[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3? signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. ?-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.Molecular Therapy (2014); doi:10.1038/mt.2014.219.
Molecular therapy : the journal of the American Society of Gene Therapy. 11/2014;
[Show abstract][Hide abstract] ABSTRACT: Abstract Purpose: To investigate chromosomal instability and radiation response mechanisms in glioblastoma cells. Methods and Materials: We undertook a comparative analysis of two patient-derived glioblastoma cell lines. Their resistance to low and high linear energy transfer (LET) radiation was assessed using clonogenic survival assay and their intrinsic chromosome instability status using fluorescence in situ hybridization. DNA damage was analyzed by pulsed-field gel electrophoresis and by γ-H2AX foci quantification. Expression of DNA damage response proteins was assessed by immunoblot. Results: Increased radioresistance to X-rays as well as carbon ions was observed in glioblastoma cells exhibiting high levels of naturally occurring chromosomal instability and impaired Ataxia-telangiectasia mutated (ATM) signaling, as reflected by lack of phosphorylation of ATM, CHK2 and p53 after double-strand breaks induction. Conclusion: Our results indicate the existence of highly radioresistant glioblastoma cells, characterized by dysfunctional ATM signaling and high levels of intrinsic chromosomal instability.
International journal of radiation biology. 07/2014;
[Show abstract][Hide abstract] ABSTRACT: Segmental Xp22.2 monosomy or a heterozygous HCCS mutation is associated with the microphthalmia with linear skin defects (MLS) or MIDAS (microphthalmia, dermal aplasia, and sclerocornea) syndrome, an X-linked disorder with male lethality. HCCS encodes the holocytochrome c-type synthase involved in mitochondrial oxidative phosphorylation (OXPHOS) and programmed cell death.
We characterized the X-chromosomal abnormality encompassing HCCS or an intragenic mutation in this gene in six new female patients with an MLS phenotype by cytogenetic analysis, fluorescence in situ hybridization, sequencing, and quantitative real-time PCR. The X chromosome inactivation (XCI) pattern was determined and clinical data of the patients were reviewed.
Two terminal Xp deletions of >=11.2 Mb, two submicroscopic copy number losses, one of ~850 kb and one of >=3 Mb, all covering HCCS, 1 nonsense, and one mosaic 2-bp deletion in HCCS are reported. All females had a completely (>98:2) or slightly skewed (82:18) XCI pattern. The most consistent clinical features were microphthalmia/anophthalmia and sclerocornea/corneal opacity in all patients and congenital linear skin defects in 4/6. Additional manifestations included various ocular anomalies, cardiac defects, brain imaging abnormalities, microcephaly, postnatal growth retardation, and facial dysmorphism. However, no obvious clinical sign was observed in three female carriers who were relatives of one patient.
Our findings showed a wide phenotypic spectrum ranging from asymptomatic females with an HCCS mutation to patients with a neonatal lethal MLS form. Somatic mosaicism and the different ability of embryonic cells to cope with an OXPHOS defect and/or enhanced cell death upon HCCS deficiency likely underlie the great variability in phenotypes.
[Show abstract][Hide abstract] ABSTRACT: Previous studies demonstrated the relevance of focal lesions (FL) in whole-body magnetic resonance imaging (wb-MRI) at the initial workup of patients with smoldering multiple myeloma (SMM). The aim of this study was to assess the effects of longitudinal wb-MRIs on progression into multiple myeloma (MM). 63 patients with SMM were analyzed who received at least 2 wb-MRIs for follow-up before progression into MM. Radiological progressive disease (MRI-PD) was defined as detection of new FL or increase in diameter of existing FL and a novel or progressive diffuse infiltration. Radiological stable disease (MRI-SD) was defined by no change compared to the prior MRI. Patients were followed-up every 3-6 months, including a serological and clinical evaluation. 182 wb-MRIs were analyzed. MRI-PD occurred in 31 patients (49%) and 25 (40%) patients developed MM. MRI-PD was highly significantly associated with progression into MM, regardless of findings at the initial MRI. In multivariate analysis MRI-PD remained a risk factor, independent of relevant baseline parameters like serum M-Protein or 95% aberrant plasma cells in the bone marrow. Patients with MRI-SD had no higher risk of progression, even when FL were present at the initial MRI. Therefore, MRI is suitable for the follow-up of patients with SMM.Leukemia accepted article preview online, 18 February 2014; doi:10.1038/leu.2014.75.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 02/2014; · 10.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Chromosomal aberrations of plasma cells are well established pathogenetic and prognostic factors in multiple myeloma, but their prognostic implication in systemic light chain (AL) amyloidosis is unclear. Therefore, the aim of this study was to identify prognostic cytogenetic risk factors by interphase FISH in a series of 103 consecutive AL amyloidosis patients treated uniformly with melphalan/dexamethasone as first-line therapy. Detection of gain of 1q21 was predictive for a poor overall survival (OS) (median 12.5 versus 38.2 months, p = 0.002). Hematologic event free survival (hem EFS) for gain of 1q21 was 5.0 versus 8.5 months in median (p = 0.08) and haematologic remission rates (≥VGPR) after three cycles were 5% versus 25% (p = 0.06). Most important, in multivariate concordance analyses the adverse prognosis carried by gain of 1q21 was retained as an independent prognostic factor (OS: p = 0.003, average hazard ratio (AHR) = 3.64, hemEFS: p = 0.008, AHR = 2.35), along with the well established Mayo cardiac staging. Patients with t(11;14) had a longer median OS with 38.2 months versus 17.5 months, though no statistical significance was reached. Deletion 13q14 and hyperdiploidy turned out to be prognostically neutral. In conclusion, we have identified gain of 1q21 as an independent adverse prognostic factor in AL amyloidosis patients treated with standard chemotherapy.
Amyloid: the international journal of experimental and clinical investigation: the official journal of the International Society of Amyloidosis 01/2014; · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Balanced chromosome abnormalities (BCAs) occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD) and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14) that gave rise to abnormal mRNAs and could be assumed as disease-causing. However, unbiased genome-wide analysis of the sequencing data for cryptic structural variation was key to reveal an additional submicroscopic inversion that truncates the schizophrenia- and bipolar disorder-associated brain transcription factor ZNF804A as an equally likely NDD-driving gene. Deep sequencing of fluorescent-sorted wild-type and derivative chromosomes confirmed the clinically undetected BCA. Moreover, deep sequencing further validated a high accuracy of mate-pair library sequencing to detect structural variants larger than 10 kB, proposing that this approach is powerful for clinical-grade genome-wide structural variant detection. Our study supports previous evidence for a role of ZNF804A in NDD and highlights the need for a more comprehensive assessment of structural variation in karyotypically abnormal individuals and patients with neurocognitive disease to avoid diagnostic deception.
PLoS ONE 01/2014; 9(3):e90894. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to analyze chromosomal aberrations in terms of frequency and impact on time to progression in patients with smoldering multiple myeloma (SMM) on the background of clinical prognostic factors.
The chromosomal abnormalities 1q21, 5p15/5q35, 9q34, 13q14.3, 15q22, 17p13, t(11;14)(q13;q32), and t(4;14)(p16.3;q32) were assessed in CD138-purified myeloma cells by interphase fluorescent in situ hybridization (iFISH) alongside clinical parameters in a consecutive series of 248 patients with SMM.
The high-risk aberrations in active myeloma (ie, del(17p13), t(4;14), and +1q21) present in 6.1%, 8.9%, and 29.8% of patients significantly confer adverse prognosis in SMM with hazard ratios (HRs) of 2.90 (95% CI, 1.56 to 5.40), 2.28 (95% CI, 1.33 to 3.91), and 1.66 (95% CI, 1.08 to 2.54), respectively. Contrary to the conditions in active myeloma, hyperdiploidy, present in 43.3% of patients, is an adverse prognostic factor (HR, 1.67; 95% CI, 1.10 to 2.54). Percentage of malignant bone marrow plasma cells assessed by iFISH and combination of M-protein and plasma cell infiltration as surrogates of tumor load significantly confer adverse prognosis with HRs of 4.37 (95% CI, 2.79 to 6.85) and 4.27 (95% CI, 2.77 to 6.56), respectively. In multivariate analysis, high-risk aberrations, hyperdiploidy, and surrogates of tumor load are independently prognostic.
The high-risk chromosomal aberrations del(17p13), t(4;14), and +1q21 are adverse prognostic factors in SMM just as they are in active myeloma, independent of tumor mass. Hyperdiploidy is the first example for an adverse prognostic factor in SMM of opposite predictiveness in active myeloma. Risk association of chromosomal aberrations is not only a priori treatment dependent (predictive) but is also an intrinsic property of myeloma cells (prognostic).
Journal of Clinical Oncology 10/2013; · 18.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In acute myeloid leukemia (AML), studies based on whole-genome sequencing have shown genomic diversity within leukemic clones. The aim of this study was to address clonal heterogeneity in AML based on metaphase cytogenetics.
This analysis included all patients enrolled onto two consecutive, prospective, randomized multicenter trials of the Study Alliance Leukemia. Patients were newly diagnosed with non-M3 AML and were fit for intensive chemotherapy.
Cytogenetic subclones were detected in 418 (15.8%) of 2,639 patients from the whole study population and in 418 (32.8%) of 1,274 patients with aberrant karyotypes. Among those, 252 karyotypes (60.3%) displayed a defined number of distinct subclones, and 166 (39.7%) were classified as composite karyotypes. Subclone formation was particularly frequent in the cytogenetically adverse group, with subclone formation in 69.0%, 67.1%, and 64.8% of patients with complex aberrant, monosomal, and abnl(17p) karyotypes (P < .001 each). Two-subclone patterns typically followed a mother-daughter evolution, whereas for ≥ three subclones, a branched pattern prevailed. In non-core binding factor AML, subclone formation was associated with inferior event-free and overall survival and was confirmed as an independent predictor of poor prognosis in multivariate analysis. Subgroup analysis showed that subclone formation adds prognostic information particularly in the cytogenetic adverse-risk group. Allogeneic stem-cell transplantation improved the prognosis of patients with subclone karyotypes as shown in landmark analyses.
Cytogenetic subclones are frequent in AML and permit tracing of clonal evolution and architecture. They bear prognostic significance with clonal heterogeneity as an independent adverse prognostic marker in cytogenetically adverse-risk AML.
Journal of Clinical Oncology 09/2013; · 18.04 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; · 4.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The incidence of skin cancer is increasing worldwide and cutaneous squamous cell carcinomas (SCCs) are associated with considerable morbidity and mortality, particularly in immunosuppressed individuals ('carcinomatous catastrophy'). Yet, molecular mechanisms are still insufficiently understood. Besides ultraviolet (UV)-indicative mutations, chromosomal aberrations are prominent. As telomeres are essential in preserving chromosome integrity, and telomere erosion as well as aberrant spatial telomere distribution contribute to genomic instability, we first established telomere length profiles across the whole tissue and identified normal skin (10/30) harboring discrete epidermal sites (stem cell territories) of evenly short telomeres. Precancerous actinic keratoses (AKs) (17) and SCCs (27) expressed two telomere phenotypes: (i) tissue-wide evenly short to intermediate and (ii) longer and tissue-wide heterogeneous telomere lengths, suggesting two modes of initiation, with one likely to originate in the epidermal stem cells. Although tumor histotype, location, patient gender or age failed to distinguish the two SCC telomere phenotypes, as did telomerase activity, we found a trend for a higher degree of aberrant p53 and cyclin D1 expression with long/heterogeneous telomeres. In addition, we established an association for the short/homogeneous telomeres with a simpler and the heterogeneous telomeres with a more complex karyotype correlating also with distinct chromosomal changes. SCCs (13) from renal transplant recipients displayed the same telomere dichotomy, suggesting that both telomere subtypes contribute to 'carcinomatous catastrophy' under immunosuppression by selecting for a common set (3, 9p and 17q) and subtype-specific aberrations (e.g., 6p gain, 13q loss). As a second mechanism of telomere-dependent genomic instability, we investigated changes in telomere distribution with its most severe form of telomeric aggregates (TAs). We identified a telomere length-independent but progression-dependent increase in cells with small telomere associations in AKs (17/17) and additional TAs in SCCs (24/32), basal cell carcinomas (30/31) and malignant melanomas (15/15), and provide evidence for a reactive oxygen species-dependent mechanism in this UV-induced telomere organization-dependent genomic instability.Oncogene advance online publication, 19 August 2013; doi:10.1038/onc.2013.323.
[Show abstract][Hide abstract] ABSTRACT: To identify variants for multiple myeloma risk, we conducted a genome-wide association study with validation in additional series totaling 4,692 individuals with multiple myeloma (cases) and 10,990 controls. We identified four risk loci at 3q26.2 (rs10936599, P = 8.70 × 10(-14)), 6p21.33 (rs2285803, PSORS1C2, P = 9.67 × 10(-11)), 17p11.2 (rs4273077, TNFRSF13B, P = 7.67 × 10(-9)) and 22q13.1 (rs877529, CBX7, P = 7.63 × 10(-16)). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy, as well as insight into the biological basis of predisposition.
[Show abstract][Hide abstract] ABSTRACT: The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (SCNT) in a process termed 'reprogramming'. This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, e.g. in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte's reprogramming capacity is in excess of a single nucleus and that double nucleus transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived iPS cells, we have now accomplished it with enucleated oocytes. Stem Cells 2013.
[Show abstract][Hide abstract] ABSTRACT: The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (SCNT) in a process termed ‘reprogramming’. This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, e.g. in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte’s reprogramming capacity is in excess of a single nucleus and that double nucleus transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived iPS cells, we have now accomplished it with enucleated oocytes.
[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: Craniofrontonasal syndrome (CFNS) is an X-linked disorder caused by inactivating mutations in the gene for ephrin-B1 (EFNB1). Paradoxically it shows a more severe phenotype in females than in males. As a result of X inactivation cell populations with and without EFNB1 expression are found in EFNB1+/- females. This is thought to initiate a process termed cellular interference which may be responsible for the phenotype in females. We present a boy with severe clinical features of CFNS. In ~42% of his blood cells we found a supernumerary ring X chromosome containing EFNB1 but lacking XIST. Mosaicism for cell populations with different levels of EFNB1 expression can explain the severe phenotype of this patient. In vitro experiments in Xenopus tissue showed that cells over expressing ephrinB1 cluster and sort out from wild type cells. Our report provides further evidence that cellular interference contributes to the paradoxical inheritance pattern of CFNS.