Search

Einleitung: Patienten mit HPV+ Kopf-Hals-Tumoren (HNSCC) zeigen ein erheblich besseres Überleben als solche mit HPV- Tumoren. Die verbesserte Prognose scheint unabhängig von der Therapiemodalität zu sein, jedoch deuten die meisten Daten auf erhöhte Strahlenempfindlichkeit hin.[for full text, please go to the a.m. URL], 86. Jahresversammlung der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie

Einleitung: Eine Kombination des EGFR Antikörpers Cetuximab mit Strahlentherapie (RT) zeigte eine Verbesserung des Gesamtüberlebens in lokoregionär fortgeschrittenen Kopf-Hals Karzinomen. Der Raf-1 Inhibitor Sorafenib kann eine deutliche Radiosensitivierung an Kopf-Halszelllinien bewirken. Eine Kombination von klinisch angewendeter cisplatinbasierter Radiochemotherapie mit Sorafenib erscheint daher erfolgversprechend. Methoden: Zur Untersuchung des Zellwachstums kamen Proliferationsassays zur Anwendung. Koloniebildungsassays wurden zur Beurteilung des Zellüberlebens nach Kombinationsbehandlung angefertigt. Apoptose, Zellzyklus und DSB-Reparatur wurden mittels Durchflusszytometrie, Western Blot und Immunfluoreszenztechniken dargestellt. Ergebnisse: Aus einem Panel von 6 Kopf-Hals-Tumorzelllinien zeigten sich UT-SCC 42A und UT-SCC 42B relativ resistent gegenüber Cisplatin, verglichen mit Cisplatin sensiblen Zelllinien wie FaDu. Dennoch zeigt Cisplatin einen antiproliferativen Effekt, der erst bei hohen Konzentrationen (5 und 10 µM) sichtbar wird. Sorafenib radiosensitiviert beide Zelllinien mit einer Dosis von 10 µM. Die Kombination mit Cisplatin ergibt einen additiven Effekt beider Substanzen in der Zelllinie UT-SCC 42A. Schlussfolgerung: Unsere präklinischen Daten zeigen einen additiven Effekt von Sorafenib in Kombination mit cisplatinbasierter Radiochemotherapie bei einer Kopf-Halstumorzelllinie. Die Aufklärung der molekularen Mechanismen ermöglicht weitere Einblicke in die Zusammenhänge zwischen der Radiosensitivierung durch den Raf-1 Inhibitor Sorafenib in Kombination mit dem Alkylantium Cisplatin. Die Ergebnisse können zur Untersuchung eines möglichen Vorteils der verschiedenen Therapievarianten (sequentielle oder konkomitante Radiochemotherapie) genutzt werden. Unterstützt durch: UCCH Forschungsstipendium 2014 Der Erstautor weist auf folgenden Interessenkonflikt hin: Sorafenib wurde von der Firma Bayer HealthCare zur Verfügung gestellt., GMS Current Posters in Otorhinolaryngology - Head and Neck Surgery; 10:Doc092; ISSN 1865-1038

To investigate the correlation between PCC fragments and cell killing.Induction and repair of DNA fragments were measured in CHO, CHO-K1, xrs1 and xrs5 cells using the premature chromosome condensation (PCC) technique and cell survival was determined by a colony assay.The number of PCC fragments measured in cells immediately fused after X-irradiation was the same for CHO (3.4 +/- 0.16/cell/Gy) and CHO-K1 (3.6 +/- 0.12/cell/Gy) cells but significantly higher for xrs1 (4.9 +/- 0.07/cell/Gy) and xrs5 cells (7.0 +/- 0.4/cell/Gy). The repair curve of PCC fragments studied for CHO, CHO-K1 and xrs5 cells was best described by a monophasic exponential decline with a final plateau; the half-time of this decline was always about 30 min. The number of unrejoined PCC fragments, which was measured 14h after irradiation, increased linearly with dose. The steepest increase was found for xrs5 cells (5.5 +/- 0.3 fragments per cell and per Gy), the lowest for CHO/CHO-K1 (0.9 +/- 0.1; 1.0 +/- 0.1) and for xrs1 in between (3.3 +/- 0.1). For all four cell lines the relationship between cell killing and unrejoined fragments could be described by a single curve with a D0 of 2.5 +/- 0.4 unrejoined PCC fragments per lethal event.The data showed that the number of unrejoined PCC fragments can be used as an indicator of cellular radiosensitivity.

Induction and repair of double-strand breaks (dsb) were measured in exponentially growing CHO-10A cells using the constant- and graded-field gel electrophoresis. Dsb repair was studied after an X-ray dose of 60 Gy. The repair curve obtained was biphasic with the respective half-times of tau 1 = 3.8 +/- 0.9 and tau 2 = 118 +/- 30 min. The number of non-reparable dsb was measured for X-ray doses up to 180 Gy and was found to be only a small fraction (14%) of all non-rejoinable breaks determined previously using the alkaline unwinding technique. The ratio of non-reparable dsb to the number of lethal events calculated from survival curves is 0.14:1. This result indicates that for CHO cells nonreparable dsb represent only a small fraction of lethal damage. This is in line with the cytogenetic observation that cell killing mainly results from mis-rejoined events (i.e. exchange aberrations, translocations, interstitial deletions). The kinetics of dsb rejoining were found to be independent of the size of the fragments involved (between 1 and 10 Mbp). In addition, the rejoining kinetics of DNA fragmentsor = 1 Mbp did not show the formation of new DNA fragments with time after irradiation indicating the absence of programmed cell death in irradiated CHO cells.

Induction and repair of X-ray-induced DNA double-strand breaks (dsbs) was compared for normal broncho-alveolar macrophages and human peripheral blood lymphocytes, using CHO cells as a reference cell model. The cells, upon their separation, were processed in a similar manner. After X-irradiation, cell lysis and proteinase K treatment, the DNA samples were subjected to constant-field gel electrophoresis (CFGE) followed by fluorimetric densitometry for quantification of released DNA. Induction of dsbs after X-ray doses of 5-100 Gy was found to show no gross differences for all cell systems used. Repair of dsbs was studied after X-ray dose of 60 Gy for up to 24 h after irradiation. The repair curves obtained proved to be similar for bronchoalveolar macrophages and CHO cells (97% of all dsbs rejoined after 24 h). However, in blood lymphocytes from normal subjects and from bone marrow recipients, dsb repair proceeded rapidly only for 0.5-1 h post-irradiation, being followed by the gradual degradation of DNA at longer intervals. The kinetics of DNA degradation correlated with cytological features of pyknosis and necrosis.

Introduction Results from preclinical and clinical trials indicate that combination of the Epidermal Growth Factor Receptor (EGFR) specific antibody Cetuximab (C225) with classical radiotherapy improves local tumor control and overall survival of patients with head and neck cancers1-4. First results of Meller et al. showed a synergistic effect of 131I-labelled C225 in combination with external irradiation in vitro5. These data indicate that the combination of internal radiation dose delivered by radiolabelled C225 plus the therapeutic effect of C225 in combination with radiotherapy might significantly lower the overall external dose applied to patients, reducing undesired side effects on healthy tissue. Thus, bimodal cancer therapy seems a promising approach. This encouraged us to investigate the combined effect of 90Y[Y]-CHX-A"-DTPA-Cetuximab (90Y-C225) and radiation dose delivered by external beam irradiation in vitro and in vivo in our joint project. Materials and Methods CHX-A´´-DTPA was conjugated to C225 via thiourea bridging. Radiolabelling was performed under mild conditions using [90Y]YCl3. Binding affinity of 90Y-C225 was studied by flow cytometric analysis as well as by competitive binding assays using cells in 2D and 3D culture or cell membrane preparations. Distribution in spheroids (FaDu) was studied in a concentration- and time-dependent manner. Cellular uptake, EGFR binding specificity and kinetics, retention of 90Y-C225 as well as clonogenic activity of 90Y-C225 treated cells in combination with external irradiation were studied. The capacity of 90Y-C225 to induce DNA double-strand breaks (DSBs) was assessed through the Gamma-H2AX/53BP1 foci technique and cell survival by colony formation. Ex vivo autoradiography was performed with 90Y-C225. Biodistribution and in vivo kinetics were measured with PET using 86Y as radiolabel. FaDu tumour bearing nude mice were treated with 90Y-C225 (2.8 MBq 13 μg C225 / mouse, i.v.) and external beam irradiation (20 Gy single dose, 1.1 Gy/min). Experimental endpoints are the tumour growth delay and the local tumour control after 180 d. Results Radiolabelling of the conjugate resulted in specific activities up to 9 GBq/mg. For the present study, a labelling protocol to achieve a reproducible specific activity of 1.2 GBq/mg was established. in vitro: Flow cytometric analysis showed that affinity of 90Y-C225 is not significantly reduced compared to native C225. A Ki of 0.4 nM vs 0.22 nM for native C225 was determined by a competitive binding assay. Saturation of FaDu spheroids with 90Y-C225 was achieved after 24 h of incubation at 5 μg/ml. A saturation test showed that the optimal concentration to block all EGFR (SAS and UT5 cells) by C225 in monolayer culture is about 3-5 nM. No unspecific binding on an EGFR-negative CHO cell line was observed. Binding 90Y-C225 to EGFR in HNSCC cells occurred time dependently with a maximum after 24 h. 24 h after treatment approximately 10% of bound activity was found in the supernatant. Conjugation of CHX-A"-DTPA to C225 does not alter the cellular and biological function of C225. Combination of single doses of 2 or 4 Gy with 90Y-C225 reduced clonogenic survival in the UT5 and SAS cell lines. For UT5 cells the D37 value for non-treated control cells was 3.7 Gy with X-rays. Treatment with C225 reduced D37 of UT5 cells to 2.7 Gy. When 90Y-C225 was combined with X-rays, the D37 of UT5 cells dropped to 1.8 Gy. Thus, in comparison to C225, a decrease of D37 by about 33 % was observed in cells treated with 90Y-C225. D37 of untreated cells was only reduced from 3.9 Gy to 3.7 Gy after incubation with native C225. A D37 of 3.3 Gy resulted upon treatment with 90Y-C225 which reflects only a reduction of about 11%, compared to C225. Induction of DNA DSBs by 90Y-C225 in cell lines with varying EGFR expression showed that the amount of 90Y-C225 binding and the number of induced DSBs is proportional to the EGFR on the cell membrane and that the cytotoxicity is dependent on the number of residual DSB (clonogenic survival). in vivo: Autoradiography revealed high tumour accumulation 48 h p.i., also PET showed an increasing accumulation of activity in the tumour, which was abundant after 24 h p.i. The combined treatment was well tolerated by all mice and no histological alterations in organs were found. A clear dose-dependent effect of the external irradiation was observed. A significant improvement of the local tumour control after X-ray irradiation with 20 Gy was achieved after application of 13 μg of 90Y-C225 compared to native C225 or to the treatment by external irradiation alone. Conclusion The results from in depth investigations of the effects of 90Y-C225 on various EGFR expressing cell lines validate it as a powerful tool for in vivo studies. The combined treatment of tumours in our experimental mouse model permits a reduction of the external radiation dose of 12 Gy. Our in-vivo data support the concept that bimodal cancer treatment results in a potentially relevant improvement of local tumour control. This encourages us to follow this promising scientific concept. Research Support: The Bundesministerium für Bildung und Forschung (grant 02NUK006, framework “Kompetenzverbund Strahlenforschung” (KVSF)) is gratefully acknowledged for the financial support. References 1 Bonner JA, Harari PM, Giralt J et al. [2010] Lancet Oncol.; 11: 21-8. 2 Bonner JA, Harari PM, Giralt J et al. [2006] N Engl J Med.; 354: 567-78. 3 Curran D, Giralt J, Harari PM et al. [2007] J Clin Oncol.; 25: 2191-7. 4 Zhang N, Erjala K, Kulmala J et al. [2009] Radiother Oncol.; 92: 388-92. 5 Meller B, Rades D, Wolff C et al. [2009] IJROBP 75: 1226-1231.

Einleitung: Bei der Behandlung fortgeschrittener HNSCC ist die Strahlentherapie von großer Bedeutung. Eine Erhöhung der Strahlenempfindlichkeit ist notwendig, um bei ausreichender Tumordosis die Normalgewebstoxizität zu reduzieren. Die Hemmung des epidermalen Wachstumsfaktor Rezeptors[for full text, please go to the a.m. URL], 83. Jahresversammlung der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie

kein Abstract verfügbar

Backround: Molecular targeting is a novel approach in radiotherapy with great potential to increase local tumour control. The epidermal growth factor receptor (EGFR) is a suitable target for such strategies, due to its overexpression in many solid tumours. The aim of the project is an increased inactivation of tumour cells, including EGFR overexpressing metastases. Therefore the radioactive isotope 90Yttrium was bound to the EGFR directed antibody Cetuximab (C225). This allows a specific induction of DNA damage in EGFR overexpressing tumour cells with low side effects in normal tissue expressing low EGFR. In this study double strand breaks (DSB) induced by 90YDTPA-Cetuximab were determined. Therefore γH2AX foci formation in human squamous cell carcinoma cells of head and neck differing in EGFR expression were detected after treatment with 90Y-DTPA-Cetuximab. Summary & Conclusion: • The binding of Cetuximab to cells depends on their membranous EGFR expression level. • The chelator DTPA attached to the antibody Cetuximab reduces the affinity to EGFR about 30% compared to native Cetuximab. This effect can be avoided by using saturating concentrations of antibody. • 90Y-DTPA-Cetuximab induces DNA double strand breaks in human squamous cell carcinoma cell lines. • The dose applied by 90Y-DTPA-Cetuximab, and thereby the amount of DNA double strand breaks induced, depends on the time of treatment and seems to be related to the membranous EGFR expression. • The repair of DNA double strand breaks in human squamous cell carcinoma cell lines shows comparable kinetics after treatment with 90Y-DTPA-Cetuximab and irradiation with X-ray. __> 90Y-DTPA-Cetuximab can be used to generate DNA-double strand breaks in human cell lines expressing high EGFR. The data shows that this EGFR directed targeting strategy using a radioactive labelled antibody has the potential to increase tumour control. Experiments in tumour model systems (respective xenografts) to investigate the therapeutic benefit (with and without external radiation) and possible side effects are currently done by our cooperation partners. Acknowledgements This project is supported by BMBF (02 NUK 006 C) & DFG (PAK-190)