Oliver Riesterer

Tumor Recovery by Angiogenic Switch from Sprouting to Intussusceptive Angiogenesis after Treatment with PTK787/ZK222584 or Ionizing Radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.

Degradation of PKB/Akt protein by inhibition of the VEGF receptor/mTOR pathway in endothelial cells.

An intact VEGF receptor/PI3K/PKB/Akt signaling cascade protects endothelial cells from apoptotic stress-stimuli and mediates the formation of new blood vessels in pathological conditions such as cancer. Therefore, downregulation of this signaling cascade is of clinical interest for antiangiogenic cancer therapy. In this report, we demonstrate that VEGF controls the protein stability of the serine–threonine kinase PKB/Akt via inhibition of PKB/Akt protein degradation. VEGF deprivation or blockage of the VEGF signal transduction cascade with the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 resulted in a specific decrease of the PKB/Akt protein level and subsequent cellular restimulation with VEGF rescued its stability. Real-time quantitative RT–PCR analysis demonstrated that VEGF does not regulate PKB/Akt gene expression. On the other hand, broad range inhibitors of caspases and the proteasome complex prevented VEGF-dependent downregulation of the PKB/Akt protein level indicating that PKB/Akt protein stability is regulated by VEGF-controlled proteolysis. Inhibition of the VEGF receptor and PKB/Akt-downstream PIK-related mTOR-kinase by rapamycin also neutralized the VEGF-protective effect in an PKB/Akt gene expression-independent way but results in proteolysis-dependent reduction of PKB/Akt protein stability. These results demonstrate a novel regulatory mechanism of the activated VEGF receptor/mTOR-signal transduction pathway to control the protein stability of PKB/Akt and survival threshold in endothelial cells.

Neoadjuvant chemotherapy and extrapleural pneumonectomy of malignant pleural mesothelioma with or without hemithoracic radiotherapy (SAKK 17/04): a randomised, international, multicentre phase 2 trial

BACKGROUND Postoperative hemithoracic radiotherapy has been used to treat malignant pleural mesothelioma, but it has not been assessed in a randomised trial. We assessed high-dose hemithoracic radiotherapy after neoadjuvant chemotherapy and extrapleural pneumonectomy in patients with malignant pleural mesothelioma. METHODS We did this phase 2 trial in two parts at 14 hospitals in Switzerland, Belgium, and Germany. We enrolled patients with pathologically confirmed malignant pleural mesothelioma; resectable TNM stages T1-3 N0-2, M0; WHO performance status 0-1; age 18-70 years. In part 1, patients were given three cycles of neoadjuvant chemotherapy (cisplatin 75 mg/m(2) and pemetrexed 500 mg/m(2) on day 1 given every 3 weeks) and extrapleural pneumonectomy; the primary endpoint was complete macroscopic resection (R0-1). In part 2, participants with complete macroscopic resection were randomly assigned (1:1) to receive high-dose radiotherapy or not. The target volume for radiotherapy encompassed the entire hemithorax, the thoracotomy channel, and mediastinal nodal stations if affected by the disease or violated surgically. A boost was given to areas at high risk for locoregional relapse. The allocation was stratified by centre, histology (sarcomatoid vs epithelioid or mixed), mediastinal lymph node involvement (N0-1 vs N2), and T stage (T1-2 vs T3). The primary endpoint of part 1 was the proportion of patients achieving complete macroscopic resection (R0 and R1). The primary endpoint in part 2 was locoregional relapse-free survival, analysed by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00334594. FINDINGS We enrolled patients between Dec 7, 2005, and Oct 17, 2012. Overall, we analysed 151 patients receiving neoadjuvant chemotherapy, of whom 113 (75%) had extrapleural pneumonectomy. Median follow-up was 54·2 months (IQR 32-66). 52 (34%) of 151 patients achieved an objective response. The most common grade 3 or 4 toxic effects were neutropenia (21 [14%] of 151 patients), anaemia (11 [7%]), and nausea or vomiting (eight [5%]). 113 patients had extrapleural pneumonectomy, with complete macroscopic resection achieved in 96 (64%) of 151 patients. We enrolled 54 patients in part 2; 27 in each group. The main reasons for exclusion were patient refusal (n=20) and ineligibility (n=10). 25 of 27 patients completed radiotherapy. Median total radiotherapy dose was 55·9 Gy (IQR 46·8-56·0). Median locoregional relapse-free survival from surgery, was 7·6 months (95% CI 4·5-10·7) in the no radiotherapy group and 9·4 months (6·5-11·9) in the radiotherapy group. The most common grade 3 or higher toxic effects related to radiotherapy were nausea or vomiting (three [11%] of 27 patients), oesophagitis (two [7%]), and pneumonitis (two [7%]). One patient died of pneumonitis. We recorded no toxic effects data for the control group. INTERPRETATION Our findings do not support the routine use of hemithoracic radiotherapy for malignant pleural mesothelioma after neoadjuvant chemotherapy and extrapleural pneumonectomy. FUNDING Swiss Group for Clinical Cancer Research, Swiss State Secretariat for Education, Research and Innovation, Eli Lilly.

Ionizing Radiation Antagonizes Tumor Hypoxia Induced by Antiangiogenic Treatment

Purpose: The combined treatment modality of ionizing radiation with inhibitors of angiogenesis is effective despite the supposition that inhibition of angiogenesis might increase tumor hypoxia and thereby negatively affect radiation sensitivity. To directly assess this still controversial issue, we analyzed treatment-dependent alterations of tumor oxygenation in response to inhibition of angiogenesis alone, ionizing radiation, and combined treatment. Experimental Design: Serial measurements with high-resolution [18F]fluoromisonidazole positron emission tomography and immunohistochemical detection of the endogenous hypoxia marker glucose transporter-1 were done to determine tumor hypoxia in a murine mammary carcinoma allograft model. Results: Inhibition of angiogenesis with the clinically relevant vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584 reduced microvessel density but had only minimal effects on tumor growth, tumor cell apoptosis, and proliferation. However, PTK787/ZK222584 treatment increased overall and local tumor hypoxia as revealed by extended expression of the hypoxia marker glucose transporter-1 and increased uptake of [18F]fluoromisonidazole. Fractionated irradiation induced a strong growth delay, which was associated with enhanced apoptosis and reduced proliferation of tumor cells but only minor effects on microvessel density and allograft oxygenation. Combined treatment with fractionated irradiation resulted in extended tumor growth delay and tumor cell apoptosis but no increase in tumor hypoxia. Conclusions: These results show that irradiation antagonizes the increase of hypoxia by vascular endothelial growth factor receptor tyrosine kinase inhibition and abrogates the potential negative effect on tumor hypoxia. Thus, the risk of treatment-induced hypoxia by inhibitors of angiogenesis exists but is kept minimal when combined with a cytotoxic treatment modality.

Differential Activation of the Phosphatidylinositol 3′-Kinase/Akt Survival Pathway by Ionizing Radiation in Tumor and Primary Endothelial Cells

Ionizing radiation induces an intracellular stress response via activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt survival pathway. In tumor cells, the PI3K/Akt pathway is induced through activation of members of ErbB receptor tyrosine kinases. Here, we investigated the receptor dependence of radiation-induced PI3K/Akt activation in tumor cells and in endothelial cells. The integrity of both the ErbB and the vascular endothelial growth factor (VEGF) ligand-activated PI3K/Akt pathway in endothelial cells was demonstrated using specific ErbB and VEGF receptor tyrosine kinase inhibitors. Irradiation of endothelial cells resulted in protein kinase B (PKB)/Akt activation in a similar time course as observed in response to VEGF. More importantly, radiation-induced PKB/Akt phosphorylation in endothelial cells was strongly down-regulated by the VEGF receptor tyrosine kinase inhibitor, whereas the ErbB receptor tyrosine kinase inhibitor did not affect PKB/Akt stimulation in response to irradiation. An opposite receptor dependence for radiation-induced PKB/Akt phosphorylation was observed in ErbB receptor-overexpressing A431 tumor cells. Furthermore, direct VEGF receptor phosphorylation was detected after irradiation in endothelial cells in absence of VEGF, which was almost completely inhibited after irradiation in presence of the VEGF receptor tyrosine kinase inhibitor. These data demonstrate that ionizing radiation induces VEGF ligand-independent but VEGF receptor-dependent PKB/Akt activation in endothelial cells and that PI3K/Akt pathway activation by radiation occurs in a differential cell type and receptor-dependent pattern.

Clinical application of flattening filter free beams for extracranial stereotactic radiotherapy.

PURPOSE To investigate the clinical application of flattening filter free (FFF) beams at maximum dose rate for stereotactic body radiotherapy (SBRT). METHODS AND MATERIALS Patients with tumors in the lung or abdomen were subjected to SBRT using 6 MV FFF or 10 MV FFF beams. For each patient, three plans were calculated using 6 MV flattened, 6 MV FFF, and 10 MV FFF beams. Treatment times were recorded and analyzed, and tumor displacements were assessed by pre- and post-treatment cone beam computed tomography (CBCT). RESULTS Altogether, 26 patients (16 lung, 10 abdominal tumors) were treated. The average dose rate per patient ranged from 442 to 1860 MU/min. Beam-on time was on average 1.6 min (1SD=0.6 min), with the total treatment times recorded at 18.5 min (1SD=3.5 min). The time advantage of using FFF beams was dose-dependent and started at 4 Gy for 6 MV FFF and at 10 Gy for 10 MV FFF beams. The average of the tumor displacements during treatment was 2.0mm (1SD = 1.0mm). CONCLUSIONS SBRT using FFF beams is time efficient and associated with excellent patient stability. According to Van Herks formula, ITV-PTV margins of 6mm are sufficient in our patient cohort. Further studies are necessary to assess clinical outcome and toxicity.

Development and evaluation of a prototype tracking system using the treatment couch

PURPOSE Tumor motion increases safety margins around the clinical target volume and leads to an increased dose to the surrounding healthy tissue. The authors have developed and evaluated a one-dimensional treatment couch tracking system to counter steer respiratory tumor motion. Three different motion detection sensors with different lag times were evaluated. METHODS The couch tracking system consists of a motion detection sensor, which can be the topometrical system Topos (Cyber Technologies, Germany), the respiratory gating system RPM (Varian Medical Systems) or a laser triangulation system (Micro Epsilon), and the Protura treatment couch (Civco Medical Systems). The control of the treatment couch was implemented in the block diagram environment Simulink (MathWorks). To achieve real time performance, the Simulink models were executed on a real time engine, provided by Real-Time Windows Target (MathWorks). A proportional-integral control system was implemented. The lag time of the couch tracking system using the three different motion detection sensors was measured. The geometrical accuracy of the system was evaluated by measuring the mean absolute deviation from the reference (static position) during motion tracking. This deviation was compared to the mean absolute deviation without tracking and a reduction factor was defined. A hexapod system was moving according to seven respiration patterns previously acquired with the RPM system as well as according to a sin(6) function with two different frequencies (0.33 and 0.17 Hz) and the treatment table compensated the motion. RESULTS A prototype system for treatment couch tracking of respiratory motion was developed. The laser based tracking system with a small lag time of 57 ms reduced the residual motion by a factor of 11.9 ± 5.5 (mean value ± standard deviation). An increase in delay time from 57 to 130 ms (RPM based system) resulted in a reduction by a factor of 4.7 ± 2.6. The Topos based tracking system with the largest lag time of 300 ms achieved a mean reduction by a factor of 3.4 ± 2.3. The increase in the penumbra of a profile (1 × 1 cm(2)) for a motion of 6 mm was 1.4 mm. With tracking applied there was no increase in the penumbra. CONCLUSIONS Couch tracking with the Protura treatment couch is achievable. To reliably track all possible respiration patterns without prediction filters a short lag time below 100 ms is needed. More scientific work is necessary to extend our prototype to tracking of internal motion.

Current Concepts for the Combined Treatment Modality of Ionizing Radiation with Anticancer Agents

In current applied radiobiology, there exists a tremendous effort in basic and translational research to identify novel treatment modalities combining ionizing radiation with anticancer agents. This is mainly due to the highly improved molecular understanding of intrinsic radioresistance and the profiling of cellular stress responses to irradiation during recent years. Ionizing radiation not only damages DNA but also affects multiple cellular components that induce a multi-layered stress response. The treatment responses can be restricted to the individual cell level but might also be part of an intercellular stress communication network. Both DNA damage-induced signaling (which results in cell cycle arrest and induction of the DNA-repair machinery) and also ionizing radiation-induced signal transduction cascades, which are generated at cellular sites distant from and independent of DNA-damage, represent interesting targets for anticancer treatment modalities to sensitize for ionizing radiation. Due to the lack of molecular knowledge classic radiobiology assembled the cellular and tissue responses into four groups (4 Rs of radiotherapy) which describe biological factors influencing the treatment response to fractionated radiotherapy. These classic 4 Rs are Repair, Reassortment, Repopulation and Reoxygenation. With the tremendous progress in molecular oncology we now begin to understand theses factors on the molecular level. At the same time this classification may guide modern molecular radiobiologists to identify novel pharmaceuticals and antisignaling agents which can modulate the treatment response to irradiation. In this review we describe current approaches to sensitize tumor cells with novel anticancer agents along the lines of these 4 Rs.

Breast reconstruction and post-mastectomy radiation practice

PurposeThe goal of this study was to explore the perspectives and practice of radiation oncologists who treat breast cancer patients who have had breast reconstruction.MethodsIn 2010, an original electronic survey was sent to all physician members of the American Society of Radiation Oncology, National Cancer Research Institute-Breast Cancer Studies Group in the United Kingdom, Thai Society of Therapeutic Radiology and Oncology, Swiss Society of Radiation Oncology, and Turkish Radiation Oncology Society. We identified factors associated with radiation oncologists who treat breast cancer patients with reconstruction performed prior to radiation and obtained information regarding radiation management of the breast reconstruction.Results358 radiation oncologists responded, and 60% of the physicians were from the United States. While 64% of participants agree or strongly agree that breast image affects a woman’s quality of life during radiation, 57% feel that reconstruction challenges their ability to deliver effective breast radiation. Compared with other countries, treatment within the United States was associated with a high reconstruction rate (>/= 50% of mastectomy patients) prior to radiation (p < 0.05). Delayed-immediate reconstruction with a temporary tissue expander was more common in the United States than in other countries (52% vs. 23%, p = 0.01). Among physicians who treat patients with tissue expanders, the majority (60%) prefer a moderately inflated implant with 150-250 cc of fluid rather than a completely deflated (13%) or inflated expander (28%) during radiation. Among radiation oncologists who treat reconstructions, 49% never use bolus and 40% never boost a breast reconstruction. United States physicians were more likely than physicians from other countries to boost or bolus the reconstruction irrespective of the type of reconstruction seen in their clinic patients (p < 0.01).ConclusionsGreat variation in practice is evident from our study of radiation treatment for breast cancer patients with reconstruction. Further research on the impact and delivery of radiation to a reconstructed breast may validate some of the observed practices, highlight the variability in treatment practice, and help create a treatment consensus.

Hypoxia modulation and radiosensitization by the novel dual EGFR and VEGFR inhibitor AEE788 in spontaneous and related allograft tumor models

Concomitant inhibition of ErbB1/2- and VEGF receptor-signaling synergizes when used in combination with DNA-damaging agents. Here, we investigated for the first time the combined treatment modality of the novel dual specific receptor tyrosine kinase inhibitor AEE788 with ionizing radiation and analyzed treatment-induced end points in situ as indicators for a potential sensitizing mechanism. Furthermore, we assessed tumor hypoxia in response to different antiangiogenic and antiproliferative treatment modalities. The combined treatment effect was investigated in a spontaneously growing mammary carcinoma model and against Her-2/neu-overexpressing mammary carcinoma allografts. In tumor allografts derived from murine mammary carcinoma cells of mouse mammary tumor virus/c-neu transgenic mice, a minimal treatment regimen with AEE788 and fractionated irradiation resulted in an at least additive tumor response. Treatment response in the corresponding spontaneous tumor model strongly exceeded the response induced in the isogenic allografts. Treatment-induced changes of tumor proliferation, apoptosis, and microvessel density were similar in the two tumor models. Treatment with AEE788 alone or in combination with IR strongly improved tumor oxygenation in both tumor models as determined by the detection of endogenous and exogenous markers of tumor hypoxia. Specific inhibition of the VEGF-receptor tyrosine kinase versus Erb1/2-receptor tyrosine kinase indicated that it is the antiproliferative and not the antiangiogenic potency of AEE788 that mediates the hypoxia-reducing effect of this dual kinase-specific inhibitor. Overall, we show that concomitant inhibition of ErbB- and VEGF-receptor signaling by AEE788, in combination with ionizing radiation, is a promising treatment approach, especially in hypoxic, oncogenic ErbB-driven tumors. [Mol Cancer Ther 2007;6(9):2496–504]