Patients and methods: Malignant supratentorial glioma (anaplastic astrocytoma, oligoastrocytoma, oligodendroglioma and glioblastoma incl. gliosarcoma), age 16-70y, KPS 50-100. Postoperative randomization to chemotherapy with either BCNU (B) (80 mg/m2 x 3 every 6 weeks) alone or additional VM 26 (V) (50 mg/m2 x 3 every 6 weeks) starting concomitant with radiotherapy. Central histopathological review was required. Primary endpoints were survival time (ST) and progression free survival (PFS) . In addition confirmative analysis of prognostic factors and their interaction with therapy was performed. Results: Eligible: 501 of 522 randomized pts: 82% WHO grade IV gliomas, 18% grade III gliomas. 57% male, mean KPS 74, mean age 50.9 years. The high incidence of lung toxicity – with a cumulative risk of 19% during the first year - was alarming. Survival was not significantly different ( median 50.3 (B) versus 52.4 (V) (weeks), but an increase in long term survivors was observed (18 months: 29% B, 34% V, 5 years 5% B, 12% V) and PFS showed a significant difference with a median of 31.4 (B) versus 34.3 (V) weeks. Qualitative interaction between KPS and therapy (p < 0.01) was demonstrated: pts with a KPS ≥ 70 benefited from additional VM26, those with reduced KPS < 70 did better with BCNU-monotherapy. Conclusion: Adding VM26 to BCNU is effective in the chemotherapy of malignant gliomas. Because of the demonstrated interaction with therapy performance status, not tumor grade is the crucial factor to determine application and aggressiveness of chemotherapy. With risk adapted therapy a significant proportion of patients even with glioblastoma survive for years in good general condition. BCNU should be replaced by an equipotent alkylans to avoid the unacceptable high rate of lung toxicity.

Like other branches, the health sector is also searching for new organisational forms in view of competitiveness. More and more, hospitals see themselves as integrated health care and service centres. This new view calls for structural and organisational consequences. This paper is to demonstrate how an architect can find systematic answers to these new requirements through his planning work. This paper presents a systematic strategy for the development of a concept for a hospital extension and also of the design of an integrated private practice for radiology and radiotherapy carrying out a fictitious conceptual study using the example of St. Elizabeth Hospital in Lörrach, Germany.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Objective: In this study, 22 cases of locally recurrent urological malignant fibrous histiocytoma were reviewed considering therapeutic options, follow-up and prognosis. Patients and Methods: In the available literature on this topic we identified 19 cases of locally recurrent genitourinary malignant fibrous histiocytoma. Three additional cases are discussed, primarily arising from the kidney, the bladder and the paratesticular region. Results: The prognosis of locally recurrent urological malignant fibrous histiocytoma was found to be extraordinarily poor. Only 2 of 22 patients have survived for longer than 3.5 years. One of them reported herein is still alive 10 years after extensive lymphatic spread accompanying the first local recurrence. In this case, late local recurrence occurred after an 8-year interval free of disease. Conclusion: Malignant fibrous histiocytoma is an unusual urological malignancy with a high rate of local recurrence. The latter is frequently accompanied by metastatic disease and unrelenting progression. Despite the poor prognosis early detection of local failure and aggressive salvage therapy might offer the chance of long-term survival in selected cases. Close and life-long follow-up is advisable for patients once treated for recurrent urological malignant fibrous histiocytoma. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Background: Advancement in diagnosis and treatment of various cancer entities led to an increasing incidence of brain metastases in the last decades. Surgical excision of single and multiple brain metastases is one of the central treatment options beside radiotherapy, radiosurgery and chemotherapy. To evaluate the benefit of surgery with/without whole-brain radiation therapy (WBRT) in single brain metastases and the influence of image guidance for brain metastases resection, 104 patients were retrospectively evaluated for post-operative outcome. Patients and Methods: Between January 1994 and December 1999 150 patients were surgically treated for brain metastases at the Department of Neurosurgery at the Technical University of Dresden. Outcome could be evaluated in 104 patients with respect to special treatment strategies and survival time (69 patients with single and 35 patients with multiple lesions). Results: Most metastases originated from primary lung and breast tumours. Karnofsky performance score improved on average by 10 after surgery. The extent of the extracerebral tumour burden was the main influence on survival time. Patients’ age below 70 years was combined with prolonged survival time (median survival time, MST: 4.5 months vs. 7 months). Patients with solitary cerebral metastasis had a MST of 16 months, whereas patients with singular lesions had a MST of 7 and 4 months, depending on the extent of the extracerebral tumour growth. Additional post-operative WBRT with 30 Gy was combined with an increase in MST in patients with single brain metastasis (surgery + WBRT: MST 13 months; surgery only: MST 8 months). In addition, the rate of recurrent cerebral tumour growth was distinctly higher in the non-WBRT group. Neuronavigation did not significantly improve post-operative survival time. In 80% of patients extracerebral tumour growth limited patients’ survival. Conclusion: Surgery is an initial treatment option in patients with single and multiple brain metastases especially with large tumours (> 3 cm). Post-operative WBRT seems to prolong survival time in patients with single brain metastasis by decreasing local and distant tumour recurrence. Neuronavigational devices permit a targeted approach. Multiple processes can be extirpated in one session without prolonging the hospitalisation time for the patient. However, neuronavigational devices cannot assure complete tumour resection. / Hintergrund: Fortschritte in der Diagnostik und Therapie von Krebserkrankungen haben in den letzten Jahrzehnten zu einer steigenden Inzidenz von Hirnmetastasen geführt. Die chirurgische Entfernung singulärer und multipler Hirnmetastasen stellt neben Strahlentherapie, Radiochirurgie und Chemotherapie eine zentrale Therapieoption dar. Um die Wertigkeit der chirurgischen Behandlung von singulären Hirnmetastasen mit/ohne Ganzhirnbestrahlung (WBRT) und den Einfluss der Neuronavigation zu untersuchen, wurden 104 Patienten retrospektiv bezüglich ihres postoperativen «Outcomes» untersucht. Patienten und Methoden: Zwischen Januar 1994 und Dezember 1999 wurden 150 Patienten mit Hirnmetastasen in der Klinik für Neurochirurgie der Technischen Universität Dresden operiert. Das «Outcome » von 104 Patienten konnte bezüglich der verschiedenen Behandlungsstrategien und Überlebenszeit ausgewertet werden (69 Patienten mit singulären und 35 Patienten mit multiplen Läsionen). Ergebnisse: Die meisten Metastasen stammen von Lungen- und Mammakarzinomen. Nach operativer Behandlung verbesserte sich der Karnofsky-Index um durchschnittlich 10. Das Ausmaß der extrazerebralen Tumormasse stellte die Haupteinflussgröße für die Überlebenszeit dar. Ein Lebensalter unter 70 Jahren war mit einer verlängerten Überlebenszeit verbunden (mittlere Überlebenszeit, MÜZ: 4,5 Monate vs. 7 Monate). Patienten mit solitären Metastasen hatten eine MÜZ von 16 Monaten, während Patienten mit singulären Läsionen, abhängig vom Ausmaß des extrazerebralen Tumorwachstums, eine MÜZ von 7 bzw. 4 Monaten aufweisen. Eine zusätzliche postoperative WBRT mit 30 Gy zeigte eine Verbesserung der MÜZ bei Patienten mit singulären Hirnmetastasen (OP + WBRT: MÜZ 13 Monate; OP allein: MÜZ 8 Monate). Gleichzeitig war die Rate der zerebralen Tumorrezidive in der Nicht-WBRT Gruppe deutlich höher. Die postoperative Überlebenszeit wurde durch Verwendung der Neuronavigation nicht signifikant verbessert. In 80% der Patienten limitierte das extrazerebrale Tumorwachstum die Überlebenszeit. Fazit: Bei Patienten mit singulären und multiplen Metastasen stellt die initiale chirurgische Tumorentfernung eine Therapiealternative insbesondere bei großen Tumoren (> 3 cm) dar. Eine postoperative WBRT scheint die ÜLZ der Patienten mit singulären Hirnmetastasen durch Begrenzung des Auftretens von Rezidivtumoren zu verlängern. Die Neuronavigation erlaubt eine gezielte Zugangsplanung. Multiple Prozesse können einzeitig operiert werden, ohne dass die postoperative stationäre Verweildauer verlängert wird. Hingegen wird eine radikale Tumorentfernung durch Verwendung der Neuronavigation nicht gewährleistet. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Brain metastases are treated with surgery, radiotherapy, radiosurgery, and chemotherapy. In this review, recently published studies concerning different treatment strategies are presented with respect to solitary lesions, multiple metastases, and recurrent tumor growth. Selection criteria for the appropriate therapy are: control of the primary tumor, extent of extracerebral metastases, time interval between diagnosis of the primary tumor and the development of cerebral lesions, number of cerebral metastases, Karnofsky performance scale score, and age. Treatment approaches were evaluated with respect to median survival time and quality of life. A singular brain metastasis can be treated with surgery or with radiosurgery. Especially when the primary tumor is under control, there are few extracerebral lesions which are stable, the Karnofsky performance scale score is above 70, the lesion is larger than 3 cm in diameter and surgically accessible surgery is the treatment of choice. Postoperative adjuvant radiotherapy may delay relapse. Median survival time ranges between 10 to 18 months. Radiosurgery can be applied in lesions smaller than 3 cm in diameter and is the treatment of choice in lesions which are surgically not accessible. Multiple metastases are treated either by conventional radiotherapy, radiosurgery or surgery. Commonly, no more than 3 lesions are approached by either surgery or radiosurgery. Median survival time ranges between 6 to 9 months for both treatment concepts, but without therapy only is 4–6 weeks. According to the clinical and neurological condition of the patient, recurrent brain metastases can be treated by operation, reirradiation, or radiosurgery. The efficacy of chemotherapy depends on the chemosensitivity of the primary tumor and the ability to penetrate the blood-brain barrier. Long-term survivors with cancer disease encourage to perform active treatment strategies. / Hirnmetastasen werden durch Operation, Ganzhirnbestrahlung, Radiochirurgie und Chemotherapie behandelt. In dieser Übersichtsarbeit werden kürzlich publizierte Studien bezüglich der Therapiekonzepte für solitäre Läsionen, multiple Metastasen und Tumorrezidive vorgestellt. Auswahlkriterien für eine angemessene Behandlung sind: Kontrolle des Primärtumors, Ausmaß der extrakraniellen Metastasen, Zeitintervall zwischen Diagnose des Primärtumors und dem Auftreten der Hirntumoren, Anzahl der zerebralen Metastasen, Karnofsky-Performance-Scale-Score und Lebensalter. Behandlungskonzepte wurden nach der medianen Überlebenszeit und Lebensqualität ausgewertet. Singuläre Hirnmetastasen können operativ oder radiochirurgisch behandelt werden. Insbesondere wenn der Primärtumor unter Kontrolle ist, wenige extrazerebrale Läsionen bestehen und diese stabil sind, der Karnofsky-Performance-Scale-Score über 70 ist, die Tumoren größer als 3 cm im Durchmesser und chirurgisch erreichbar sind, ist die Operation die Methode der Wahl. Postoperative adjuvante Strahlentherapie kann erneute Progression verzögern. Die mediane Überlebenszeit liegt zwischen 10 und 18 Monaten. Für Läsionen, die kleiner als 3 cm sind und chirurgisch nicht erreicht werden können, ist die Radiochirurgie die Therapie der Wahl. Multiple Metastasen können durch konventionelle Ganzhirnbestrahlung, Radiochirurgie oder Operation behandelt werden. Im allgemeinen werden nicht mehr als 3 Herde operativ oder radiochirurgisch angegangen. Die mediane Überlebenszeit liegt bei beiden Therapieformen zwischen 6 und 9 Monaten, ohne Behandlung hingegen bei nur 4–6 Wochen. Entsprechend dem klinischen und neurologischen Zustand der Patienten können Rezidive von Hirnmetastasen durch chirurgische Entfernung, erneute Bestrahlung oder durch Radiochirurgie therapiert werden. Die Wirkung der Chemotherapie hängt von der Chemosensitivität des Primärtumors und der Durchlässigkeit der Blut-Hirn-Schranke für das Chemotherapeutikum ab. Langzeitüberleber motivieren zu aktiven Behandlungsstrategien. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Hintergrund und Fragestellung/Hypothese Trotz verbesserter Diagnose und Therapiestrategien in der Behandlung von Plattenepithelkarzinomen des Kopf-Hals-Bereichs (HNSCC) in den letzten Jahrzehnten liegt das 5-Jahres-Überleben der Patienten bei nur etwa 60 %. Die hohe Mortalität ist zurückzuführen auf eine hohe Rezidivrate nach Radio- und Radiochemotherapie. Experimentelle und klinische Daten zeigen in dieser Tumorentität eine häufige Dysregulation des PI3K/AKT-Signalweges, der an verschiedenen Resistenzmechanismen beteiligt ist. Die Zielmoleküle des Signalweges sind involviert in DNA-Reparatur, Reoxygenierung und Repopulierung. Aufgrund der bekannten Beteiligung des PI3K/AKT-Signalweges an diesen wichtigen radiobiologischen Resistenzmechanismen beschäftigt sich die vorliegende Arbeit mit folgenden Fragestellungen: 1. Charakterisierung des Aktivierungsstatus des PI3K/AKT-Signalweges in Korrelation mit radiobiologischen Parametern in HNSCC-Experimentaltumoren 2. Bestimmung der Wirksamkeit des selektiven PI3K-Inhibitors Bay 80-6946 in HNSCC-Zelllinien in vitro 3. Untersuchung der Wirksamkeit von Bay 80-6946 in einem Panel von HNSCC-Experimentaltumoren in vivo mit und ohne Bestrahlung sowie des Einflusses der PI3K-Inhibition auf radiobiologische Parameter Material und Methode/Ergebnisse 1. Charakterisierung des Aktivierungsstatus des PI3K/AKT-Signalweges in Korrelation mit radiobiologischen Parametern in HNSCC-Experimentaltumoren Für die vorliegende Untersuchung wurde auf Tumormaterial von sechs verschiedenen Plattenepithelkarzinomzelllinien (FaDu, SAS, XF354, UT-SCC-5, UT-SCC-14, UT-SCC-15) aus einer Biobank zurückgegriffen. Analysiert wurden Xenografttumoren, die mit 0, 3, 5 oder 10 Fraktionen bestrahlt worden waren. Die Aktivierung des PI3K/AKT-Signalweges wurde mittels Western Blot-Analyse untersucht. Die publizierten Daten der lokalen Tumorkontrolle nach 30 Fraktionen in sechs Wochen, der Pimonidazol hypoxischen Fraktion und der HIF-1α-Proteinexpression wurden mit der pAKT-Expression korreliert. In allen unbehandelten Tumoren konnte pAKT nachgewiesen werden, wobei sich eine intratumorale Heterogenität in der Expression zeigte. Unter fraktionierter Strahlentherapie konnte in dem Panel von sechs Tumorlinien keine einheitliche Aktivierung des PI3K/AKT-Signalweges nachgewiesen werden. Die Expression des endogenen Hypoxiemarkers HIF 1α korrelierte in unbehandelten Tumoren mit der pAKT-Proteinexpression. Kein Zusammenhang wurde zwischen dem Gehalt von pAKT und der Pimonidazol hypoxischen Fraktion sowie der lokalen Tumorkontrolle nach 30 Fraktionen in sechs Wochen gefunden. 2. Bestimmung der Wirksamkeit des selektiven PI3K-Inhibitors Bay 80-6946 in HNSCC-Zelllinien in vitro Die Experimente wurden an den humanen Plattenepithelkarzinomzelllinien Cal33, FaDu, UT-SCC-5 und UT-SCC-14 durchgeführt. In Proliferationsassays und weiteren Zellkultur-experimenten wurde der Effekt des selektiven PI3K-Inhibitors Bay 80-6946 auf die Proliferation, die Phosphorylierung von AKT sowie die Dauer des inhibitorischen Effektes untersucht. Das klonogene Zellüberleben wurde bestimmt, indem die Tumorzellen mit 0, 2, 4, 6 oder 8 Gy bestrahlt, 24 Stunden mit dem Inhibitor inkubiert und die entstandenen Kolonien nach 14 Tagen ausgezählt wurden. Eine deutliche Hemmung der Proliferation konnte an den Plattenepithelkarzinomzelllinien schon bei geringen Konzentrationen von Bay 80-6946 gezeigt werden. Die Zelllinie Cal33, welche eine bekannte PI3K Mutation besitzt, reagierte am empfindlichsten auf die Inhibition von PI3K, gefolgt von UT-SCC-14 und FaDu. Nach Inkubation mit 1 µM Bay 80 6946 konnte in keiner der drei Tumorlinien phosphoryliertes AKT nachgewiesen werden. Die Dauer der Inhibition hielt allerdings nur für die Dauer der Inkubation mit Bay 80-6946 an. Das klonogene Zellüberleben nach Bestrahlung wurde durch die Behandlung mit dem Inhibitor nicht signifikant verändert. 3. Untersuchung der Wirksamkeit von Bay 80-6946 in einem Panel von HNSCC-Experimentaltumoren in vivo mit und ohne Bestrahlung sowie des Einflusses der PI3K-Inhibition auf radiobiologische Parameter Die Wirksamkeit von Bay 80-6946 wurde in einer Gruppe von neun verschiedenen Kopf-Hals-Tumorlinien sowie zwei NSCLC-Tumorlinien untersucht (A549, Cal33, FaDu, GLF, H460, SAS, SAT, UT-SCC-5, UT-SCC-8, UT-SCC-14 und XF354). Experimenteller Endpunkt war die Tumorwachstumsverzögerung. Bay 80-6946 wurde insgesamt fünfmal im Abstand von 48 Stunden in einer Konzentration von 20 mg/kg KG intravenös injiziert. Die Behandlung begann bei einem Tumordurchmesser von 7 mm. In den drei Tumorlinien Cal33, FaDu und UT-SCC-5 wurde Bay 80-6946 zusätzlich zur Monotherapie auch in Kombination mit fraktionierter Bestrahlung untersucht. Diese Tumormodelle wurden ausgewählt, da sie die intertumorale Heterogenität im Ansprechen auf die alleinige PI3K-Inhibition widerspiegeln. Die Tiere wurden mit 5 x 2 Gy täglich (Gesamtdosis 10 Gy) bestrahlt. Bay 80-6946 wurde einmal wöchentlich in einer Konzentration von 20 mg/kg KG über einen Zeitraum von 29 Tagen verabreicht. Die Behandlung begann eine Woche vor der ersten Bestrahlungsfraktion. Aufgrund des verlängerten Gesamtbehandlungs-zeitraums begann die Behandlung bereits bei einem Tumordurchmesser von 4 mm. In allen Experimentalarmen wurden zu verschiedenen Zeitpunkten Tumoren entnommen und immunhistochemisch auf die radiobiologischen Parameter Hypoxie, Anzahl der Gefäße und Perfusion untersucht. Hinsichtlich der Wirksamkeit von Bay 80-6946 auf unbehandelte Plattenepithelkarzinome lassen sich drei Gruppen abgrenzen: starkes Ansprechen bei Cal33, intermediäres Ansprechen (SAS, UT-SCC-8, SAT, FaDu) und fehlendes Ansprechen (XF354, GLF, UT SCC-5, UT-SCC-14). Die NSCLC-Modelle A549 und H460 sprachen moderat auf Bay 80 6946 an. Bei der Kombinationstherapie von Bay 80-6946 mit fraktionierter Bestrahlung deutet sich bei Cal33 ein additiver und bei FaDu ein supraadditiver Effekt an. Bei UT-SCC-5 war in der Kombinationstherapie kein signifikanter Effekt auf das Tumorwachstum nachweisbar. Die immunhistochemischen Analysen zeigten in allen drei Tumorlinien weder bei der Mono- noch bei der Kombinationstherapie einen Einfluss des Inhibitors auf die Parameter Hypoxie, Anzahl der Gefäße und Perfusion im Vergleich zur Kontrolle. Schlussfolgerung Die in einem Panel von Experimentaltumoren gewonnen Daten unterstützen derzeit nicht den Einsatz von pAKT als Biomarker, um das Ansprechen auf eine fraktionierte Strahlentherapie vorherzusagen. Vielmehr scheinen die gewonnenen Ergebnisse im Einklang zu den kontroversen und teilweise inkonsistenten Daten anderer Arbeitsgruppen zu stehen. Somit sind trotz klarer radiobiologischer Rationale weitere methodische und translationale Untersuchungen zum potenziellen Stellenwert des AKT-Signalweges als prognostischer Biomarker für die Strahlentherapie von Kopf-Hals-Tumoren notwendig. Durch die Kombination einer fraktionierten Strahlentherapie mit einem selektiven PI3K-Inhibitior konnte in der vorliegenden experimentellen Arbeit ein deutlicher Effekt auf die Tumorwachstumsverzögerung von Kopf-Hals-Tumoren gezeigt werden. Das Ausmaß des Ansprechens unterschied sich jedoch zwischen den drei untersuchten Tumorlinien und wirft die Frage nach der Ursache dieser Variabilität auf. Vor dem Einsatz eines PI3K-Inhibitors unter fraktionierter Strahlentherapie sollte in weiteren Experimenten der zugrundeliegende Mechanismus der Strahlensensibilisierung geklärt werden. Als zentrale Mechanismen sollten dabei vertiefend der Einfluss der selektiven PI3K-Inhibition auf das Tumormikromilieu sowie die Beteiligung des PI3K-Inhibitors an der Reparatur strahleninduzierter Schäden untersucht werden.

Radiation therapy is afflicted by a multitude of geometric uncertainties, which must be compensated to ensure treatment success. Such mitigation is currently achieved by enlarging the apparent target volume by various safety margins. This thesis investigated uncertainty sources relating to target position and extent in oesophageal carcinoma, both static and dynamic, and evaluated their impact in a combined model. The first were errors inherent to delineation of the gross tumour volume (GTV), where computed tomography (CT) imaging, the overall modality of choice for target volume delineation (TVD), has a tendency to overestimate target extent. Two rival modalities, [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) and endoscopic ultrasound (EUS), are generally expected to yield more accurate assessments. EUS has previously suffered from a difficulty in transferring its findings to the spatial domain in which TVD is undertaken. This limitation was overcome here through the use of endoscopically implanted fiducial markers visible on the planning CT. This has enabled their inclusion in TVD and allowed a direct comparison of FDG-PET and EUS based target extents, which were found to agree quite well on average, but showed occasional discrepancies on the order of a few cm. Recently published reports on inter-observer variability (IOV) in TVD of oesophageal carcinoma were summarised with a particular focus on its reduction afforded by the use of fiducial markers. The influence of IOV was investigated more widely in other tumour entities, where it was shown to increase during the course of treatment, mostly due to differing adaptation practices. Microscopic disease extension (MDE), undetectable prior to treatment with current imaging techniques, constituted the second uncertainty source. Reports on histopathological measurements of MDE incidence and its distance from the main tumour were reviewed and spatial measurements extracted to derive a combined estimate of the distribution of extension distances. The overall incidence was estimated as 14.6%, with individual studies reporting widely differing values. Conventional margin widths to compensate for MDE were extracted from the pooled distribution and found to largely agree with the common clinical choice of 3–5 cm, but associated with broad confidence intervals. The addition of such margins to the GTV defines the clinical target volume (CTV). Most studies acknowledged tissue deformations as a major problem, but not all implemented means to prevent or correct it. Preliminary measurements on oesophageal resection specimens were presented, wherein fiducial markers were used to measure tissue deformations, and might ultimately be used to correct spatial measurements of MDE. Fiducial markers also facilitated the study of inter-fractional target mobility in a cohort (n=23) receiving daily orthogonal X-ray imaging for target positioning verification. Markers were found capable of detecting target misalignments, which were a common occurrence with 54% and 15% of analysed markers and treatment fractions showing displacements from their planned position in excess of 5 and 10mm, respectively. Mobility amplitudes were highest in the longitudinal direction and a dependence on tumour location was hinted at, with motion more restricted for proximally located lesions. Measures of systematic and random mobility components were extracted to derive safety margins, which are added to the CTV to form the planning target volume (PTV). A radiobiological model of tumour control probability (TCP) was then evaluated under different uncertainty scenarios. It simplified the tumour system to its longitudinal dimension, which is most affected by the aforementioned phenomena, and simulated positional uncertainties, as well as MDE. The differential impact of systematic and random mobility components on TCP was demonstrated and margin widths sufficient to limit TCP reduction to 1% could best be described by a quadratic combination of their magnitudes. This composition was still applicable when MDE was introduced and mitigated by a conventional margin, but the relative impact of both components shifted. The addition of a PTV margin to the CTV afforded the MDE-positive subpopulation similar protection against positional uncertainties as the same margin achieved without consideration of MDE. The MDE-negative subpopulation attained a much improved tolerance to positional uncertainties through the CTV margin, which also propagated to the overall population. An alternative mitigation of MDE was attempted by optimising the applied dose distribution to an assumed tumour cell density distribution motivated by the literature, which decreases towards the target edge. The optimisation maximised TCP while preserving the integral dose delivered with a conventional margin, under the assumption that this translates into a similar likelihood of normal tissue toxicity. Reduced doses could be delivered to lower cell density regions without sacrificing overall TCP, but this reduction was modest despite vastly diminished cell densities. When this spared dose was redistributed so as to enlarge the treated area, negligible TCP change was observed, but redistribution to the central target did result in appreciably improved TCP in both subpopulations. These effects persisted when positional uncertainties were added and when MDE incidence was increased to the most extreme value reported in the literature.

The radiosensitivity of tumour cells depends mainly on their capacity to maintain genomic integrity. This requires efficient repair of radiation-induced DNA double strand breaks, a process governed by the cell cycle. Based on their functions in cell cycle regulation and DNA damage repair, we hypothesised that targeting of CDK2 and CDK9 modifies cancer cell response to radiotherapy. Therefore, we evaluated the significance of CDK2 and CDK9 for the cellular radiation response in a panel of human head and neck squamous cell carcinoma (HNSCC) cell lines. In order to achieve our goal, we performed a series of experiments to measure several key parameters such as clonogenic radiation survival, cell cycling, DNA damage repair and apoptosis. We found that loss of CDK2 radiosensitises mouse embryonic fibroblasts (MEFs) as well as HNSCC two dimensional (2D) cell cultures. However, under more physiological three dimensional (3D) growth conditions in laminin-rich extracellular matrix, targeting of CDK2 failed to modulate the radiosensitivity of HNSCC cells. Moreover, CDK2 attenuated the repair of radiogenic double strand breaks (DSBs) in MEFs as well as SAS and FaDu HNSCC cells indicating a possible role of CDK2 in DNA damage repair. However, we found that CDK2 is dispensable for cell cycle and checkpoint regulation in response to irradiation in SAS and FaDu cells. Taken together, our results suggest that targeting of CDK2 may not provide a therapeutic benefit to overcome HNSCC cell resistance to radiotherapy. We also showed that depletion of CDK9 clearly enhances the radiosensitivity of HNSCC cultures. In addition, the ectopic expression of CDK9 has a radioprotective effect. These findings suggest a potential role of CDK9 in the radiation response of HNSCC cells. Moreover, our study indicates a possible role of CDK9 in the DNA damage repair response and cell cycling of HNSCC cells. Conclusively, on the basis of these data, targeting of CDK9 in addition to conventional radiotherapy might be a viable strategy to overcome cancer cell resistance.

Background: Head and neck squamous cell carcinomas (HNSCCs) are complex and highly aggressive tumours that develop in the mouth, throat, salivary glands and nose. HNSCCs account for more than half a million cases annually and are the sixth most common cancer worldwide. Alcohol, tobacco and human papilloma virus (HPV) infection are the well-known causes for HNSCC. The current options for treatment are surgery, radiotherapy, chemotherapy or a combination of therapies. Locally advanced HNSCC patients show heterogenous response to standard treatments and the survival after 5 years is about 50%. Therefore, there is a need to identify biomarkers to predict outcome and improve personalised therapies. The recent advancement in next generation sequencing technologies has allowed for understanding the molecular characteristics of the tumour and identify patients at high risk that are unresponsive to the standard treatment. HPV-associated oropharyngeal carcinoma have shown a very high rate of loco-regional control (LRC) and overall survival (OS) after postoperative radio- chemotherapy (PORT-C) and are being assessed for treatment de-escalation strategies to reduce toxicity in clinical trials. The treatment response of HPV-negative HNSCC, however, is still heterogeneous and novel biomarkers are required to identify subgroups of patients for treatment adaptation. Objectives: The overall aim of the thesis is to develop biomarkers to identify patients at high risk for future treatment adaptations and improve personalised radiotherapy based on the biological differences in HNSCC patients. For this purpose, novel gene signatures were developed and validated using machine learning approaches and biological information in order to predict LRC in patients with locally advanced HNSCC. The novel gene signatures will help to identify patients at high risk that do not respond to standard treatments and to further understand the molecular mechanisms involved in heterogenous treatment response. Materials and methods: The data from a total of 504 locally advanced HNSCC patients of the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG) treated with postoperative radiotherapy (PORT) or postoperative radiochemotherapy (PORT-C) were evaluated. Data from 60 mice bearing xenografts of ten established human HNSCC cell lines were also evaluated. Gene expression analyses was performed using the GeneChip Human Transcriptome Array 2.0 and nanoString analyses. Differential gene expression analysis, Cox regression analysis and machine learning algorithms were used to develop gene signatures. Models were built on the training cohort and then applied on an independent validation cohort. Results: The patients with HPV-negative HNSCC that were treated with PORT-C were classified into the four molecular subtypes basal, mesenchymal, atypical and classical that were previously reported for HNSCC patients treated with primary radio(chemo)therapy or surgery and were related to LRC. The mesenchymal subtype had the worst prognosis as compared to the other subtypes. These tumours were associated with overexpression of epithelial-mesenchymal transition genes and DNA repair genes. A novel 6-gene signature was developed and validated based on full-transcriptome data using machine-learning approaches that was prognostic for LRC in patients with HPV-negative HNSCC treated with PORT-C. The 6-gene signature consisted of four individual genes CAV1, GPX8, IGLV3-25, TGFBI and one metagene combining the highly correlated genes, INHBA and SERPINE1. The identified gene signature was combined with the clinical parameters, T stage and tumour localisation as well as the stem-cell marker CD44 and the 15-gene hypoxia- associated classifier and this improved the performance of the model. Previously identified prognostic gene signatures and molecular-subtype classification were back-translated from HNSCC patients to pre-clinical tumour models. The tumour models were classified into the four subtypes basal, mesenchymal, atypical and classical, similar to the patients. The mesenchymal tumours were significantly associated with a higher TCD50 as compared to other subtypes. A novel 2-gene signature consisting of FN1 and SERPINE1 was developed based on tumour models and patient data using differential gene expression analysis. The 2-gene signature was prognostic for the TCD50 in tumour models and was successfully validated on an independent PORT-C patient cohort for LRC. A matched-pair analysis was performed between patients that were treated with postoperative radiochemotherapy and patients that were treated with postoperative radiotherapy. A 2- metagene signature, consisting of KRT6A, KRT6B, KRT6C forming one metagene and SPRR1A, SPRR1B, SPRR2A, SPRR2C forming the second metagene, was identified. The novel predictive signature stratified patients into high and low risk groups. The high-risk group patients that received PORT-C showed higher LRC as compared to the high-risk patients that received PORT. Thus, the predictive gene signature identified patients that were considered to be at intermediate risk according to clinical factors but were at biologically high risk for the development of loco-regional recurrences after PORT. These patients might benefit from PORT-C treatment. Conclusions: In this thesis, novel gene signatures were identified by combining machine learning and biological information to stratify locally advanced HNSCC patients into high and low risk groups for loco-regional control. This information could be used in the future, e.g. to adjust radiotherapy doses based on the risk group. The developed gene signatures could be combined with other gene signatures or the molecular subtype stratification to develop potential combined treatment approaches. Within the DKTK-ROG framework, the gene signatures will be incorporated with biomarkers developed on the same cohort at the other DKTK-ROG partner sites using the data from different omics platforms in the future. This would help to better understand the molecular basis of heterogenous treatment response in HNSCC patients and uncover novel targets for therapies. The thesis also provides a valuable insight into the applicability of preclinical tumour models to study the efficacy of personalised radiotherapy treatments. Overall, the gene signatures identified in this thesis were from retrospective studies and have to be validated in prospective studies before their application in interventional clinical trials to improve personalised radiotherapy treatments. Additionally, the methods used in the thesis to identify the gene signatures could be used and applied across different cancer datasets for identification of biomarkers. Therefore, this thesis has provided a basis for future studies on personalized treatment of HNSCC based on their genetic profile.:Content Abbreviations VII Tables XII 1 Introduction 1 2 Biological and statistical background 6 2.1 Head and neck squamous cell carcinoma 6 2.1.1 Tumourigenesis 6 2.1.2 Biomarkers: clinical and genomics 9 2.2 Statistics 12 General statistical analyses 17 2.3 Gene expression analyses 18 3 Molecular subtypes and mechanisms of radioresistance 20 3.1 Introduction and motivation 20 3.2 Patient cohort and experimental design 21 3.2.1 Patient cohort 21 3.2.2 Clinical endpoints and statistical analysis 23 3.2.3 Experimental design 23 3.3 Results 26 3.3.1 Prognostic factors for LRC and OS 26 3.3.2 Death as competing risk 26 3.3.3 Multivariable Cox regression for improved prognosis 29 3.3.4 Molecular subtypes in HPV-negative HNSCC patients 31 3.3.5 Molecular subtypes are prognostic for LRC after PORT-C 33 3.4 Discussion 36 4 A novel 6-gene signature for LRC prognosis 39 4.1 Introduction and motivation 39 4.2 Patient cohort and experimental design 40 4.2.1 Patient cohorts 40 4.2.2 Clinical endpoints and statistical analysis 41 4.2.3 Experimental design 41 4.3 Results 44 4.3.1 Characteristics of the patient cohorts 44 4.3.2 Development of the 6-gene signature prognostic for LRC 45 4.3.3 Combination of the 6-gene signature and clinical parameters 47 4.3.4 Extension with CD44 and the 15-gene hypoxia signature 48 4.3.5 Prognostic for secondary endpoints 49 4.3.6 Technical validation using nanoString technology 52 4.3.7 Death as competing risk 56 4.4 Discussion 58 5 Biomarker development in preclinical tumour models and HNSCC patients 62 5.1 Introduction and motivation 62 5.2 Patient cohort and experimental design 64 5.2.1 Patient derived xenograft tumour models 64 5.2.2 Patient cohorts 64 5.2.3 Clinical endpoints and statistical analysis 65 5.2.4 Experimental design 65 5.3 Results 68 5.3.1 Molecular subtypes 68 5.3.2 Development of the 2-gene signature 70 5.3.3 Technical validation using the nanoString technology 71 5.3.4 Back-translation of gene signatures in xenograft models 75 5.4 Discussion 79 6 PORT-C improves LRC in intermediate-risk patients 82 6.1 Introduction and motivation 82 6.2 Patient cohort and experimental design 83 6.2.1 Patient cohorts 84 6.2.2 Clinical endpoints and statistical analysis 84 6.2.3 Experimental design 84 6.3 Results 87 6.3.1 Characteristics of the patient cohorts 87 6.3.2 Propensity score matching analysis 88 6.3.3 Development of the predictive 2-metagene signature 90 6.4 Discussion 93 7 Conclusion and future perspectives 96 8 Summary 99 9 Zusammenfassung 102 Appendix 105 A. Supplementary Figures 105 B. Supplementary Tables 110 Bibliography 116 Erklärungen 149