Andreas E. Kulozik

Profiling and functional annotation of mRNA gene expression in pediatric rhabdomyosarcoma and Ewing's sarcoma.

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewings sarcomas (EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t‐test, SAM, k‐nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS. We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). Expression levels for selected candidate genes were validated by quantitative real‐time reverse‐transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t‐test). Genes involved in protein biosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor‐specific genes reveals detailed insights into tumor biology and differentiation‐specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html.

Splicing and 3′ end formation in the definition of nonsense‐mediated decay‐competent human β‐globin mRNPs

Premature translation termination codons are common causes of genetic disorders. mRNAs with such mutations are degraded by a surveillance mechanism termed nonsense‐mediated decay (NMD), which represents a phylogenetically widely conserved post‐transcriptional mechanism for the quality control of gene expression. How NMD‐competent mRNPs are formed and specified remains a central question. Here, we have used human β‐globin mRNA as a model system to address the role of splicing and polyadenylation for human NMD. We show that (i) splicing is an indispensable component of the human β‐globin NMD pathway, which cannot be compensated for by exonic β‐globin ‘failsafe’ sequences; (ii) the spatial requirements of human β‐globin NMD, as signified by the maximal distance of the nonsense mutation to the final exon–exon junction, are less constrained than in yeast; and (iii) non‐polyadenylated mRNAs with a histone 3′ end are NMD competent. Thus, the formation of NMD‐competent mRNP particles critically depends on splicing but does not require the presence of a poly(A) tail.

Non-randomized therapy trial to determine the safety and efficacy of heavy ion radiotherapy in patients with non-resectable osteosarcoma

BackgroundOsteosarcoma is the most common primary malignant bone tumor in children and adolescents. For effective treatment, local control of the tumor is absolutely critical, because the chances of long term survival are <10% and might effectively approach zero if a complete surgical resection of the tumor is not possible. Up to date there is no curative treatment protocol for patients with non-resectable osteosarcomas, who are excluded from current osteosarcoma trials, e.g. EURAMOS1. Local photon radiotherapy has previously been used in small series and in an uncontrolled, highly individualized fashion, which, however, documented that high dose radiotherapy can, in principle, be used to achieve local control. Generally the radiation dose that is necessary for a curative approach can hardly be achieved with conventional photon radiotherapy in patients with non-resectable tumors that are usually located near radiosensitive critical organs such as the brain, the spine or the pelvis. In these cases particle Radiotherapy (proton therapy (PT)/heavy ion therapy (HIT) may offer a promising new alternative. Moreover, compared with photons, heavy ion beams provide a higher physical selectivity because of their finite depth coverage in tissue. They achieve a higher relative biological effectiveness. Phase I/II dose escalation studies of HIT in adults with non-resectable bone and soft tissue sarcomas have already shown favorable results.Methods/DesignThis is a monocenter, single-arm study for patients ≥ 6 years of age with non-resectable osteosarcoma. Desired target dose is 60-66 Cobalt Gray Equivalent (Gy E) with 45 Gy PT (proton therapy) and a carbon ion boost of 15-21 GyE. Weekly fractionation of 5-6 × 3 Gy E is used. PT/HIT will be administered exclusively at the Ion Radiotherapy Center in Heidelberg. Furthermore, FDG-PET imaging characteristics of non-resectable osteosarcoma before and after PT/HIT will be investigated prospectively. Systemic disease before and after PT/HIT is targeted by standard chemotherapy protocols and is not part of this trial.DiscussionThe primary objectives of this trial are the determination of feasibility and toxicity of HIT. Secondary objectives are tumor response, disease free survival and overall survival. The aim is to improve outcome for patients with non-resectable osteosarcoma.Trail RegistrationRegistration number (ClinicalTrials.gov): NCT01005043

Radiotherapy in the treatment of primary osteosarcoma - A single center experience

Purpose To analyze our experiences concerning radiation treatment in patients with osteosarcoma. Materials and methods Since 1981, 40 patients with osteosarcoma have undergone radiotherapy in Heidelberg; 3 of them were immediately lost to follow-up. Twenty patients with metastases were treated palliatively and 17 patients were treated with a curative intent. Results Interestingly, 14 of the 17 patients treated with a curative intent were referred to our clinic during the last 8 years, whereas the number of patients referred for palliation decreased. The mean dose applied for palliation was 47 Gy (range, 26 Gy to >70 GyE), for cure was 59 Gy (range, 45 Gy to >70 GyE). Local control until death could be achieved in 15 of the 20 palliatively treated patients, with a mean survival of 7 months after radiation. Five patients experienced local failure with symptom recurrence, and 3 of them had received doses >60 Gy. At last follow-up, 3 of the 17 curatively treated patients had experienced local recurrence. Median follow-up was 32 months (range, 3-144). Estimated 5-year overall survival and local control rates were 38% and 68%, respectively. Local disease-free survival was shorter in patients treated for recurrent, inoperable or incompletely resected tumors and doses below 60 Gy. Conclusions With adequate doses, long-term local control is possible even in inoperable or incompletely resected tumors. Improvements of systemic therapy and modern radiation techniques have begun to bring the possibly curative role of radiation treatment back to the fore. However, in disseminated tumors, even doses beyond 60 Gy do not guarantee local control, suggesting an extremely low radiosensitivity of certain kinds of osteosarcoma.

Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.

PurposeOsteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines.MethodsClonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis.ResultsSAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24xa0h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type.ConclusionSAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.ZusammenfassungZielsetzungOsteosarkome und atypische teratoide Rhabdoidtumore sind Tumorentitäten mit sehr variablem Ansprechen auf aktuelle Standardtherapien. Die Acetylierung von Histonen beeinflusst die Regulierung von Chromatinstruktur und Genexpression, beides Parameter, die eine wichtige Rolle bei der Strahlenempfindlichkeit von Zellen bzw. Geweben spielen. Die vorliegende Studie untersucht den Effekt des Histon-Deacetylasehemmers SAHA in Kombination mit einer Strahlentherapie auf die o.g. Tumorentitäten im Vergleich zu Normalgewebszelllinien (Osteoblasten, Fibroblasten) in vitro.MethodenDas zelluläre Überleben wurde im klonogenen Assay, DNA-Doppelstrangbrüche (DSB) mittels Durchflusszytometrie, Mikroskopie, Immunoblot und Pulsfeld-Gelelektrophorese (PFGE) untersucht.ErgebnisseSAHA führte zu einer signifikant erhöhten Strahlenempfindlichkeit in den Tumor-zelllinien, nicht aber in den Normalgewebszellen. Die γH2AX-Expression als Marker für DSB erhöhte sich signifikant, wenn die Tumorzellen 24xa0h vor der Bestrahlung SAHA ausgesetzt wurden. Im Gegensatz dazu kam es in den Normalgewebszellen zu einer signifikant niedrigeren γH2AX-Expression, wenn die Zellen nicht nur bestrahlt, sondern kombiniert mit SAHA behandelt wurden. Die Analyse der initialen DNA-Fragmentierung bzw. deren Reparatur mittels PFGE zeigte jedoch keine Unterschiede zwischen SAHA- und nicht-SAHA-behandelten Zellen, sowohl in den Normalgewebs- als auch in den Tumorzelllinien.SchlussfolgerungSAHA führt zu einer Radiosensitivierung von Tumor-, nicht aber von Normalgewebszellen. Der gesteigerte H2AX-Phosphorylierungsstatus nach Bestrahlung bei den SAHA-behandelten Tumorzellen scheint am ehesten durch eine verzögerte Dephosphorylierung nach DNA-Schädigung bedingt zu sein, weniger durch eine Chromatinacetylierungs-modifizierte DSB-Induktion oder Reparatur. Die Ergebnisse unterstützen die Hypothese, dass die Kombination von Bestrahlung mit dem Histon-Deacetylasehemmer SAHA eine neue, potenziell entwicklungsfähige Strategie in der Behandlung von soliden Tumoren darstellt.

Radiosensitization by histone deacetylase inhibition in an osteosarcoma mouse model

BackgroundOsteosarcomas (OS) are highly malignant and radioresistant tumors. Histone deacetylase inhibitors (HDACi) constitute a novel class of anticancer agents. We sought to investigate the effect of combined treatment with suberoylanilide hydroxamic acid (SAHA) and radiotherapy in OS in vivo.MethodsClonogenic survival of human OS cell lines as well as tumor growth delay of OS xenografts were tested after treatment with either vehicle, radiotherapy (XRT), SAHA, or XRT and SAHA. Tumor proliferation, necrosis, microvascular density, apoptosis, and p53/p21 were monitored by immunohistochemistry. The CD95 pathway was performed by flow cytometry, caspase (3/7/8) activity measurements, and functional inhibition of CD95 death signaling.ResultsCombined treatment with SAHA and XRT markedly reduced the surviving fraction of OS cells as compared to XRT alone. Likewise, dual therapy significantly inhibited OS tumor growth in vivo as compared to XRT alone, reflected by reduced tumor proliferation, impaired angiogenesis, and increased apoptosis. Addition of HDACi to XRT led to elevated p53, p21, CD95, and CD95L expression. Inhibition of CD95 signaling reduced HDACi- and XRT-induced apoptosis.ConclusionOur data show that HDACi increases the radiosensitivity of osteosarcoma cells at least in part via ligand-induced apoptosis. HDACi thus emerge as potentially useful treatment components of OS.ZusammenfassungZielsetzungOsteosarkome (OS) sind hochmaligne und relative radioresistente Tumore. Histon-Deacetylase-Inhibitoren (HDACi) stellen eine neue Substanzklasse in der Tumortherapie dar, insbesondere in Kombination mit anderen antineoplastischen Therapiestrategien. Wir haben deshalb die Effekte der Kombinationstherapie von Suberoylanilide Hydroxamic Acid (SAHA) und konventioneller Radiotherapie im OS-Mausmodell untersucht.MethodenEs wurde die Tumorwachstumsverzögerung in OS-Xenografts nach Behandlung mit Radiotherapie (XRT), SAHA alleine oder XRT und SAHA untersucht. Die Tumorproliferation, Tumornekrose, Tumorvaskularisation, Apoptose und p53/p21-Expression wurden immunohistochemisch analysiert. Die Bestimmung der CD95-vermittelten Apoptose erfolgte durchflusszytometrisch sowie durch Messung der Caspase-(3-/7-/8-)Aktivität und funktionaler Inhibition des CD95-Liganden.ErgebnisseDie Kombinationsbehandlung führt zu einer signifikanten Reduktion des Tumorwachstums in vivo im Vergleich zur alleinigen Radiotherapie, reflektiert durch eine verminderte Tumorproliferation sowie reduzierte Angiogenese und gesteigerte Apoptoseinduktion. Die Kombination von HDACi und XRT resultiert in einer gesteigerten p53-, p21-, CD95- und CD95L-Expression. Eine Inhibition des CD95-Liganden führt zu einer reduzierten HDACi- und XRT-induzierten Apoptoserate nach Kombinationstherapie.SchlußfolgerungDie Ergebnisse zeigen, dass eine HDACi die Radiosensitivität von Osteosarkomzellen signifikant erhöht. Dies erfolgt – zumindest teilweise – durch die CD95-abhängige Apoptoseinduktion. Die Autoren sind daher der Meinung, dass HDACi eine potenziell wirksame Therapiestrategie beim OS darstellt.

In vivo efficacy of the histone deacetylase inhibitor suberoylanilide hydroxamic acid in combination with radiotherapy in a malignant rhabdoid tumor mouse model

PurposeHistone deacetylase inhibitors are promising new substances in cancer therapy and have also been shown to sensitize different tumor cells to irradiation (XRT). We explored the effect as well as the radiosensitizing properties of suberoylanilide hydroxamic acid (SAHA) in vivo in a malignant rhabdoid tumor (MRT) mouse model.Methods and materialPotential radiosensitization by SAHA was assessed in MRT xenografts by analysis of tumor growth delay, necrosis (HE), apoptosis (TUNEL), proliferation (ki-67) and γH2AX expression as well as dynamic 18F-Fluorodeoxyglucose Positron Emission Tomography (18F-FDG -PET) after treatment with either SAHA alone, single-dose (10 Gy) or fractionated XRT (3 × 3Gy) solely as well as in combination with SAHA compared to controls.ResultsSAHA only had no significant effect on tumor growth. Combination of SAHA for 8 days with single-dose XRT resulted in a higher number of complete remissions, but failed to prove a significant growth delay compared to XRT only. In contrast fractionated XRT plus SAHA for 3 weeks did induce significant tumor growth delay in MRT-xenografts.The histological examination showed a significant effect of XRT in tumor necrosis, expression of Ki-67, γH2AX and apoptosis. SAHA only had no significant effect in the histological examination. Comparison of xenografts treated with XRT and XRT plus SAHA revealed a significantly increased γH2AX expression and apoptosis induction in the mice tumors after combination treatment with single-dose as well as fractionated XRT. The combination of SAHA with XRT showed a tendency to increased necrosis and decrease of proliferation compared to XRT only, which, however, was not significant. The 18F-FDG-PET results showed no significant differences in the standard uptake value or glucose transport kinetics after either treatment.ConclusionSAHA did not have a significant effect alone, but proved to enhance the effect of XRT in our MRT in vivo model.

Combination of suberoylanilide hydroxamic acid with heavy ion therapy shows promising effects in infantile sarcoma cell lines

IntroductionThe pan-HDAC inhibitor (HDACI) suberoylanilide hydroxamic acid (SAHA) has previously shown to be a radio-sensitizer to conventional photon radiotherapy (XRT) in pediatric sarcoma cell lines. Here, we investigate its effect on the response of two sarcoma cell lines and a normal tissue cell line to heavy ion irradiation (HIT).Materials and methodsClonogenic assays after different doses of heavy ions were performed. DNA damage and repair were evaluated by measuring γH2AX via flow-cytometry. Apoptosis and cell cycle analysis were also measured via flow cytometry. Protein expression of repair proteins, p53 and p21 were measured using immunoblot analysis. Changes of nuclear architecture after treatment with SAHA and HIT were observed in one of the sarcoma cell lines via light microscopy after staining towards chromatin and γH2AX.ResultsCorresponding with previously reported photon data, SAHA lead to an increase of sensitivity to heavy ions along with an increase of DSB and apoptosis in the two sarcoma cell lines. In contrast, in the osteoblast cell line (hFOB 1.19), the combination of SAHA and HIT showed a significant radio-protective effect. Laser scanning microscopy revealed no significant morphologic changes after HIT compared to the combined treatment with SAHA. Immunoblot analysis revealed no significant up or down regulation of p53. However, p21 was significantly increased by SAHA and combination treatment as compared to HIT only in the two sarcoma cell lines - again in contrast to the osteoblast cell line. Changes in the repair kinetics of DSB p53-independent apoptosis with p21 involvement may be part of the underlying mechanisms for radio-sensitization by SAHA.ConclusionOur in vitro data suggest an increase of the therapeutic ratio by the combination of SAHA with HIT in infantile sarcoma cell lines.

Childhood acute lymphoblastic leukemia in the Middle East and neighboring countries: A prospective multi-institutional international collaborative study (CALLME1) by the Middle East Childhood Cancer Alliance (MECCA)

Little is known about childhood ALL in the Middle East. This study was undertaken by MECCA as initial efforts in collaborative data collection to provide clinical and demographic information on children with ALL in the Middle East.

Screening for the beta-thalassaemia trait: hazards among populations of West African Ancestry

The aim of this study was to examine the accuracy and characteristics of detecting the beta-thalassaemia trait in populations of West African ancestry. School children, aged 16–19xa0years, in Manchester Parish, Jamaica were screened to detect the genes which could give rise to offspring with sickle cell disease. Haematological indices and HbA2 levels in subjects with an MCHu2009≤u200926xa0pg and an RDWu2009<u200918.0 with DNA analysis in those with indices consistent with the beta thalassaemia trait were measured. The performance of published discriminant indices in distinguishing iron deficiency and beta-thalassaemia trait in this population was assessed. Of 10,148 subjects, 1,739 (17.1%) had an AA haemoglobin phenotype and red cell indices consistent with beta-thalassaemia (MCH valuesu2009≤u200926xa0pg, RDWu2009<u200918.0) requiring estimations of HbA2 levels. HbA2 levels were ≥3.5% in 112 and beta-thalassaemia mutations were identified in 77 of these including the −88 C>T mutation in 35 (45%), −29 A>G in 19 (25%), −90 C>T in 7 (9%), the IVS II-849 A>G in 5 (6%) with smaller contributions from five other mutations. Discriminant indices performed poorly in the differentiation of iron deficiency and the beta-thalassaemia trait. Detection of the beta-thalassaemia trait is relatively insensitive in populations of West African ancestry partly because of the mild defects characterising beta-thalassaemia in this population and also the high prevalence of deletional alpha thalassaemia. More sensitive indicators are required for beta-thalassaemia detection to inform such populations at risk of offspring with sickle cell disease.