Karl-Ludwig Schäfer

Diagnostic value of the molecular genetic detection of the t(11;22) translocation in Ewing's tumours

One consistent feature of the Ewings tumour family is the presence of a balanced translocation involving band q12 and band q24 of chromosome 22 and chromosome 11. Recent cloning of the chromosome breakpoint regions of t(11;22)(q24;q12) Ewings sarcoma translocation has revealed that the breakpoints were localized within the Ewings sarcoma gene (EWS gene) on chromosome 22 and the Fli-1 gene on chromosome 11. Molecular genetic techniques can thus be applied to the detection of the t(11;22) translocation in Ewings tumours. By reverse transcription and polymerase chain reaction technique (RT-PCR) 11 Ewings tumour derived cell lines, 12 primary Ewings tumours, and 11 tumours after treatment were analysed for the occurence of the t(11;22) translocation. Furthermore, blood and bone marrow samples from 5 patients were available for RT-PCR. In 78% of the cell lines and 91% of the primary Ewings tumours the t(11;22) translocation was detectable by RT-PCR. In bone marrow samples from a Ewings sarcoma patient presenting in relapse tumour cells were detected by molecular genetic analysis. Our results indicate that molecular genetic detection of the t(11;22) translocation is valuable in the differential diagnosis of small round cell tumours and will provide important information for the staging and prognosis of Ewings tumour.

Selective and nonselective toxicity of TRAIL/Apo2L combined with chemotherapy in human bone tumour cells vs. normal human cells

Although TRAIL/Apo2L preferably induces apoptosis in tumour cells without toxicity in normal cells, many tumour cell types display TRAIL/Apo2L resistance. Whether TRAIL/Apo2L in combination with chemotherapy may overcome TRAIL/Apo2L resistance while maintaining tumour selectivity remains to be determined. Here, we report that while ActD, DOX and CDDP sensitised both OS and Ewings tumour cell lines and normal cells (hOBs, synovial cells, fibroblasts) to TRAIL/Apo2L‐induced apoptosis, the combination of etoposide (VP16) and TRAIL/Apo2L was selectively active on tumour cells without affecting normal cells. Sensitisation of OS cells and hOBs to TRAIL/Apo2L did not correlate with a compatible change in the gene expression profile of the receptors for TRAIL/Apo2L determined by quantitative real‐time RT‐PCR. Also, sensitisation of the TRAIL/Apo2L death pathway did not rely entirely on the chemotherapy‐induced, caspase‐dependent cytotoxicity. Further, chemotherapy did not cause a compatible change in expression levels of proteins such as Bcl‐2, Bcl‐xL, Bax, cIAP2, XIAP and survivin. However, ActD, DOX and CDDP downregulated expression of cFLIP in OS cells as well as expression of p21 in normal hOBs. Interestingly, while VP16 also extinguished cFLIP in OS cells, which were sensitised for TRAIL/Apo2L by VP16, VP16 induced cFLIP and enhanced p21 levels in normal hOBs, which remained refractory to VP16 plus TRAIL/Apo2L. Together, our data reveal that TRAIL/Apo2L combined with certain chemotherapeutic drugs is toxic to bone tumour and normal human cells and suggest that cotreatment with TRAIL/Apo2L and VP16 provides an attractive approach for selective killing of tumour cells while leaving unaffected normal cells.

Telomerase Is a Highly Sensitive and Specific Molecular Marker in Fine-Needle Aspirates of Breast Lesions

PURPOSE Telomerase has been detected in a majority of human malignant tumors, making telomerase activity (TA) one key difference between mortal and immortal cells. In this study, we evaluated in blind-trial fashion the association of TA with cytologic and final clinical/pathologic diagnosis in fine-needle aspirates (FNAs) of breast lesions. MATERIALS AND METHODS In 172 FNAs, including 80 samples that were cytologically malignant, 18 that were atypical but not diagnostic for malignancy, and 74 that were cytologically benign, TA was determined by a modified nonradioactive telomeric repeat amplification protocol (TRAP) assay. Final diagnosis was made by pathologic examination of follow-up surgical material available for all the cytologically malignant samples, a majority of the cytologically atypical samples, and a portion of the cytologically benign samples. RESULTS TA was detected in 85 of 172 samples. Comparison of the cytologic and histologic diagnoses with TA showed that 80 of 87 samples from patients with breast cancer were telomerase-positive, resulting in a sensitivity of 92%. TA was found in four of five FNAs from carcinomas that were considered cytologically atypical but not diagnostic for malignancy. Eighty of 85 samples from patients with benign breast lesions were telomerase-negative, revealing a specificity of 94%. The five positive cases in this group were all fibroadenomas with low TA. Among the 18 cases with a cytologic diagnosis of atypia, there was a strong positive relationship between TRAP findings and histologic diagnosis. CONCLUSION The detection of TA in FNAs of breast lesions is a highly sensitive and specific marker of malignancy and may be used as an adjunct in cases with an equivocal cytologic diagnosis.

Chromosomal alterations in osteosarcoma cell lines revealed by comparative genomic hybridization and multicolor karyotyping

We characterized the chromosomal alterations in eight osteosarcoma cell lines (OST, HOS, U-2 OS, ZK-58, MG-63, SJSA-1, Saos-2, and MNNG) by comparative genomic hybridization (CGH); gains and losses of DNA sequences were defined as chromosomal regions with a fluorescence ratio, wherein all of the 95% confidence interval was above 1.25 and below 0.75, respectively. In four of 8 cell lines, multicolor karyotyping (MK) was added. CGH revealed the average number of aberrations per cell line was 20.8 (range: 10-31); the average numbers of gains and losses were 11.1 and 9.6, respectively. The frequent gains were identified on 1p21 approximately q24, 1q25-q31, 7p21, 7q31, 8q23 approximately q24, and 14q21; frequent losses were at 18q21 approximately q22, 18q12, 19p, and 3p12 approximately p14. High-level gains were observed on 8q23 approximately q24, 5p, and 1p21 approximately p22. MK revealed the most common translocations in the four cell lines were t(8;9), t(1;3), t(3;5), t(1;13), t(2;6), t(3; 17), t(1;15), t(10;20), and t(6;20). Chromosomes 1, 3, 8, 9, and 20 were most frequently involved in translocation events. The concordance rate of aberrations in CGH and translocations in MK was 76%. MK was useful to identify the chromosomal alterations and as a supplement to the CGH results in three of four chromosomes.

Establishment of an In Vivo Model for Pediatric Ewing Tumors by Transplantation into NOD/ scid Mice

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection. NOD/ scid mice that carry a complex immune deficiency and thus nearly completely lack the ability to reject human cells were used as recipients. Overall, 26 of 52 mice (50%) transplanted with VH-64, WE-68, CADO-ES1, TC-71, and RM-82 cells and 4 of 10 mice (40%) transplanted with primary tumor cells engrafted. Moreover, primary cells that did not grow in vitro proliferated in mice. The pattern of metastasis was similar to that in patients with frequent metastases in lungs (62%), bone marrow (30%), and bone (23%). Using limiting dilution experiments, the frequency of the engraftment unit was estimated at 1 Ewing tumor-initiating cell in 3 × 105 VH-64 cells. These data demonstrate that we have been able to establish an in vivo model that recapitulates many aspects of growth and progression of human Ewing tumors. For the first time, this model provides the opportunity to identify and characterize primitive in vivo clonogenic solid tumor stem cells. This model will, therefore, be instrumental in studying many aspects of tumor cell biology, including organ-selective metastasis and tumor angiogenesis.

Association of telomerase activity with radio- and chemosensitivity of neuroblastomas

BackgroundTelomerase activity compensates shortening of telomeres during cell division and enables cancer cells to escape senescent processes. It is also supposed, that telomerase is associated with radio- and chemoresistance. In the here described study we systematically investigated the influence of telomerase activity (TA) and telomere length on the outcome of radio- and chemotherapy in neuroblastoma.MethodsWe studied the effects on dominant negative (DN) mutant, wild type (WT) of the telomerase catalytic unit (hTERT) using neuroblastoma cell lines. The cells were irradiated with 60Co and treated with doxorubicin, etoposide, cisplatin and ifosfamide, respectively. Viability was determined by MTS/MTT-test and the GI50 was calculated. Telomere length was measured by southernblot analysis and TA by Trap-Assay.ResultsCompared to the hTERT expressing cells the dominant negative cells showed increased radiosensitivity with decreased telomere length. Independent of telomere length, telomerase negative cells are significantly more sensitive to irradiation. The effect of TA knock-down or overexpression on chemosensitivity were dependent on TA, the anticancer drug, and the chemosensitivity of the maternal cell line.ConclusionsOur results supported the concept of telomerase inhibition as an antiproliferative treatment approach in neuroblastomas. Telomerase inhibition increases the outcome of radiotherapy while in combination with chemotherapy the outcome depends on drug- and cell line and can be additive/synergistic or antagonistic. High telomerase activity is one distinct cancer stem cell feature and the here described cellular constructs in combination with stem cell markers like CD133, Aldehyddehydrogenase-1 (ALDH-1) or Side population (SP) may help to investigate the impact of telomerase activity on cancer stem cell survival under therapy.

Primitive neuroectodermal tumor (PNET) in the differential diagnosis of malignant kidney tumors.

Primitive neuroectodermal tumors (PNETs) of the kidney, a rare neoplastic disease of high malignancy with a tendency towards early metastasis, affect young adults (26-30 years) irrespective of the gender. Differential diagnosis from other renal tumors is very important for an effective therapy. Herein, we report on a 24-year-old male patient with a renal tumor consisting of small, round cells, and summarize the diagnostic procedures that establish the diagnosis of PNET. Light microscopy revealed not only areas containing small, round cells forming rosettes and pseudorosettes, but also areas containing spindle cells. Expression of CD 99 in combination with neural markers, such as NSE, was detected by immunohistochemistry, and further evidence of neural differentiation was provided by electron microscopy. Image cytometry revealed a peridiploid DNA-stemline. A reciprocal translocation of the chromosomes 11 and 22 [t(11;22)(q24;q12)] with expression of a EWS/FLI-1 fusion transcript was demonstrated by molecular pathology. Using these methods, the diagnosis of PNET was firmly established, and the tumor was treated by surgical resection and subsequent adjuvant chemotherapy. Eighteen months after therapy, the patient is in excellent health condition without any evidence of tumor recurrence.

Radiation-induced changes of telomerase activity in a human Ewing xenograft tumor.

Aim: The effect of ionizing irradiation on telomerase activity and further associated biological factors was evaluated in a human Ewing tumor xenograft model on nude mice. Material and Methods: The human Ewing tumor cell line STA-ET-1 was established in a nude mouse model. Initially, the dose-response relationship for the tumor model was established. For the radiation experiments two dose levels were chosen: 5 Gy and 30 Gy. After 5 Gy, there was no significant growth delay whereas after 30 Gy there was a marked growth delay without the induction of a complete remission. Tumors were examinated 6, 12, 24, 48, 72, and 96 hours post irradiation. After irradiation with 30 Gy further time points were 6, 9, 12 and 15 days. For each dose and time group, three tumors were evaluated. Results: There was a reduction of telomerase activity after 5 Gy to 50% (not statistically significant) after 3 days; however, after 30 Gy there was a reduction of telomerase activity to 23% of the initial value after 6 days (p = 0.001). Telomerase activity correlated with the expression of human telomerase reverse transcriptase (hTERT), but not with the expression of telomerase-associated protein (TP1) and human telomerase RNA (hTR). The maximal amounts of necrisis or apoptosis after 30 Gy were 19% and 6.9%, respectively. Conclusions: Ionizing radiation reduces telomerase activity and the expression of hTERT which cannot be explained by the induction of necrosis or apoptosis alone. The reduction of telomerase activity may contribute to delayed cell death after radiotherapy. The combined use of radiation and specific telomerase inhibitors may be a potentially synergistic treatment strategy.Ziel: Die Wirkung von ionisierender Strahlung auf die Telomeraseaktivität und weiterer assoziierter biologischer Parameter wurde an einem humanen Ewing-Tumor-Xenograft-Modell auf der Nacktmaus untersucht. Material und Methode: Die humane Ewing-Tumor-Zelllinie STA-ET-1 wurde auf der Nacktmaus etabliert. Zuerst wurde die Dosis-Wirkungs-Beziehung für den Tumor etabliert. Für die Bestrahlungsexperimente wurden zwei Bestrahlungsdosen gewählt: 5 Gy und 30 Gy. Nach 5 Gy wurde keine Wachstumsverzögerung beobachtet, nach 30 Gy trat eine deutliche Wachstumsverzögerung auf, ohne eine komplette Remission zu induzieren. Die Tumoren wurden 6, 12, 24, 48, 72 und 96 Stunden nach Bestrahlung untersucht. Nach 30 Gy wurden zusätzliche Zeitpunkte nach 6, 9, 12 und 15 Tagen gewählt. Für jede Dosis- und Zeitgruppe wurden drei Tumoren untersucht. Ergebnisse: Nach 5 Gy kam es zu einer statistisch nicht signifikanten Reduktion der Telomeraseaktivität auf 50% nach 3 Tagen (Abbildung 3a). Nach 30 Gy war die Telomerasekativität nach 6 Tagen auf 23% reduziert (p = 0.001, Abbildung 4a). Die Telomeraseaktivität korrelierte mit der Expression von hTERT (Human Telomerase Reverse Transcriptase, Abbildungen 3b und 4b), nicht jedoch mit der Expression von TP1 (Telomerase-assoziiertes Protein) und hTR (Human Telomerase RNA). Der maximale Nekrose- und Apoptoseanteil betrug 19% und 6,9% (Abbildung 6). Schlussfolgerungen: Ionisierende Strahlung reduziert die Expression von hTERT und Telomeraseaktivität. Diese Reduktion ist nicht allein auf die Induktion von Apoptose oder Nekrose zurückzuführen. Die Reduktion der Telomeraseaktivität kann zu einem verzögerten Zelluntergang nach Bestrahlung beitragen. Die kombinierte Verwendung von Bestrahlung und spezifischen Telomeraseinhibitoren ist ein potentiell synergistischer Therapieansatz.

Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease

Receptor tyrosine kinases (RTKs) have provided molecular targets for the development of novel, prognosis‐improving agents in many cancers; however, resistances to these therapies occur. On the cellular level, one resistance mechanism is attributed to functional RTK redundancies and compensatory cross‐signaling, leading to perception of RTKs as signaling and target networks. To provide a basis for better exploitation of this network in Ewing sarcoma, we generated comprehensive qPCR gene expression profiles of RTKs in Ewing sarcoma cell lines and 21 untreated primary tumors. Key findings confirm broad‐spectrum RTK expressions with potential for signaling redundancy. Profile analyses with regard to patient risk‐group further revealed several individual RTKs of interest. Among them, VEGFR3 and TIE1 showed high‐level expressions and also were suggestive of poor prognosis in localized tumors; underscoring the relevance of angiogenic signaling pathways and tumor‐stroma interactions in Ewing sarcoma. Of note, compared to localized disease, tumors derived from metastatic disease were marked by global high‐level RTK expressions. Nine individual RTKs were significantly over‐expressed, suggesting contributions to molecular mechanisms of metastasis. Of these, ROR1 is being pursued as therapeutic target in leukemias and carcinomas, but un‐characterized in sarcomas. We demonstrate expression of ROR1 and its putative ligand Wnt5a in Ewing sarcomas, and of an active ROR1 protein variant in cell lines. ROR1 silencing impaired cell migration in vitro. Therefore, ROR1 calls for further evaluation as a therapeutic target in metastatic Ewing sarcoma; and described as a pseudo‐kinase with several isoforms, underlines these additional complexities arising in our understanding of RTK signaling networks.

Nachweis einer strahleninduzierten Produktion von Tumor-Nekrose-Faktor alpha im Ewing-Sarkom RM 82 in vitro und in vivo

ZusammenfassungFragestellungDie Expression von Zytokinen aus Tumor- und Normalgewebszellen spielt bei der Vermittlung der Strahlenwirkung auf Tumoren eine wichtige Rolle. Tumor-Nekrose-Faktor alpha (TNF α), ein pleiotropes, primär als Monokin beschriebenes Polypeptid, ist durch seine zytotoxische Wirkung auf Tumorzellen sowie durch die Induktion hämorrhagischer Nekrosen von Tumoren von besonderem Interesse. Die vorliegende Arbeit untersucht den Einfluß ionisierender Strahlen auf die tumorzelleigene Expression von TNF α an einer humanen Ewing-Sarkom-Zellinie in vitro und in vivo am Xenograft-Tumor der Nacktmaus.Material und MethodeDer TNF-α-Protein-und-mRNA-Gehalt der Zellinie RM 82 wurde in vitro mittles “Enhanced Amplified Sensitivity Immunoassay” (EASIA) und semiquantitativer RT-PCR vor und nach Bestrahlung mit Einzeldosen zwischen 2 und 40 Gy zu unterschiedlichen Zeitpunkten (1 bis 72 Stunden nach Bestrahlung) bestimmt. Nach Etablierung der Zellinie auf der Nacktmaus wurde in vivo ebenfalls eine Zeit- und Dosiskorrelation zwischen Bestrahlung und TNF-α-mRNA-Produktion vorgenommen.ErgebnisseDie Zellinie RM 82 exprimiert in vitro einen TNF-α-Basisproteinspiegel von 20,1±4,3 pg/ml/106 Zellen. Nach Bestrahlung zeigt sich ein zeit- und dosisabhängiger Anstieg der TNF-α-Expression um das maximal 5,9 fache 24 Stunden nach Bestrahlung mit 20 Gy. Die TNF-α-Produktion auf mRNA-Ebene in vitro zeigt ihr Maximum nach bereits sechs bis zwölf Stunden. Die Eigenschaft der TNF-α-Expression von RM 82 blieb in vivo im Xenograft-Tumor erhalten. Die TNF-α-mRNA nimmt hier ebenfalls mit der Zeit und der Dosis zu: Die maximale Zunahme der Produktion liegt bei zwölf Stunden nach Bestrahlung mit 10 Gy.SchlußfolgerungenDie vorliegende Untersuchung zeigt in vitro eine zeit- und dosisabhängige Mehrproduktion von TNF α der Ewing-Sarkom-Linie RM 82 auf Protein- und mRNA-Ebene. Erstmals wird dieses Phänomen auch am In-vivo-System beschrieben. Damit steht ein Modell zur näheren Untersuchung der Bedeutung von TNF α als tumoreigenem “radiation response modifier” zur Verfügung.AbstractAimThe expression of cytokines plays an important role in the transmission of the effects of ionizing radiation to tumor cells and normal tissue. Tumor necrosis factor alpha (TNF α), a pleiotropic monokine, is of special interest because of its cytotoxic effect on tumor cells and the induction of hemorrhagic necrosis in tumors. We examined the influence of ionizing radiation on TNF α production in a human Ewing’s sarcoma cell line in vitro and in vivo.MethodsThe protein and mRNA levels of the Ewing’s sarcoma cell line RM 82 were examined in vitro with „Enhanced Amplified Sensitivity Immunoassay” (EASIA) and semiquantitative RT-PCR before and after treatment with single doses of 2 to 40 Gy, 1 to 72 hours after irradiation. After successful transplantation to nude mice, the time and dose correlation of TNF α mRNA production was examined in vivo.ResultsIn vitro, RM 82 had a basal protein level of TNF α of 20.1±4.3 pg/ml/106 cells. We observed a time- and dose-dependent increase of TNF α expression with a maximum of 125 pg/ml/106 (5.9fold) 24 hours after irradiation with 20 Gy. At the mRNA level, the maximal up-regulation occurred 6 to 12 hours after 10 Gy. In vivo, the xenograft tumor maintained the capacity of TNF α expression. Time-and dose-dependency in mRNA production showed a maximum increase 6 hours after treatment with 10 Gy.ConclusionsThe presented experiments show in vitro a dose- and time-dependent up-regulation of TNF α in the Ewing’s sarcoma cell line RM 82 on protein and mRNA level. For the first time this phenomenon was also observed in vivo in a human xenograft tumor. This tumor model could be used for further experiments to examine the role of TNF α as a biologic radiation response modifier in human tumors.