Thymidine-phosphorothioate oligonucleotides induce activation and apoptosis of CLL cells independently of CpG motifs or BCL-2 gene interference.

We compared antisense phosphorothioate oligonucleotides (PS-ODN) that target BCL-2 such as Genasense (G3139-PS), with other PS-ODN or phosphodiester-ODN (PO-ODN) in their relative capacity to induce apoptosis of chronic lymphocytic leukemia (CLL) B cells in vitro. Surprisingly, we found that thymidine-containing PS-ODN, but not PO-ODN, induced activation and apoptosis of CLL cells independent of BCL-2 antisense sequence or CpG motifs. All tested thimidine-containing PS-ODN, irrespective of their primary sequences, reduced the expression of Bcl-2 protein and increased the levels of the proapoptotic molecules p53, Bid, Bax in CLL cells. Apoptosis induced by thymidine-containing PS-ODN was preceded by cellular activation, could be blocked by the tyrosine-kinase inhibitor imatinib mesylate (Gleevec), and was dependent on ABL kinase. We conclude that thymidine-containing PS-ODN can activate CLL cells and induce apoptosis via a mechanism that is independent of BCL-2 gene interference or CpG motifs.

Genetic characterization of MYD88-mutated lymphoplasmacytic lymphoma in comparison with MYD88-mutated chronic lymphocytic leukemia.

MYD88 (myeloid differentiation primary response 88) is mutated in the majority of Waldenström macroglobulinemia/lymphoplasmacytic lymphoma (LPL); but also, albeit less frequently, in other B-cell malignancies, including chronic lymphocytic leukemia (CLL). This suggests MYD88 as a central regulator of pathogenesis, but requests a broader approach to define diagnostically relevant genetic profiles for LPL and CLL. We identified the L265P hotspot mutation in 86% (n=67/78) of our LPL and 2% (n=12/767) of our CLL cohort. Importantly, in CLL (n=5), but also in LPL (n=4) other MYD88 mutations were identified. MYD88-mutated LPL was characterized by CXCR4 mutations (25%) and del(6q) (19%), whereas both aberrations were absent in the MYD88-unmutated LPL cases. MYD88-mutated CLL formed a prognostically favorable subset with a high frequency of del(13q), mutated IGHV status and no adverse aberrations (del(11q), del(17p), TP53 mutations). MYD88-mutated CLL differed from LPL with respect to cytogenetic aberrations and the absence of CXCR4 mutations. In both entities, based on mutation load evaluation, MYD88 mutations were found to be present in the stem clone in each case, whereas CXCR4 (LPL) and SF3B1 (CLL) mutations also occurred in subclones only.

WT1 mutations are secondary events in AML, show varying frequencies and impact on prognosis between genetic subgroups.

To investigate frequency and prognostic impact of Wilms tumor 1 (WT1) mutations (mut), we analyzed 3157 unselected acute myeloid leukemia patients for WT1mut in exons 7 and 9. In total, 188 WT1 mutations were detected (exon 7: n=150, exon 9: n=38); 141 were frameshift, 24 missense, 14 non-sense, 7 splice site and 2 indel mutations. In 175/3157 (5.5%) patients, a WT1mut was found. Higher frequencies were detected in patients with biallelic CEBPAmut (13.6%; P=0.001), followed by t(15;17)/PML-RARA (11.0%, P=0.004), and FLT3-ITD (8.5%, P<0.001). WT1mut were rare in DNMT3Amut (4.4%, P=0.014), ASXL1mut (1.7%, P<0.001), IDH2R140 (1.7%, P=0.001) and IDH1R132 (0.9%, P<0.001), and not detected in complex karyotypes (P=0.047). They were more frequent in females than in males (6.6 vs 4.7%; P=0.014) and in patients <60 years (P<0.001). Analysis of paired samples of 35 patients revealed a relatively unstable character of WT1mut (65.7% retained, 34.3% lost WT1mut at relapse). In the total cohort and subgroups with high WT1mut incidences (biallelic CEBPAmut, PML-RARA), WT1mut had no impact on prognosis. In normal karyotype AML, WT1mut patients had shorter event-free survival (EFS) (10.8 vs 17.9 m, P=0.008). In multivariate analysis, WT1mut had an independent adverse impact on EFS (P=0.002, hazard ratio (HR): 1.64) besides FLT3-ITD status (P<0.001, HR: 1.71) and age (P<0.001, HR: 1.28).

Digital imaging microscopy of firefly luciferase activity to directly monitor differences in cell transduction efficiencies between AdCMVLuc and Ad5LucRGD vectors having different cell binding properties.

The luciferase reporter gene incorporated into adenoviral vectors is very useful for monitoring viral transduction of different cell types or for comparing the transduction efficiency of different viral constructs of one cell type. Luciferase protein expression can be detected and quantified with very high sensitivity from whole cells or organ extracts. However, its disadvantages become obvious when aiming at evaluation of transduction events at the single cell level. The results obtained from whole cell extracts cannot be directly correlated to single cell events. In this paper direct cellular luciferase imaging using cell permeable luciferin substrates is applied for comparative analysis of cellular transduction events by two adenoviral vectors with different cell binding properties. Using digital imaging microscopy we show a more than ten-fold increase in transduction efficiency by Ad5LucRGD vectors versus AdCMVLuc vectors on human A549 cells.

Monitoring of residual disease by next-generation deep-sequencing of RUNX1 mutations can identify acute myeloid leukemia patients with resistant disease.

We studied the utility and clinical relevance of RUNX1 (runt-related transcription factor 1) mutations and their application as residual disease detection markers using next-generation deep-sequencing. Mutation screening was prospectively performed in 814 acute myeloid leukemia patients. At diagnosis, 211/814 (25.9%) patients harbored mutations with a median clone size of 39% (range: 2-96%). Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed. In 47/57 (82.5%) cases the same alterations detected at diagnosis were present at relapse, whereas in 1/57 (1.8%) cases the mutation from the diagnostic sample was no longer detectable. Discrepancies were observed in 9/57 (15.8%) cases, also including the occurrence of novel RUNX1 mutations not restricted to those regions affected at diagnosis. Moreover, in 103 patients the prognostic impact of residual levels of RUNX1 mutations during complete remission was studied. Separation of patients according to median residual mutation burden into 'good responders' and 'poor responders' (median: 3.61%; range: 0.03-48.0%) resulted in significant differences of both event-free (median 21.0 vs. 5.7 months, P<0.001) and overall survival (OS; median 56.9 vs. 32.0 months, P=0.002). In conclusion, deep-sequencing revealed that RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. The measurement of mutation load may refine the assignment into distinct risk categories and treatment strategies.

SF3B1 mutations correlated to cytogenetics and mutations in NOTCH1, FBXW7, MYD88, XPO1 and TP53 in 1160 untreated CLL patients.

We analyzed a large cohort of 1160 untreated CLL patients for novel genetic markers (SF3B1, NOTCH1, FBXW7, MYD88, XPO1) in the context of molecular, immunophenotypic and cytogenetic data. NOTCH1 mutations (mut) (12.3%), SF3B1mut (9.0%) and TP53mut (7.1%) were more frequent than XPO1mut (3.4%), FBXW7mut (2.5%) and MYD88mut (1.5%). SF3B1mut, NOTCH1mut, TP53mut and XPO1mut were highly correlated to unmutated, whereas MYD88mut were associated with mutated IGHV status. Associations of diverse cytogenetic aberrations and mutations emerged: (1) SF3B1mut with del(11q), (2) NOTCH1mut and FBXW7mut with trisomy 12 and nearly exclusiveness of SF3B1mut, (3) MYD88mut with del(13q) sole and low frequencies of SF3B1mut, NOTCH1mut and FBXW7mut. In patients with normal karyotype only SF3B1mut were frequent, whereas NOTCH1mut rarely occurred. An adverse prognostic impact on time to treatment (TTT) and overall survival (OS) was observed for SF3B1mut, NOTCH1mut and TP53 disruption. In multivariate analyses SF3B1mut, IGHV mutational status and del(11q) were the only independent genetic markers for TTT, whereas for OS SF3B1mut, IGHV mutational status and TP53 disruption presented with significant impact. Finally, our data suggest that analysis of gene mutations refines the risk stratification of cytogenetic prognostic subgroups and confirms data of a recently proposed model integrating molecular and cytogenetic data.

The role of different genetic subtypes of CEBPA mutated AML.

The prognostic impact of mutations in the CCAAT/enhancer binding protein α (CEBPA) gene was evaluated in the context of concomitant molecular mutations and cytogenetic aberrations in acute myeloid leukemia (AML). CEBPA was screened in a cohort of 2296 adult AML cases. Of 244 patients (10.6%) with CEBPA mutations, 140 cases (6.1%) were single-mutated (CEBPAsm) and 104 cases (4.5%) were double-mutated (CEBPAdm). Cytogenetic analysis revealed normal karyotype in 172/244 (70.5%) of CEBPAmut cases, whereas in 72/244 cases (29.5%) at least one cytogenetic aberration was detected. Concurrent molecular mutations were seen less frequently in CEBPAdm than in CEBPAsm AML cases (69.2% vs 88.6% P<0.001). In detail, the spectrum of concurrent mutations was different in both groups with the frequent occurrence of GATA1 and WT1 mutations in CEBPAdm patients. In contrast, FLT3-ITD, NPM1, ASXL1 and RUNX1 mutations were detected more frequently in CEBPAsm cases. Favorable outcome was restricted to CEBPAdm cases and remained an independent prognostic factor for a favorable outcome in multivariate analysis (hazard ratio: 0.438, P=0.020). Outcome in CEBPAsm cases strongly depended on concurrent FLT3-ITD. In conclusion, we propose that only CEBPAdm should be considered as an entity in the WHO classification of AML and should be clearly distinguished from CEBPAsm AML.

BAALC expression: a suitable marker for prognostic risk stratification and detection of residual disease in cytogenetically normal acute myeloid leukemia.

High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). The prognostic value of BAALC expression in relation to other molecular prognosticators was analyzed in 326 CN-AML patients (<65 years). At diagnosis, high BAALC expression was associated with prognostically adverse mutations: FLT3 internal tandem duplication (FLT3-ITD) with an FLT3-ITD/FLT3 wild-type (wt) ratio of 0.5 (P=0.001), partial tandem duplications within the MLL gene (MLL-PTD) (P=0.002), RUNX1 mutations (mut) (P<0.001) and WT1mut (P=0.001), while it was negatively associated with NPM1mut (P<0.001). However, high BAALC expression was also associated with prognostically favorable biallelic CEBPA (P=0.001). Survival analysis revealed an independent adverse prognostic impact of high BAALC expression on overall survival (OS) and event-free survival (EFS), and also on OS when eliminating the effect of allogeneic stem cell transplantation (SCT) (OS(TXcens)). Furthermore, we analyzed BAALC expression in 416 diagnostic and follow-up samples of 66 patients. During follow-up, BAALC expression correlated with mutational load or expression levels, respectively, of other minimal residual disease markers: FLT3-ITD (r=0.650, P<0.001), MLL-PTD (r=0.728, P<0.001), NPM1mut (r=0.599, P<0.001) and RUNX1mut (r=0.889, P<0.001). Moreover, a reduction in BAALC expression after the second cycle of induction chemotherapy was associated with improved EFS. Thus, our data underline the utility of BAALC expression as a marker for prognostic risk stratification and detection of residual disease in CN-AML.

Landscape of TET2 mutations in acute myeloid leukemia.

We investigated ten-eleven translocation 2 (TET2) mutations in acute myeloid leukemia (AML), their correlation with other gene mutations and prognostic value. By deep-sequencing, 131 somatic TET2 mutations were identified in 87/318 (27.4%) patients. Of 87 mutated cases, 44 (50.6%) carried two mutations. TET2 mutations were concomitantly observed with mutations in NPM1, FLT3-ITD, FLT3-TKD, JAK2, RUNX1, CEBPA, CBL and KRAS. However, TET2 mutations rarely concomitantly occurred with IDH1mut or IDH2mut (2/251 or 0/184; P=0.046 and P=0.003, respectively). TET2 mutations were associated with normal karyotype AML (CN-AML) (62/206 (30.1%) CN-AML vs 20/107 (18.7%) aberrant karyotype; P=0.031), higher white blood cell count (mean 65.3 vs 40.3 × 10(9)/l, P=0.023), lower platelet count (mean 68.6 vs 92.4 × 10(9)/l, P=0.03) and higher age (67.5 vs 65.2 years, P<0.001). Survival analyses were restricted to de novo CN-AML patients (n=165) and showed inferior event-free survival (EFS) of TET2 mutations compared with TET2wt (median: 6.7 vs 18.7 months, P=0.009). This negative effect of TET2 mutation on EFS was particularly observed in patients 65 years (median: 8.9 months vs not reached (n.r.), P=0.027) as well as in patients of the European LeukemiaNet favorable-risk subgroup, that is, patients harboring mutated CEBPA and/or mutated NPM1 without FLT3-ITD (median: 10.3 vs 41.3 months, P=0.048). These data support a role for TET2 as an important prognostic biomarker in AML.

Targeted next-generation sequencing detects point mutations, insertions, deletions and balanced chromosomal rearrangements as well as identifies novel leukemia-specific fusion genes in a single procedure.

DNA sequence enrichment from complex genomic samples using microarrays enables targeted next-generation sequencing (NGS). In this study, we combined 454 shotgun pyrosequencing with long oligonucleotide sequence capture arrays. We demonstrate the detection of mutations including point mutations, deletions and insertions in a cohort of 22 patients presenting with acute leukemias and myeloid neoplasms. Importantly, this one-step methodological procedure also allowed the detection of balanced chromosomal aberrations, including translocations and inversions. Moreover, the genomic representation of only one of the partner genes of a chimeric fusion on the capture platform also permitted identification of the novel fusion partner genes. Using acute myeloid leukemias harboring RUNX1 abnormalities as a model system, three novel chromosomal fusion sequences and KCNMA1 as a novel RUNX1 fusion partner gene were detected. This assay has the strong potential to become an important method for the comprehensive genetic characterization of particular leukemias and other malignancies harboring complex genomes.

Characterization of NPM1-mutated AML with a history of myelodysplastic syndromes or myeloproliferative neoplasms.

The role of the nucleophosmin (NPM1) mutations in de novo acute myeloid leukemia (AML) is well analyzed, but the impact in secondary AML (s-AML) following myelodysplastic syndromes (MDS) or transformed myeloproliferative neoplasms (MPNs) remains unclear. We investigated 350 patients-283 s-AML after MDS and 67 transformed MPNs-for NPM1mut. NPM1mut was detected in 43/350 patients (12.3%) at diagnosis of s-AML (transformed MDS: 37/283; 13.1%; transformed MPNs: 6/67; 9.0%). Cytogenetic alterations were present in 12/40 cases (30.0%) with available karyotypes. Additional molecular mutations were found in 23/43 NPM1mut s-AML after MDS (53.5%) and in transformed MPN in 18/37 (48.6%): FLT3-ITD: 14/37 (37.8%); FLT3-TKD: 3/28 (10.7%); NRASmut: 4/37 (10.8%), RUNX1mut: 1/16 (6.3%). In NPM1mut-transformed MPNs, five out of six cases showed 1-2 additional molecular mutations (2 × KITD816V, ETV6-PDGFRB, 2 × JAK2V617F, 2 × FLT3-ITD). Backtracking of nine of these cases by quantitative real time PCR showed the NPM1mut already at diagnosis of MDS/MPN, at variable levels and up to 14 months before diagnosis of AML, and at transformation often being preceded or accompanied by other genetic alterations. Thus, NPM1 mutations are involved in the transformation from MDS to AML or MPN to blast phase in single cases, which should be further confirmed in larger studies.

The detection of TP53 mutations in chronic lymphocytic leukemia independently predicts rapid disease progression and is highly correlated with a complex aberrant karyotype.

The poor prognosis of chronic lymphocytic leukemia (CLL) patients with del (17p) is well established. We analyzed whether mutation of TP53 on the remaining allele adds to the poor prognosis or whether even TP53 mutation alone may be an adverse prognostic factor. We analyzed TP53 mutations in 193 CLL patients by denaturing high performance liquid chromatography in combination with direct DNA sequencing and a TP53 resequencing research microarray. Mutations were correlated to chromosomal aberrations defined by interphase fluorescent in situ hybridization and chromosome banding analyses and to the clinical course of patients. TP53 mutations were detected in 13.5% (26 of 193) of samples, whereas the incidence of del (17p) was 9.3% (18 of 193). TP53 mutations were significantly associated with del (17p) (concordance 94%, P<0.001) and complex cytogenetic abnormalities (concordance 50%, P<0.001). Among 147 patients whose clinical data were available, patients with TP53 abnormalities (n=20) had a significantly decreased time to treatment compared to patients without TP53 aberration (P<0.001). Median time to treatment was short in patients with isolated TP53 mutation (n=6, 2.0 months) and in those with del (17p) (n=14, 21.3 months) as compared to patients without TP53 aberration (n=127, 64.9 months, P<0.001). In multivariate Cox regression analysis, VH status, TP53 mutations and also isolated TP53 mutations independently predicted rapid disease progression.

Mutations of the TP53 gene in acute myeloid leukemia are strongly associated with a complex aberrant karyotype.

In acute myeloid leukemia (AML) with complex aberrant karyotype, a loss of one TP53 allele is frequently observed. We analyzed the incidence of TP53 mutations and deletions in 107 AML with complex aberrant karyotype. In 50 of 57 cases showing a loss of one TP53 allele, a TP53 mutation was detected in the remaining allele. In addition, in 33 of 50 cases with two TP53 copies, a TP53 mutation was found. Therefore, the frequency of TP53 mutations in AML with complex aberrant karyotype was 78%. In a second step, we analyzed TP53 mutations in a cohort of AML comprising different cytogenetic subgroups. TP53 mutations were detected in 33 of 235 cases (14%). Coincidences with other molecular mutations were rare. We confirmed a high incidence of TP53 mutations in AML with a complex aberrant karyotype (29/42, 69%) and demonstrated that TP53 mutations are very rare in AML without a complex aberrant karyotype (4/193, 2.1%).

Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping.

In CLL data from chromosome banding analysis (CBA) have been scarce due to the low proliferative activity of CLL cells in vitro. We improved the cultivation technique using an immunostimulatory CpG-oligonucleotide DSP30 and IL-2. A total of 506 CLL samples were analysed with CBA and interphase FISH using probes for the detection of trisomy 12, IgH rearrangements and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). A total of 500 of 506 (98.8%) cases were successfully stimulated for metaphase generation and are subject to this study. Aberrations were detected in 415 of 500 (83.0%) cases by CBA and in 392 of 500 (78.4%) cases by FISH. CBA detected 832 abnormalities and FISH only 502. Therefore, CBA offers important information in addition to FISH. (1) CLL is characterized mainly by genomic imbalances and reciprocal translocations are rare. (2) A subgroup with complex aberrant karyotype (16.4%) is identified which is associated with an unmutated IgV(H) status and CD38 expression (P=0.034 and 0.02, respectively). (3) Additional abnormalities are detectable providing new biological insights into different CLL subclasses revealing a much more heterogeneous pattern of cytogenetic abnormalities as assumed so far based on FISH data only. Therefore, prospective clinical trials should evaluate the prognostic impact of newly available CBA data.