway through which these agents could induce apoptosis in CTCL. Finally, we show that SS and MF respond differently to treatment, verifying their distinct nature and further emphasizing the need for discrete treatment approaches.

Analysis of FLT3 gene mutations in de novo myelodysplastic syndromes. FLT3 internal tandem duplication detected in a case of refractory anemia

FLT3 (FMS-like receptor tyrosine kinase) gene, a member of the class III receptor tyrosine kinase family (RTK), is expressed on hematopoietic progenitor cells and mediates stem cell proliferation and differentiation [1]. In recent years, an internal tandem duplication (ITD) within JM/TK-1 domains as a somatic mutation of the FLT3 gene has been reported in 20 – 25% of adult patients with acute myeloid leukemia (AML) and 3% of adult MDS [2 – 7]. FLT3/ITDs have been detected in all FAB subtypes of AML and correlated with poor prognosis [3,8 – 9]. This mutation results in constitutive activation of FLT3 kinase with subsequent activation of proliferative pathways [1]. In addition, point mutations at aspartic acid within the activation loop of FLT3 have also been described in AML (7%), MDS (3.4%), and acute lymphoblastic leukemia (ALL) (2.8%) [10]. The most common is the D835 mutation (G!T!Asp!Tyr), which has also been associated with constitutive activation of receptor. The aim of the present study was the concurrent detection and clinical significance of FLT3 mutations (ITD and D835) in the bone marrow of 64 adult patients with de novo MDS at diagnosis. This study included 64 newly diagnosed patients with de novo MDS. Clinical characteristics of the patients are shown in Table I. Morphologic subtypes of MDS were classified according to the FAB criteria [11]. The karyotype was regularly performed and was normal in 36/48 examined cases. Clonal karyotypic abnormalities were found in 12/48 (25%) of cases including 3 RA, 7 RAEB, and 2 RAEB-t as depicted in Table I. Data allowing the calculation of risk scores according to the International Prognostic Scoring System (IPSS) [12] were available in 39 cases as shown in Table I. Bone marrow mononuclear cells from patients with MDS before the institution of any treatment were obtained after informed consent. Bone marrow mononuclear cells from patients with lymphoma without bone marrow involvement were used as a normal control. Bone marrow mononuclear cells were separated on Histopaque gradient. High molecular weight DNA was extracted using the phenol chloroform method. FLT3/ITD was examined by amplification of the JM domain from exon14 to exon15, using primers 14F: 50-GCAATTTAGGTATGAAAGC CAGC-30 and 15R: 50-CTTTCAGCATTTTGA CGGCAAC C-30, as previously described [2]. DNA from an AML patient known to carry the FLT3/ITD as demonstrated by direct sequencing was used as a positive control for the presence of the FLT3/ITD mutation. Exon 17 of FLT3 gene was amplified by PCR using the primers 17F: 50-CCG CCAGGAACG TGCTTG-30 and 17R: 50-GCAG CCTCACATTGCCCC-30 and digested with restriction enzyme EcoRV [10]. Normal products produce two bands of 68 bp and 46 bp following

Quantitative and qualitative analysis of regulatory T cells in B cell chronic lymphocytic leukemia

Accumulated data indicate a significant role of T cell dysfunction in the pathogenesis of chronic lymphocytic leukemia. In CLL, regulatory T cells are significantly higher and show lower apoptotic levels compared to healthy donors. We demonstrate that CLL derived CD4+CD25-CD127- and CD4+CD25lowCD127- subpopulations share a common immunophenotypic profile with conventional Tregs and are associated with advanced stage disease. We further provide evidence that the increased number of Tregs contributes indirectly to the proliferation of the CLL clone, by suppressing the proliferation of Teffs which in turn suppress CLL cells. These data are further supported by our observations that CLL derived Tregs appear rather incapable of inducing apoptosis of both normal B cells and CLL cells, in contrast to normal Tregs, suggesting an immunoediting effect of CLL cells on Tregs which negatively affects the functionality of the latter and contributes to the failure of Tregs in CLL to efficiently eliminate the abnormal clone.