[Show abstract][Hide abstract] ABSTRACT: Mutations in TP53, NOTCH1, and SF3B1 were analyzed in the CLL8 study evaluating first-line therapy with fludarabine and cyclophosphamide (FC) or FC with rituximab (FCR). TP53, NOTCH1, and SF3B1 were mutated in 11.5%, 10.0%, and 18.4% of patients, respectively. While NOTCH1(mut) and SF3B1(mut) virtually showed mutual exclusivity (0.6% concurrence), TP53(mut) was frequently found in NOTCH1(mut) (16.1%) and in SF3B1(mut) (14.0%) patients. There were few significant associations with clinical and laboratory characteristics but genetic markers had strong influence on response and survival. In multivariable analyses, an independent prognostic impact was found for FCR, thymidine kinase (TK) ≥10, unmutated IGHV, 11q deletion, 17p deletion, TP53(mut), and SF3B1(mut) on progression-free survival; and of FCR, age ≥65 years, ECOG performance status ≥0, β2-microglobulin ≥3.5, TK ≥10, unmutated IGHV, 17p deletion, and TP53(mut) on overall survival. Notably, predictive marker analysis identified an interaction of NOTCH1 mutational status and treatment, in that rituximab failed to improve response and survival in patients with NOTCH1(mut). In conclusion, TP53 and SF3B1 mutations appear among the strongest prognostic markers in CLL patients receiving current standard first-line therapy. NOTCH1(mut) was identified as a predictive marker for decreased benefit from the addition of rituximab to FC (FCR). This study is registered at ClinicalTrials.gov, identifier: NCT00281918.
[Show abstract][Hide abstract] ABSTRACT: In a single-center retrospective donor versus no-donor comparison, we investigated if allogeneic stem cell transplantation (alloSCT) can improve the dismal course of poor-risk chronic lymphocytic leukemia (CLL).
All patients with CLL who were referred for evaluation of alloSCT within a 7-year time frame and had a donor search indication according to the EBMT criteria or because of Richter's transformation were included. Patients for whom a matched donor could be found within 3 months (matches) were compared with patients without such a donor (controls). Primary end point was overall survival measured from the 3-month landmark after search initiation.
Of 105 patients with donor search, 97 (matches 83; controls 14) were assessable at the 3-month landmark. Matches and controls were comparable for age, gender, time from diagnosis, number of previous regimens, and remission status. Disregarding if alloSCT was actually carried out or not, survival from the 3-month landmark was significantly better in matches versus controls [hazard ratio 0.38, 95% confidence interval (CI) 0.17-0.85; P = 0.014]. The survival benefit of matches remained significant on multivariate analysis.
This study provides first comparative evidence that alloSCT may have the potential to improve the natural course of poor-risk CLL as defined by the EBMT criteria.
Annals of Oncology 01/2014; 25(1):200-6. DOI:10.1093/annonc/mdt511 · 7.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although clonal selection by genetic driver aberrations in cancer is well documented, the ability of epigenetic alterations to promote tumor evolution is undefined. We used 450k arrays and next-generation sequencing to evaluate intra-tumor heterogeneity and evolution of DNA methylation and genetic aberrations in chronic lymphocytic leukemia (CLL). CLL cases exhibit vast inter-patient differences in intra-tumor methylation heterogeneity, with genetically clonal cases maintaining low methylation heterogeneity and up to 10 percent of total CpGs in a monoallelically methylated state. Increasing methylation heterogeneity correlates with advanced genetic subclonal complexity. Selection of novel DNA methylation patterns is observed only in cases that undergo genetic evolution, and independent genetic evolution is uncommon and is restricted to low-risk alterations. These results reveal that although evolution of DNA methylation occurs in high-risk, clinically-progressive cases, positive selection of novel methylation patterns entail co-evolution of genetic alteration(s) in CLL.
Cancer Discovery 12/2013; 4(3). DOI:10.1158/2159-8290.CD-13-0349 · 19.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B cells. One of the major challenges in treatment of CLL is the achievement of a complete remission to prevent relapse of disease originating from cells within lymphoid tissues and subsequent chemoresistance. In search for novel drugs that target CLL cells also in protective microenvironments, we performed a fungal extract screen using cocultures of primary CLL cells with bone marrow-derived stromal cells. A metabolite produced by Penicillium aquamarinium was identified as Chaetoglobosin A, a member of the cytochalasan family that showed preferential induction of apoptosis in CLL cells, even under culture conditions that mimic lymphoid tissues. In vitro testing of 89 CLL cases revealed effective targeting of CLL cells by Chaetoglobosin A, independent of bad prognosis characteristics, like 17p deletion or TP53 mutation. To provide insight into its mechanism of action, we showed that ChA targets filamentous actin in CLL cells and thereby induces cell cycle arrest and inhibits membrane ruffling and cell migration. Our data further revealed that Chaetoglobosin A prevents CLL cell activation and sensitizes them for treatment with PI3K and BTK inhibitors, suggesting this compound as a novel potential drug for CLL.Leukemia accepted article preview online, 27 November 2013. doi:10.1038/leu.2013.360.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 11/2013; 28(6). DOI:10.1038/leu.2013.360 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recurrent gene mutations contribute to the pathogenesis of chronic lymphocytic leukaemia (CLL). We developed a next-generation sequencing (NGS) platform to determine the genetic profile, intratumoural heterogeneity, and clonal structure of two independent CLL cohorts. TP53, SF3B1, and NOTCH1 were most frequently mutated (16·3%, 16·9%, 10·7%). We found evidence for subclonal mutations in 67·5% of CLL cases with mutations of cancer consensus genes. We observed selection of subclones and found initial evidence for convergent mutations in CLL. Our data suggest that assessment of (sub)clonal structure may need to be integrated into analysis of the mutational profile in CLL.
British Journal of Haematology 08/2013; 163(4). DOI:10.1111/bjh.12539 · 4.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aminoferrocene-based prodrugs are activated at cancer specific conditions (high concentration of reactive oxygen species, ROS) with formation of glutathione scavengers (p-quinone methide) and ROS-generating iron complexes. Herein we explored three structural modifications of these prodrugs with an attempt to improve their properties: (a) attachment of a -COOH function to the ferrocene fragment leads to the improvement of water solubility and reactivity in vitro, but also decreases cell membrane permeability and biological activity; (b) alkylation of the N-benzyl residue does not show any significant affect; (c) attachment of the second arylboronic acid fragment improves toxicity (IC50) of the prodrugs towards human promyelocytic leukemia cells (HL-60) from 52 to 12 µM. Finally, we demonstrated that the prodrugs are active against primary chronic lymphotic leukemia (CLL) cells with the best compounds exhibiting IC50 value of 1.5 µM. These most active compounds were found not to affect mononuclear cells and representative bacterial cells.
[Show abstract][Hide abstract] ABSTRACT: Abstract Extracellular vesicles (EVs) are membrane-enclosed nanoparticles 30 to 1000 nm in size and represent a novel mechanism of cell communication. By transferring RNA and protein from their cell of origin, they can reprogram target cells and thus are involved in changes within the cellular microenvironment - a key player in CLL pathogenesis. In the current study, we were able to isolate EVs of 20 to 300 nm from blood plasma of CLL patients as well as from supernatant of primary CLL cells in culture. Further, proteomic profiling by Coomassie staining of SDS-PAGE gels and by mass spectrometry revealed an EV-specific protein profile. These findings suggest that EVs represent an important mean of CLL cells to interact with other cells, which might contribute to the establishment of a pro-survival microenvironment for CLL cells.
[Show abstract][Hide abstract] ABSTRACT: The ATM–p53 DNA damage response pathway plays a crucial role in chemoresistance in chronic lymphocytic leukemia, as indicated by the adverse prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM) detected by fluorescence in situ hybridization (FISH) analysis. In addition to deletions, mutations in these respective genes are also associated with chemoresistance, and add to the prognostic information provided by FISH. In order to explore the possibility that dysfunction of the ATM–p53 pathway might also result from mechanisms other than ATM/TP53 deletion/mutation, assays have been developed that probe the functional integrity of the ATM–p53 pathway. Currently, four different p53 function assays have been developed that are based on the measurement of p53 and p53-dependent genes at the RNA (real-time polymerase chain reaction [RT-PCR]p21; RT-PCRmiR34a; reverse transcription-multiplex ligation-dependent probe amplification assay [RT-MLPA]p21, bax, puma and CD95) or protein (fluorescence activated cell sorting [FACS]p53-p21) level in untreated cells or following irradiation or drug treatment. Here we provide an overview of these assays based on the available literature.
Leukemia and Lymphoma 07/2013; 54(8). DOI:10.3109/10428194.2013.796058 · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied the incidences, associations and prognostic roles of NOTCH1 and SF3B1 mutations (NOTCH1(mut), SF3B1(mut)) as compared to TP53(mut) in fludarabine-refractory chronic lymphocytic leukemia (CLL) patients treated with alemtuzumab on the CLL2H trial. We found NOTCH1(mut), SF3B1(mut) and TP53(mut) in 13.4%, 17.5% and 37.4% of patients, respectively. NOTCH1(mut) and SF3B1(mut) were mutually exclusive, whereas TP53(mut) were evenly distributed within both subgroups. Apart from correlation of SF3B1(mut) with 11q deletion (p=.029), there were no other significant associations of the mutations with any baseline characteristics or response rates. However, NOTCH1(mut) cases had a significantly longer progression-free survival (PFS) compared to wild type (WT) cases (15.47 vs. 6.74 months; p=.025) while there was no significant difference regarding OS. SF3B1(mut) had no significant impact on PFS and overall survival (OS). In multivariable analyses, NOTCH1(mut) was identified as an independent favorable marker for PFS. This clinical trial is registered at www.clinicaltrials.gov as #NCT00274976.
[Show abstract][Hide abstract] ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 06/2013; 27(9). DOI:10.1038/leu.2013.190 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Sensitive identification of mutations in genes related to the pathogenesis of cancer is a prerequisite for risk-stratified therapies. Next-generation sequencing (NGS) in lymphoma revealed genetic heterogeneity which makes clinical translation challenging. We established a 454-based targeted resequencing platform for robust high-throughput sequencing from limited material of lymphoma patients. Hotspot mutations in the most frequently mutated cancer consensus genes were amplified in a two-step multiplex-PCR which was optimized for homogenous coverage of all regions of interest. We show that targeted resequencing based on NGS technologies allows highly sensitive detection of mutations and assessment of clone size. The application of this or similar techniques will help develop genotype-specific treatment approaches in lymphoma.
[Show abstract][Hide abstract] ABSTRACT: In multiple myeloma, there has been little progress in the specific therapeutic targeting of oncogenic mutations. Whole-genome sequencing data has recently revealed that a subset of patients carry an activating mutation (V600E) in BRAF kinase. To uncover the clinical relevance of this mutation in multiple myeloma, we correlated the mutation status in primary tumor samples from 379 myeloma patients with disease outcome. We found a significantly higher incidence of extramedullary disease and a shorter overall survival in mutation carriers when compared to controls. Most importantly, we report on a patient with confirmed BRAF V600E mutation and relapsed myeloma with extensive extramedullary disease, refractory to all approved therapeutic options, who has rapidly and durably responded to low doses of the mutation-specific BRAF inhibitor, vermurafenib. Collectively, we provide evidence for the development of the BRAF V600E mutation in the context of clonal evolution and describe a prognostic and therapeutic relevance of this targetable mutation.
Cancer Discovery 04/2013; 3(8). DOI:10.1158/2159-8290.CD-13-0014 · 19.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this issue of Blood, Riches and colleagues provide an in-depth characterization of T cells and particularly CD8+ T cells from patients with chronic lymphocytic leukemia (CLL). They demonstrate that CD8+ T cells exhibit defects in proliferation, cytotoxicity, and increased expression of inhibitory receptors and thus exhibit features of T-cell exhaustion.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this analysis was to provide six-year follow-up of the GCLLSG CLL3X trial which studied reduced-intensity allogeneic hematopoietic stem cell transplantation (HSCT) in patients with poor-risk chronic lymphocytic leukemia (CLL), and to investigate the impact of TP53, SF3B1, and NOTCH1 mutations on HSCT outcome. Six-year overall survival (OS) and event-free survival (EFS) of 90 allografted patients was 58% and 38%, respectively. TP53, SF3B1, and NOTCH1 mutations were found in 30%, 26%, and 14% of the trial population. By univariate and multivariate analyses, the mutational status of the TP53, SF3B1, and NOTCH1 genes had no significant impact on OS and EFS. Studies of minimal residual disease confirmed durability of CLL eradication in mutated patients. We conclude that HSCT can provide long-term disease control in patients with poor-risk CLL independent of the presence of TP53, SF3B1, and NOTCH1 mutations. The trial has been registered at the US National Cancer Institute (Protocol Identity # EU-20554, NCT00281983).
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental stimuli for their survival, provided for example by monocyte-derived nurse-like cells (NLCs). The immunomodulatory drug lenalidomide shows therapeutic effects in subgroups of CLL patients, and is believed to act via the microenvironment. To investigate the effects of lenalidomide on the survival support of NLCs, co-cultures of monocytes and CLL cells were treated for 14 days with lenalidomide, which resulted in significantly decreased viability of CLL cells. Among the changes induced by this drug, we observed reduced expression of HLA-DR in NLCs, as well as increased secretion of IL-10, indicating an altered inflammatory milieu in the co-cultures. The increase in IL-10 levels lead to an induction of STAT1 phosphorylation in CLL cells and to enhanced cell surface expression of ICAM-1 and altered expression of cytoskeletal and migration-related genes. Chemotaxis assays with lenalidomide-treated CLL cells revealed an impaired migration capability. Our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug affects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide acts on the migratory potential of CLL cells, which may affect circulation and homing of CLL cells in vivo.