A conserved long-distance telomeric silencing mechanism suppresses mTOR signaling in aging human fibroblasts.

Telomeres are repetitive nucleotide sequences at the ends of each chromosome. It has been hypothesized that telomere attrition evolved as a tumor suppressor mechanism in large long-lived species. Long telomeres can silence genes millions of bases away through a looping mechanism called telomere position effect over long distances (TPE-OLD). The function of this silencing mechanism is unknown. We determined a set of 2322 genes with high positional conservation across replicatively aging species that includes known and candidate TPE-OLD genes that may mitigate potentially harmful effects of replicative aging. Notably, we identified PPP2R2C as a tumor suppressor gene, whose up-regulation by TPE-OLD in aged human fibroblasts leads to dephosphorylation of p70S6 kinase and mammalian target of rapamycin suppression. A mechanistic link between telomeres and a tumor suppressor mechanism supports the hypothesis that replicative aging fulfills a tumor suppressor function and motivates previously unknown antitumor and antiaging strategies.

Method Optimization of Skin Biopsy-Derived Fibroblast Culture for Reprogramming Into Induced Pluripotent Stem Cells.

Background: Fibroblasts can be isolated from skin biopsies using a chemical dissociation, a physical dissociation, or a combination of both techniques. They can be reprogrammed into induced pluripotent stem cells (iPSCs) through the introduction of defined sets of key transcription factors. This study aimed to identify the optimal protocol for skin biopsy dissociation, fibroblast culture, and fibroblast cryopreservation in the scope of reprogramming into iPSCs and in the context of biobank accreditation. Methods: First, four dissociation techniques typically used in the laboratory (explant based, enzymatic, and/or mechanical) and two cryopreservation media containing 10% dimethyl sulfoxide, either commercial or homemade, were evaluated in terms of post-thaw recovery, viability, growth curves, and karyotyping analyses of the fibroblasts. Next, the clones reprogrammed from the fibroblasts isolated with the two optimal dissociation methods and cryopreservation media were further assessed by reprogramming quality before cryopreservation and post-thaw pluripotency comparison. Results: Fibroblasts isolated from skin biopsies using an explant-based or enzymatic dissociation method showed higher viability, higher proliferative potential, and higher genome stability post-thaw compared to the other dissociation techniques. Fibroblasts obtained by the explant-based dissociation technique showed a slightly higher reprogramming quality. The iPSC reprogrammed from explant-based dissociated fibroblasts showed successful recovery of iPSC clones. No difference between the two cryopreservation media was detected for the tested endpoints, with the exception of a higher visual count of colonies at the end of the reprogramming for the explant-based dissociation method. Conclusions: This article presents a formal method optimization for biospecimen processing in the context of accreditation in laboratories and biobanks. We validated skin biopsy-derived fibroblast isolation, culture, and cryopreservation for downstream mRNA reprogramming into iPSCs. The explant-based dissociation technique and homemade medium are selected as optimal to isolate and cryopreserve fibroblasts from skin biopsies in the scope of reprogramming into iPSCs.

Acute myeloid leukemia: negative prognostic impact of early blast persistence can be in part overcome by a later remission prior to post-induction therapy.

In acute myeloid leukemia, there is an ongoing debate on the prognostic value of the early bone marrow assessment in patients receiving intensive therapy. In this retrospective study, we analyzed the prognostic impact of the early response in 1,008 patients with newly diagnosed acute myeloid leukemia, who were treated at our institution with intensive chemotherapy followed by consolidation chemotherapy and/or allogeneic hematopoietic stem cell transplantation (HSCT). We found that early blast persistence has an independent negative prognostic impact on overall survival, eventfree survival and relapse-free survival. This negative prognostic impact may only be overcome in patients showing at least a partial remission at the early bone marrow assessment and who subsequently achieve blast clearance by additional induction chemotherapy prior to consolidation therapy with allogeneic HSCT. In accordance, we propose that the time slope of remission is an additional leukemia-related dynamic parameter that reflects chemosensitivity and thus may inform post-induction therapy decision-making. In addition to patient-related factors, European LeukemiaNet risk group, measurable residual disease monitoring and donor availability, this may particularly apply to European LeukemiaNet intermediate-risk patients, for whom a decision between consolidation chemotherapy and allogeneic HSCT remains challenging in many cases.

Clinical outcome of older adults with acute myeloid Leukemia: An analysis of a large tertiary referral Center over two decades.

OBJECTIVE: In older adults with acute myeloid leukemia (AML), the overall outcome is still dismal and long-term data on survival are scarce, particularly outside of clinical trials. Here, we assess characteristics, prognostic factors and long-term survival in patients ≥60 years who were treated for AML at our center over the past 17 years. METHODS: 590 older adults with newly diagnosed AML were characterized according to Eastern Cooperative Oncology Group (ECOG) score, Charlson comorbidity index (CCI), European LeukemiaNet (ELN) risk, type of therapy, serum ferritin (SF) and further baseline characteristics. Survival analysis was performed accordingly. RESULTS: Median age was 68 years and most patients were in good general condition. Median follow-up was 55.8 months. Of all patients, 66% received intensive chemotherapy (IC) +/- allogeneic hematopoietic stem cell transplantation (allo-HSCT). The remaining cohort received palliative chemotherapy (PC, 26%) or best supportive care only (BSC, 8%). Enrollment rate for interventional clinical trials was 26%. 5-year overall survival (OS) and relapse-free survival (RFS) were 18% (median 12.5 months) and 11,5% (median 10.0 months). Long-term survival was independently influenced by ECOG score, ELN risk group, baseline SF, previous myocardial infarction, and choice of therapy, but not consistently by age or CCI. Considering therapeutic subgroups, the contribution of particular parameters in predicting OS was most compelling in IC patients, but less consistent with PC or BSC. CONCLUSION: Our results provide thorough insights into prognostication within therapeutic subgroups and emphasize the need for more detailed prognostic algorithms and routine geriatric assessment in the treatment of older adults with AML.

Aneuploidy in children with relapsed B-cell precursor acute lymphoblastic leukaemia: clinical importance of detecting a hypodiploid origin of relapse.

Aneuploidy is common in paediatric B-cell precursor acute lymphoblastic leukaemia (ALL). Specific subgroups, such as high hyperdiploidy (>50 chromosomes or DNA Index ≥1·16) and hypodiploidy (<45 chromosomes), predict outcome of patients after primary treatment. Whether aneuploidy has a prognostic value for relapsed disease is yet to be determined. Using DNA index and centromere screening by multiplex ligation-dependent probe amplification, we investigated aneuploidy in 413 children treated for first relapse of B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. Ten-year event-free survival of patients with high hyperdiploid relapses approached 70%, whereas it was only 40% in low hyperdiploid relapses. Three patients with apparent hyperdiploid relapse had TP53 mutations. In these cases, array-based allelotyping revealed a hypodiploid origin with absence of the hypodiploid founder clone (masked hypodiploidy). Collectively, patients with evident or masked hypodiploid relapses showed an extremely low event-free survival rate of 9%. Importantly, the current relapse risk stratification did not identify cases with masked hypodiploidy as high-risk patients, due to their favourable clinical presentation. In multivariate analysis, hypodiploidy proved to be an independent prognostic factor. This finding supports stratification of relapses with hypodiploid origin into high-risk arms in future trials or allocation of patients to alternative treatment approaches.

AML: high serum ferritin at initial diagnosis has a negative impact on long-term survival.

Increased serum ferritin (SF) is common in hematologic malignancies; however, its prognostic role in acute myeloid leukemia (AML) is not clearly established. We examined the impact of baseline SF on long-term survival in 137 intensively treated AML patients. Patients and baseline characteristics were retrieved from an AML database at Charité University Medical Center Berlin, Campus Virchow Clinic. After c-reactive protein (CRP)-based adjustment for inflammation, patients were grouped according to their baseline SF level. Survival analysis was performed accordingly. A significant decline in overall survival and relapse-free survival was observed in patients with high SF as compared to those with low SF. Furthermore, elevated baseline SF remained an independent poor prognostic factor within the multivariate analysis and was associated with a significant higher risk of relapse and non-relapse mortality (NRM). In conclusion, our data show that elevated baseline SF has a negative impact on long-term survival in intensively treated AML patients.

Multilayered Omics-Based Analysis of a Head and Neck Cancer Model of Cisplatin Resistance Reveals Intratumoral Heterogeneity and Treatment-Induced Clonal Selection.

Purpose: Platinum-based drugs, in particular cisplatin (cis-diamminedichloridoplatinum(II), CDDP), are used for treatment of squamous cell carcinoma of the head and neck (SCCHN). Despite initial responses, CDDP treatment often results in chemoresistance, leading to therapeutic failure. The role of primary resistance at subclonal level and treatment-induced clonal selection in the development of CDDP resistance remains unknown.Experimental Design: By applying targeted next-generation sequencing, fluorescence in situ hybridization, microarray-based transcriptome, and mass spectrometry-based phosphoproteome analysis to the CDDP-sensitive SCCHN cell line FaDu, a CDDP-resistant subline, and single-cell derived subclones, the molecular basis of CDDP resistance was elucidated. The causal relationship between molecular features and resistant phenotypes was determined by siRNA-based gene silencing. The clinical relevance of molecular findings was validated in patients with SCCHN with recurrence after CDDP-based chemoradiation and the TCGA SCCHN dataset.Results: Evidence of primary resistance at clonal level and clonal selection by long-term CDDP treatment was established in the FaDu model. Resistance was associated with aneuploidy of chromosome 17, increased TP53 copy-numbers and overexpression of the gain-of-function (GOF) mutant variant p53R248L siRNA-mediated knockdown established a causal relationship between mutant p53R248L and CDDP resistance. Resistant clones were also characterized by increased activity of the PI3K-AKT-mTOR pathway. The poor prognostic value of GOF TP53 variants and mTOR pathway upregulation was confirmed in the TCGA SCCHN cohort.Conclusions: Our study demonstrates a link of intratumoral heterogeneity and clonal evolution as important mechanisms of drug resistance in SCCHN and establishes mutant GOF TP53 variants and the PI3K/mTOR pathway as molecular targets for treatment optimization. Clin Cancer Res; 24(1); 158-68. ©2017 AACR.

A Novel de novo FZD2 Mutation in a Patient with Autosomal Dominant Omodysplasia.

We described a heterozygous de novo mutation (G434V) in the frizzled class receptor 2 (FZD2) gene in a patient with distinct facial features including hypertelorism, bilateral cleft lip/palate, short nose with a broad nasal bridge, microretrognathia, and bilateral shortness of the upper limbs, first metacarpal bones, and middle phalanges of the 5th digits. The findings of our patient were compared to an autosomal dominant omodysplasia (OMOD2) family with FZD2 mutation reported in the literature. OMOD2 is a rare skeletal dysplasia and characterized by facial dysmorphism and shortness of the upper extremities and first metacarpal bones. This is the second report which supports the findings of the first family described and points out that heterozygous FZD2 mutations may be disease-causing for OMOD2.

A Rare Case of Acute Myeloid Leukemia with a t(2;3) Chromosomal Translocation Characterized by Thrombophilia and Chemoresistance.

We hereby report a case of acute myeloid leukemia with translocation t(2;3) and involvement of the ectopic virus integration site-1 (EVI1) gene. Like most other 3q26-related disorders reported thus far, we describe a phenotype with elevated platelet counts and dysmegakaryopoesis. The clinical course of our patient was complicated by symptomatic thrombophilia and chemoresistance. In addition, our case exhibited FLT3 (Fms-related tyrosine kinase 3) internal tandem duplication. Although anagrelide was successful in controlling elevated platelet counts, allogeneic stem cell transplantation failed to overcome chemoresistance due to simultaneous graft-versus-host-disease and relapse of acute myeloid leukemia. Given the dismal outcome of our case and previously reported cases, we propagate the implementation of targeted therapies to newly diagnosed patients with acute myeloid leukemia t(2;3). Preclinical models indicate drugs that plausibly target the EVI1-related molecular vulnerability as candidates for basket trials. Anagrelide exhibited a hopeful signal of activity in 3q26-related thrombocytosis and should be evaluated for implementation as supportive care.

Flow cytometric maturity score as a novel prognostic parameter in patients with acute myeloid leukemia.

The European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). In order to establish immunophenotypic features that predict prognosis, the expression of single AML blast cell antigens has been evaluated with partly conflicting results; however, the influence of immunophenotypic blast maturity is largely unknown. In our study, 300 AML patients diagnosed at our institution between January 2003 and April 2012 were analyzed. A flow cytometric maturity score was developed in order to distinguish "mature" AML (AML-ma) from "immature" AML (AML-im) by quantitative expression levels of early progenitor cell antigens (CD34, CD117, and TdT). AML-ma showed significantly longer relapse-free survival (RFS) and overall survival (OS) than AML-im (p < 0.001). Interestingly, statistically significant differences in RFS and OS were maintained within the "intermediate-risk" group according to ELN (RFS, 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS, 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome. It remains to be clarified whether these results simply reflect an accumulation of favorable molecular phenotypes in the AML-ma subgroup or whether they rely on biological differences such as a higher proportion of leukemia stem cells and/or a higher degree of genetic instability within the AML-im subgroup.

BCR-ABL1(+) acute myeloid leukemia: clonal selection of a BCR-ABL1(-) subclone as a cause of refractory disease with nilotinib treatment.

The presence of a Philadelphia chromosome with a corresponding BCR-ABL1 rearrangement is the hallmark of chronic myeloid leukemia, but is considered a very rare event in de novo acute myeloid leukemia (AML). Here, we report the first case in which a dominant Philadelphia chromosome-positive subclone was detected upon relapse in a formerly Philadelphia chromosome-negative MLL-AF6(+) AML. Due to refractory disease under salvage chemotherapy, the patient was started on nilotinib treatment. As a result, the Philadelphia chromosome-positive subclone was eradicated within 1 month; however, disease progressed and was again dominated by the Philadelphia chromosome-negative founding clone, demonstrating rapid clonal expansion under nilotinib-induced selection pressure.

High prevalence of immunoglobulin light chain gene aberrations as revealed by FISH in multiple myeloma and MGUS.

Multiple myeloma (MM) is a malignant B-cell neoplasm characterized by an uncontrolled proliferation of aberrant plasma cells in the bone marrow. Chromosome aberrations in MM are complex and represent a hallmark of the disease, involving many chromosomes that are altered both numerically and structurally. Nearly half of the cases are nonhyperdiploid and show IGH translocations with the following partner genes: CCND1, FGFR3 and MMSET, MAF, MAFB, and CCND3. The remaining 50% are grouped into a hyperdiploid group that is characterized by multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. In this study, we analyzed the immunoglobulin light chain kappa (IGK, 2p12) and lambda (IGL, 22q11) loci in 150 cases, mostly with MM but in a few cases monoclonal gammopathy of undetermined significance (MGUS), without IGH translocations. We identified aberrations in 27% (= 40 patients) including rearrangements (12%), gains (12%), and deletions (4.6%). In 6 of 18 patients with IGK or/and IGL rearrangements, we detected a MYC rearrangement which suggests that MYC is the translocation partner in the majority of these cases.

Copy number genome alterations are associated with treatment response and outcome in relapsed childhood ETV6/RUNX1-positive acute lymphoblastic leukemia.

The clinical heterogeneity among first relapses of childhood ETV6/RUNX1-positive acute lymphoblastic leukemia indicates that further genetic alterations in leukemic cells might affect the course of salvage therapy and be of prognostic relevance. To assess the incidence and prognostic relevance of additional copy number alterations at relapse of the disease, we performed whole genome array comparative genomic hybridization of leukemic cell DNA from 51 patients with first ETV6/RUNX1-positive relapse enrolled in and treated according to the relapse trials ALL-REZ of the Berlin-Frankfurt-Münster Study Group. Within this cohort of patients with relapsed ETV6/RUNX1-positive acute lymphoblastic leukemia, the largest analyzed for genome wide DNA copy number alterations to date, alterations were present in every ETV6/RUNX1-positive relapse and a high proportion of them occurred in recurrent overlapping chromosomal regions. Recurrent losses affected chromosomal regions 12p13, 6q21, 15q15.1, 9p21, 3p21, 5q and 3p14.2, whereas gains occurred in regions 21q22 and 12p. Loss of 12p13 including CDKN1B was associated with a shorter remission duration (P=0.009) and a lower probability of event-free survival (P=0.001). Distribution of X-chromosomal copy number alterations was gender-specific: whole X-chromosome loss occurred exclusively in females, gain of Xq only in males. Loss of the glucocorticoid receptor gene NR3C1 (5q31.3) was associated with a poor response to induction treatment (P=0.003), possibly accounting for the adverse prognosis of some of the ETV6/RUNX1-positive relapses.

Involvement of the MLL gene in adult T-lymphoblastic leukemia.

While the MLL "recombinome" is relatively well characterized in B-cell precursor acute lymphoblastic leukemia (BCP ALL), available data for adult acute T-lymphoblastic leukemia (T-ALL) are scarce. We performed fluorescence in situ hybridization (FISH) for an MLL split signal on 223 adult T-ALL samples obtained within the framework of the German Multicenter ALL 07/2003 therapy trial. Three biphenotypic leukemias (T-ALL/AML) were also included in the analysis. Samples showing any alteration by FISH were further investigated to characterize the MLL aberration. In addition, they were investigated for common genetic lesions known in T-ALL. Twenty-two cases (9.5%) showed an abnormal MLL signal by FISH analysis. Most of these appeared to be deletions or gains but in five cases (2.1%) a chromosomal translocation involving the MLL gene was identified. The translocation partners and chromosomal breakpoints were molecularly characterized. Three T-ALLs had an MLL-AF6/t(6;11) and two biphenotypic leukemias had an MLL-ELL/t(11;19). The chromosomal breakpoints in two of the MLL-AF6-positive cases were located outside the classical MLL major breakpoint cluster known from BCP ALL. In conclusion, the spectrum of MLL translocation partners in adult T-ALL much more resembles that of AML than that of BCP ALL and thus the mechanisms by which MLL contributes to leukemogenesis in adult T-ALL appear to differ from those in BCP ALL. Proposals are made for the diagnostic assessment of MLL fusion genes in adult T-ALL.

Mesenchymal stromal cells of myelodysplastic syndrome and acute myeloid leukemia patients have distinct genetic abnormalities compared with leukemic blasts.

Mesenchymal stromal cells (MSCs) are an essential cell type of the hematopoietic microenvironment. Concerns have been raised about the possibility that MSCs undergo malignant transformation. Several studies, including one from our own group, have shown the presence of cytogenetic abnormalities in MSCs from leukemia patients. The aim of the present study was to compare genetic aberrations in hematopoietic cells (HCs) and MSCs of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Cytogenetic aberrations were detected in HCs from 25 of 51 AML patients (49%) and 16 of 43 MDS patients (37%). Mutations of the FLT3 and NPM1 genes were detected in leukemic blasts in 12 (23%) and 8 (16%) AML patients, respectively. Chromosomal aberrations in MSCs were detected in 15 of 94 MDS/AML patients (16%). No chromosomal abnormalities were identified in MSCs of 36 healthy subjects. We demonstrate herein that MSCs have distinct genetic abnormalities compared with leukemic blasts. We also analyzed the main characteristics of patients with MSCs carrying chromosomal aberrations. In view of these data, the genetic alterations in MSCs may constitute a particular mechanism of leukemogenesis.

A BACH2-BCL2L1 fusion gene resulting from a t(6;20)(q15;q11.2) chromosomal translocation in the lymphoma cell line BLUE-1.

Abnormalities of the long arm of chromosome 6 are a common feature in various B-cell malignancies. In most cases, the genes involved have not yet been clearly identified. We have molecularly characterized the recently established Burkitt lymphoma cell line BLUE-1 that carries a t(6;20)(q15;q11.2) rearrangement in addition to the typical t(8;14) with MYC-IGH fusion. To identify the gene loci involved on both chromosomes we applied a sequential BAC clone mapping strategy. By using RT-PCR we were finally able to detect a chimeric mRNA transcript showing a fusion of the first (non-coding) exon of BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) on 6q15 to the second exon of BCL2L1 (BCL-X) on 20q11. Various fusion transcripts were detected for different BCL2L1 (BCL-XL) isoforms. The fusion ultimately results in strong expression of the BCL2L1 (BCL-XL) anti-apoptosis protein, as demonstrated by immunoblotting. This is the first report that shows the involvement of both BCL2L1 and the transcription factor BACH2 in a chromosomal rearrangement. It points to BACH2 as a possibly important target in lymphomas with 6q aberrations, although other genes on 6q are probably also involved in these cases. Moreover, it suggests that members of the BCL2 anti-apoptosis gene family other than BCL2 itself might also be involved in lymphoma.

Homeobox genes d11-d13 and a13 control mouse autopod cortical bone and joint formation.

The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11-/-Hoxd12-/-Hoxd13-/- triple-knockout mice and Hoxd13-/-Hoxa13+/- mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2.

Cytogenetic studies in acute leukemia patients relapsing after allogeneic stem cell transplantation.

We analyzed karyotype stability in 22 patients with acute leukemia at relapse or disease progression after allogeneic stem cell transplantation (allo-SCT). Karyotypes before and at relapse after allo-SCT were different in 15 patients (68%), the most frequent type being clonal evolution either alone or combined with clonal devolution (13 patients). Patients with and without a karyotype change did not differ significantly in overall survival (OS) (median, 399 vs. 452 days; P = 0.889) and survival after relapse (median, 120 vs. 370 days; P = 0.923). However, acquisition of additional structural chromosome 1 abnormalities at relapse after allo-SCT occurred more frequently than expected and was associated with reduced OS (median, 125 vs. 478 days; P = 0.008) and shorter survival after relapse (median, 37 vs. 370 days; P = 0.002). We identified a previously undescribed clonal evolution involving t(15;17) without PML-RARA rearrangement in an AML patient. We conclude that a karyotype change is common at relapse after allo-SCT in acute leukemia patients. Moreover, our data suggest that additional structural chromosome 1 abnormalities are overrepresented at relapse after allo-SCT in these patients and, in contrast to a karyotype change per se, are associated with reduced OS and shorter survival after relapse.

Impact of additional chromosomal aberrations and BCR-ABL kinase domain mutations on the response to nilotinib in Philadelphia chromosome-positive chronic myeloid leukemia.

BACKGROUND: Additional chromosomal aberrations in Philadelphia chromosome-positive chronic myeloid leukemia are non-random and strongly associated with disease progression, but their prognostic impact and effect on treatment response is not clear. Point mutations in the BCR-ABL kinase domain are probably the most common mechanisms of imatinib resistance. DESIGN AND METHODS: We assessed the influence of additional chromosomal aberrations and BCR-ABL kinase domain mutations on the response to the second-generation tyrosine kinase inhibitor nilotinib after imatinib-failure. Standard cytogenetic analysis of metaphases was performed to detect additional chromosomal aberrations and the BCR-ABL kinase domain was sequenced to detect point mutations. RESULTS: Among 53 patients with a median follow-up of 16 months, of whom 38, 5 and 10 were in chronic phase, accelerated phase and blast crisis, respectively, 19 (36%) had additional chromosomal aberrations and 20 (38%) had BCR-ABL kinase domain mutations. The 2-year overall survival rate of all patients with-out additional chromosomal aberrations (89%) was higher than that of patients with such aberrations (54%) (P=0.0025). Among patients with chronic phase disease, overall survival at 2 years was 100% and 62% for patients without or with additional chromosomal aberrations, respectively (P=0.0024). BCR-ABL kinase domain mutations were associated with lower remission rates in response to nilotinib, with 9 of 20 (45%) of these patients achieving a major cytogenetic remission as compared to 26 of 33 (79%) patients without mutations (P<0.05). However, overall survival was not affected by BCR-ABL kinase domain mutations. CONCLUSIONS: Whereas BCR-ABL kinase domain mutations may confer more specific resistance to nilotinib, which will predominantly affect response rates, the presence of additional chromosomal aberrations may reflect genetic instability and, therefore, intrinsic aggressiveness of the disease which will be less amenable to subsequent alternative treatments and thus negatively affect overall survival. Conventional cytogenetic analyses remain mandatory during follow-up of patients with chronic myeloid leukemia under tyrosine kinase inhibitor therapy.

Synergistic action of the novel HSP90 inhibitor NVP-AUY922 with histone deacetylase inhibitors, melphalan, or doxorubicin in multiple myeloma.

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP-AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM-2, RPMI-8226, U-266, LP-1, MM1.S, and primary myeloma cells were exposed to NVP-AUY922 and one of the combination partners histone deacetylase inhibitor NVP-LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP-AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP-AUY922 with SAHA, NVP-LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI-8226 cell line and relative resistance of some primary myeloma cells against NVP-AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP-AUY922 with melphalan, doxorubicin, NVP-LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.