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.

Polymorphisms of TNF and IL-10 genes and clinical outcome of patients with chronic lymphocytic leukemia.

Genetic variations in tumor necrosis factor (TNF) and interleukin-10 (IL-10) were reported to influence susceptibility to and outcome of patients with non-Hodgkin lymphoma. Therefore, we investigated whether single nucleotide polymorphisms in TNF and IL-10 may play a role in the clinical course of patients with chronic lymphocytic leukemia (CLL). TNF-308G>A, IL-10-3575T>A, and IL-10-1082A>G seem to be functionally relevant, were genotyped in 292 previously untreated patients with CLL. The control group consisted of 192 randomly selected blood donors. The patients carrying TNF-308GG and IL-10-1082AA genotypes presented a higher 3-year treatment-free survival (56.6 vs. 40.6%, P = 0.05) as well as a 10-year overall survival (OS) rates (92.3 vs. 57.6%, P = 0.005) than those with other TNF-308 and IL-10-1082 genotype combinations. Multivariate analysis demonstrated the Rai stage (P = 0.0002), IGHV mutation status (P = 0.01), TNF-308G>A (P = 0.03), and TNF/IL-10 polymorphism-based risk groups (P = 0.05) to be independent factors predicting OS. When the mutated IGHV patients were analyzed, the homozygotes TNF-308GG and IL-10-1082AA presented a higher 10-year OS rate than those carrying other TNF-308 and IL-10-1082 genotypes (100 vs. 67.7%, P = 0.01). In the unmutated IGHV patients, only the TNF-308G>A polymorphism influenced OS. The genetic variations in TNF and IL-10 genes work as independent predictors of survival and may play a role in the clinical course of CLL. It suggests inherited ability of the host to shift the balance between the Th1 and Th2 response, which in turn might contribute to the pathogenesis and prognosis of B-cell malignancies.

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 Prognostic Value of Whole-Blood PSMB5, CXCR4, POMP, and RPL5 mRNA Expression in Patients with Multiple Myeloma Treated with Bortezomib.

Proteasome inhibitors, like bortezomib, play a key role in the treatment of multiple myeloma (MM); however, most patients eventually relapse and eventually show multiple drug resistance, and the molecular mechanisms of this resistance remain unclear. The aim of our study is to assess the expression of previously described genes that may influence the resistance to bortezomib treatment at the mRNA level (ABCB1, CXCR4, MAF, MARCKS, POMP, PSMB5, RPL5, TXN, and XBP1) and prognosis of MM patients. mRNA expression was determined in 73 MM patients treated with bortezomib-based regimens (30 bortzomib-sensitive and 43 bortezomib-refractory patients) and 11 healthy controls. RPL5 was significantly down-regulated in multiple myeloma patients as compared with healthy controls. Moreover, POMP was significantly up-regulated in MM patients refractory to bortezomib-based treatment. In multivariate analysis, high expression of PSMB5 and CXCR and autologous stem cell transplantation were independent predictors of progression-free survival, and high expression of POMP and RPL5 was associated with shorter overall survival.

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+.

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.

Different prognosis of acute myeloid leukemia harboring monosomal karyotype with total or partial monosomies determined by FISH: retrospective PALG study.

A monosomal karyotype (MK) was identified by banding techniques (BT) in acute myeloid leukemia (AML). However, BT may be insufficient to determine the actual loss of a complete chromosome, especially in complex karyotypes. We have investigated the effect of monosomy type, total (MK-t) and partial (MK-p), reevaluated by FISH, on prognosis. We have found that complete remission rate and probability of overall survival at 1 year was higher in MK-p (n=27) than MK-t (n=15) group (40% vs. 15.4%, P=0.19 and 30% vs. 9%, P=0.046, respectively). Our results indicate for the first time that monosomy type influences the prognosis of MK-AML.

Changes in the apoptotic gene expression profile in CLL patients treated with rituximab combined with cladribine and cyclophosphamide-preliminary results.

The study was aimed to investigate modifications of apoptotic gene expression profile by microarray technique in 10 patients with chronic lymphocytic leukemia by treatment with rituximab, cladribine and cyclophosphamide (RCC) according to IGHV mutational status. The TaqMan Low Density Array for 96 gene transcripts was used. Those modifications followed two distinctive patterns largely overlapping the IGHV mutational status. In the IGHV-mutated group, the expression of many proapoptotic genes increased after treatment as compared to initial value. Our results suggest that RCC drugs may act through influence on the expression of some apoptosis-involved genes dependently on the IGVH mutational status.

Chromosomal aberrations in chronic lymphocytic leukemia detected by conventional cytogenetics with DSP30 as a single agent: comparison with FISH.

The aim of our study was to estimate the usefulness for conventional cytogenetics (CC) of DSP30 as a single agent (CC-DSP30) for detecting the most important chromosomal aberrations revealed in CLL by FISH and to find other abnormalities possibly existing but undetected by FISH with standard probes. Using CC-DSP30, the metaphases suitable for analysis were obtained in 90% of patients. CC-DSP30 and FISH were similarly efficacious for detecting del(11)(q22) and trisomy 12, whereas FISH was more sensitive for del(13)(q14). Sole del(13)(q14) detected by FISH, in 50% of patients was associated with other aberrations revealed by CC-DSP30. Additionally, the most recurrent anomaly detected by CC-DSP30 were structural aberrations of chromosome 2.