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Supplemental Figure 1. Chemical structures of non-FDA approved compounds used in the Study; Supplemental Figure 2. Effect of drugs on growth and target phosphorylation; Supplemental Figure 3. mTOR inhibition does not show dose dependent Radiosensitization; Supplemental Figure 4. MEK162 + RT abrogate spheroid regrowth; Supplemental Figure 5. MEK162 radiosensitizes GBM8 primary spheroid culture and increases γH2AX levels; Supplemental Figure 6. MEK162 abrogates BrdU washout and increases sub-G1 accumulation of BrdU+ cells; Supplemental Figure 7. MEK162 + RT in vivo; Supplemental Table 1: Antibodies used; Supplemental Table 2: Gene signature MEK+RT response

Glioblastoma (GBM) is a highly aggressive and lethal brain cancer type. PI3K and MAPK inhibitors have been studied preclinically in GBM as monotherapy, but not in combination with radiotherapy, which is a key component of the current standard treatment of GBM. In our study, GBM cell lines and patient representative primary cultures were grown as multicellular spheroids. Spheroids were treated with a panel of small-molecule drugs including MK2206, RAD001, BEZ235, MLN0128, and MEK162, alone and in combination with irradiation. Following treatment, spheroid growth parameters (growth rate, volume reduction, and time to regrow), cell-cycle distribution and expression of key target proteins were evaluated. In vivo, the effect of irradiation (3 × 2 Gy) without or with MEK162 (50 mg/kg) was studied in orthotopic GBM8 brain tumor xenografts with endpoints tumor growth and animal survival. The MAPK-targeting agent MEK162 was found to enhance the effect of irradiation as demonstrated by growth inhibition of spheroids. MEK162 downregulated and dephosphorylated the cell-cycle checkpoint proteins CDK1/CDK2/WEE1 and DNA damage response proteins p-ATM/p-CHK2. When combined with radiation, this led to a prolonged DNA damage signal. In vivo data on tumor-bearing animals demonstrated a significantly reduced growth rate, increased growth delay, and prolonged survival time. In addition, RNA expression of responsive cell cultures correlated to mesenchymal stratification of patient expression data. In conclusion, the MAPK inhibitor MEK162 was identified as a radiosensitizer in GBM spheroids in vitro and in orthotopic GBM xenografts in vivo. The data are supportive for implementation of this targeted agent in an early-phase clinical study in GBM patients. Mol Cancer Ther; 17(2); 347–54. ©2017 AACR.See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”

OBJECTIVE: Oral tongue squamous cell carcinoma (OTSCC) frequently harbors non-functional p53 and depends on G2/M checkpoint mediated by WEE1. WEE1 suppression has been identified as a promising anti-tumor strategy. This study investigated the capacity of WEE1 kinase inhibitor (MK-1775) and its underlying mechanisms in enhancing radiation responses of OTSCC cells in vitro.MATERIALS AND METHODS: WEE1 kinase expression and its downstream target (CDK1) were investigated in OTSCC versus normal oral tissue. A synergistic combination of MK-1775 with radiation on OTSCC cell lines with different p53 statuses was assessed by viability assay. The radio-sensitizing effects of MK-1775 on apoptosis, cell cycle, DNA damage, and mitotic entry were also determined.RESULTS: Irradiation enhanced CDK1 expression in all tested cell lines, though the effect was far more pronounced in p53 mutated cell lines. MK-1775 exhibited inhibitory effects against the survival of all cell lines and enhanced their response to the radiation. These effects were strongly elicited by induction of apoptosis and lethal mitosis, but less likely by abrogation of radiation-induced G2 arrest.CONCLUSION: These results demonstrate the efficacy of MK-1775 in enhancing the radiation effect on OTSCC in vitro associated with a significant apoptotic death rate, identifying WEE1 inhibitor as a potent radiosensitizer in OTSCC irrespective of p53 mutational status.

Background and purpose Glioblastoma multiforme (GBM) is the most aggressive subtype of malignant gliomas, with an average survival rate of 15 months after diagnosis. More than 90% of all GBMs have activating mutations in the MAPK/ERK pathway. Recently, we showed the allosteric MEK1/2 inhibitor binimetinib (MEK162) to inhibit cell proliferation and to enhance the effect of radiation in preclinical human GBM models. Because the free drug cannot pass the blood–brain barrier (BBB), we investigated the use of nanocarriers for transport of the drug through the BBB and its efficacy when combined with radiotherapy and temozolomide (TMZ) in glioma spheroids. Methods In vitro studies were performed using multicellular U87 human GBM spheroids. Polymeric nanocarriers (polymersomes) were loaded with MEK162. The interaction between nanocarrier delivered MEK162, irradiation and TMZ was studied on the kinetics of spheroid growth and on protein expression in the MAPK/ERK pathway. BBB passaging was evaluated in a transwell system with human cerebral microvascular endothelial (hCMEC/D3) cells. Results MEK162 loaded polymersomes inhibited spheroid growth. A synergistic effect was found in combination with fractionated irradiation and an additive effect with TMZ on spheroid volume reduction. Fluorescent labeled polymersomes were taken up by human cerebral microvascular endothelial cells and passed the BBB in vitro. Conclusion MEK162 loaded polymersomes are taken up by multicellular spheroids. The nanocarrier delivered drug reduced spheroid growth and inhibited its molecular target. MEK162 delivered via polymersomes showed interaction with irradiation and TMZ. The polymersomes crossed the in vitro BBB model and therewith offer exciting challenges ahead for delivery of therapeutics agents to brain tumours.

Purpose There is progressing evidence for the anti-cancer potential of the natural compound and dietary spice curcumin. Curcumin has been ascribed to be cytotoxic for various tumour cell types, to inhibit cell proliferation and to interfere with the cellular oxidant status. The compound has been notified as a therapeutic agent with radiosensitizing potential in brain tumour therapy. We considered the rationale to combine curcumin with radiation in the treatment of human glioblastoma multiforme (GBM). Method Determination of clonogenic cell survival following exposure of U251 human glioma cells to single dose (1–6 Gy) and fractionated irradiation (5 daily fractions of 2 Gy) without and with curcumin. Additional literature search focused on the interaction between curcumin and radiotherapy in experimental and clinical studies on human glioma. Results No interaction was found on the survival of U251 human glioma cells after irradiation in combination with curcumin at clinically achievable concentrations. Experimental in vitro and in vivo data together with clinical bioavailability data from the literature do not give evidence for a radiosensitizing effect of curcumin. Reported GBM intratumoural curcumin concentrations are too low to either exert an own cytotoxic effect or to synergistically interact with radiation. Novel approaches are being explored to increase the bioavailability of curcumin and to facilitate transport over the blood–brain barrier, aimed to reach therapeutic curcumin levels at the tumour site. Conclusion There is neither a biological nor clinical rationale for using curcumin as radiosensitizer in the therapy of GBM patients.

Introduction: Advancements in cancer management and treatment are associated with strong preclinical research data, in which reliable cancer models are demanded. Indeed, inconsistent preclinical findings and stringent regulations following the 3Rs principle of reduction, refinement, and replacement of conventional animal models currently pose challenges in the development and translation of efficient technologies. The chick embryo chorioallantoic membrane (CAM) is a system for the evaluation of treatment effects on the vasculature, therefore suitable for studies on angiogenesis. Apart from vascular effects, the model is now increasingly employed as a preclinical cancer model following tumor-grafting procedures. Areas covered: The broad application of CAM tumor model is highlighted along with the methods for analyzing the neoplasm and vascular system. The presented and cited investigations focus on cancer biology and treatment, encompassing both conventional and emerging nanomaterial-based modalities. Expert opinion: The CAM tumor model finds increased significance given the influences of angiogenesis and the tumor microenvironment in cancer behavior, then providing a qualified miniature system for oncological research. Ultimately, the establishment and increased employment of such a model may resolve some of the limitations present in the standard preclinical tumor models, thereby redefining the preclinical research workflow.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor response to chemo- and (modest-dose conventionally fractionated) radio-therapy. Emerging evidence suggests that pancreatic stellate cells (PSCs) secrete deoxycytidine, which confers resistance to gemcitabine. In particular, deoxycytidine was detected by analysis of metabolites in fractionated media from different mouse PSCs, showing that it caused PDAC cells chemoresistance by reducing the capacity of deoxycytidine kinase (dCK) for gemcitabine phosphorylation. However, data on human models are missing and dCK expression was not associated with clinical efficacy of gemcitabine. We recently established co-culture models of hetero-spheroids including primary human PSCs and PDAC cells showing their importance as a platform to test the effects of cancer- and stroma-targeted drugs. Here, we discuss the limitations of previous studies and the potential use of above-mentioned models to study molecular mechanisms underlying chemo- and radio-resistance.

Personalized cancer treatments using combinations of drugs with a synergistic effect is attractive but proves to be highly challenging. Here we present an approach to uncover the efficacy of drug combinations based on the analysis of mono-drug effects. For this we used dose-response data from pharmacogenomic encyclopedias and represent these as a drug atlas. The drug atlas represents the relations between drug effects and allows to identify independent processes for which the tumor might be particularly vulnerable when attacked by two drugs. Our approach enables the prediction of combination-therapy which can be linked to tumor-driving mutations. By using this strategy, we can uncover potential effective drug combinations on a pan-cancer scale. Predicted synergies are provided and have been validated in glioblastoma, breast cancer, melanoma and leukemia mouse-models, resulting in therapeutic synergy in 75% of the tested models. This indicates that we can accurately predict effective drug combinations with translational value., Drug synergies impact the efficacy of combination therapies but are difficult to identify. Here Narayan et al. describe the drug atlas, a method to predict effective drug combinations from common exclusive drug effects providing a resource for exploring and understanding effective drug combinations.

Alpha emitters have great potential in targeted tumour therapy, especially in destroying micrometastases, due to their high linear energy transfer (LET). To prevent toxicity caused by recoiled daughter atoms in healthy tissue, alpha emitters like 225Ac can be encapsulated in polymeric nanocarriers (polymersomes), which are capable of retaining the daughter atoms to a large degree. In the translation to a (pre-)clinical setting, it is essential to evaluate their therapeutic potential. As multicellular tumour spheroids mimic a tumour microenvironment more closely than a two-dimensional cellular monolayer, this study has focussed on the interaction of the polymersomes with U87 human glioma spheroids. We have found that polymersomes distribute themselves throughout the spheroid after 4 days which, considering the long half-life of 225Ac (9.9 d) (Vaidyanathan and Zalutsky, 1996), allows for irradiation of the entire spheroid. A decrease in spheroidal growth has been observed upon the addition of only 0.1 kBq 225Ac, an effect which was more pronounced for the 225Ac in polymersomes than when only coupled to DTPA. At higher activities (5 kBq), the spheroids have been found to be destroyed completely after two days. We have thus demonstrated that 225Ac containing polymersomes effectively inhibit tumour spheroid growth, making them very promising candidates for future in vivo testing.

AIMS: Relapsed high-grade gliomas (HGGs) have poor prognoses and there is no standard treatment. HGGs have ischemia/hypoxia associated and, as such, drugs and oxygen have low access, with increased resistance to chemotherapy and radiotherapy. Tumor hypoxia modification can improve outcomes and overall survival in some patients with these tumors. In previous works, we have described that cervical spinal cord stimulation can modify tumor microenvironment in HGG by increasing tumor blood flow, oxygenation, and metabolism. The aim of this current, preliminary, nonrandomized, study was to assess the clinical effect of spinal cord stimulation during brain reirradiation and chemotherapy deployed for the treatment of recurrent HGG; the hypothesis being that an improvement in oxygenated blood supply would facilitate enhanced delivery of the scheduled therapy.MATERIALS AND METHODS: Seven patients had spinal cord stimulation applied during the scheduled reirradiation and chemotherapy for the treatment of recurrent HGG (6 anaplastic gliomas and 1 glioblastoma). Median dose of previous irradiation was 60 Gy (range = 56-72 Gy) and median dose of reirradiation was 46 Gy (range = 40-46 Gy). Primary end point of the study was overall survival (OS) following confirmation of HGG relapse.RESULTS: From the time of diagnosis of last tumor relapse before reirradiation, median OS was 39 months (95% CI = 0-93) for the overall study group: 39 months (95% CI = 9-69) for those with anaplastic gliomas and 16 months for the patient with glioblastoma. Posttreatment, doses of corticosteroids was significantly decreased (P = .026) and performance status significantly improved (P = .046).CONCLUSIONS: Spinal cord stimulation during reirradiation and chemotherapy is feasible and well tolerated. In our study, spinal cord stimulation was associated with clinical improvement and longer survival than previously reported in recurrent anaplastic gliomas. Spinal cord stimulation as adjuvant during chemotherapy and reirradiation in relapsed HGGs merits further research.

Background Glioblastoma multiforme (GBM) is the most common, invasive and deadly primary type of malignant brain tumor. The Phosphatidylinositol-3-Kinase/AKT (PI3K/AKT) pathway is highly active in GBM and has been associated with increased survival and resistance to therapy. The aim of this study is to investigate the effects of AKT inhibition in combination with the current standard of care which consists of irradiation and temozolomide (TMZ) on human malignant glioma cells growing adherent and as multicellular spheroids in vitro. Methods The effects of the allosteric inhibitor MK2206 combined with irradiation and TMZ were assessed on glioma cells growing adherent and as multicellular 3D spheroids. The interaction was studied on proliferation, clonogenic cell survival, cell invasion, −migration and on expression of key proteins in the PI3K-AKT pathway by western blot. Results A differential effect was found at low- (1 μM) and high dose (10 μM) MK2206. At 1 μM, the inhibitor reduced phosphorylation of Thr308 and Ser473 residues of AKT in both adherent cells and spheroids. Low dose MK2206 delayed spheroid growth and sensitized spheroids to both irradiation and TMZ in a synergistic way (Combination index 5 μM). Conclusions The data show that a 3D spheroid model show different sensitivity to irradiation when combined with AKT inhibition. Thereby we show that MK2206 has potential synergistic efficacy to the current standard of care for glioma patients. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3193-9) contains supplementary material, which is available to authorized users.

Glioblastoma multiforme (GMB) is the most malignant and common type of all astrocytic tumors. Current standard of care entails maximum surgical resection of the tumor, followed by radiotherapy and chemotherapy, usually by the alkylating agent Temozolomide (TMZ). Despite this aggressive combination therapy, the survival rate of GBM patients is still low. Deregulation of the phosphatidylinositol 3-kinase (PI3K) / Akt pathway is a frequent occurrence in GBM. Activation of the PI3K-Akt pathway results in disturbance of control of cell growth and cell survival, which contributes to a competitive growth advantage, metastatic competence as well as to therapy resistance. The PI3K-Akt pathway is therefore an attractive therapeutic target in GBM, because it serves as a convergence point for malignant processes and intervention might overcome resistance to chemotherapy and radiation. The present review shows the importance of Akt in GBM and its role in the DNA damage response. Furthermore, an overview is given of specific inhibitors of Akt which are currently being tested in preclinical and in early phase clinical studies.

Refereed/Peer-reviewed

Since the introduction of chemoradiotherapy with temozolomide as the new standard of care for patients with glioblastoma, there has been an increasing awareness of progressive and enhancing lesions on MRI, noted immediately after the end of treatment, which are not related to tumour progression, but which are a treatment effect. This so-called pseudoprogression can occur in up to 20% of patients who have been treated with temozolomide chemoradiotherapy, and can explain about half of all cases of increasing lesions after the end of this treatment. These lesions decrease in size or stabilise without additional treatments and often remain clinically asymptomatic. Additionally, there is evidence that treatment-related necrosis occurs more frequently and earlier after temozolomide chemotherapy than after radiotherapy alone. The mechanisms behind these events have not yet been fully elucidated, but the likelihood is that chemoradiotherapy causes a higher degree of (desired) tumour-cell and endothelial-cell killing. This increased cell kill might lead to secondary reactions, such as oedema and abnormal vessel permeability in the tumour area, mimicking tumour progression, in addition to subsequent early treatment-related necrosis in some patients and milder subacute radiotherapy reactions in others. in patients managed with temozolomide chemoradiotherapy who have clinically asymptomatic progressive lesions at the end of treatment, adjuvant temozolomide should be continued; in clinically symptomatic patients, surgery should be considered. If mainly necrosis is noted during surgery, continuation of adjuvant temozolomide is logical. Trials on the treatment of recurrent malignant glioma should exclude patients with progression within the first 3 months after temozolomide chemoradiotherapy unless histological confirmation of tumour recurrence is available. Further research is needed to establish reliable imaging parameters that distinguish between true tumour progression and pseudoprogression or treatment-related necrosis.

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0–6andnbsp;Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24–72andnbsp;h exposure to 250–750 μM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24andnbsp;h for all cell lines. Exposure to 750andnbsp;μM meloxicam for 24 h increased the fraction of cells in the radiosensitive G2/M cell cycle phase in D384 (18–27%) and U251 (17–41%) cells. 750 μM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.

Object In nearly all patients with glioblastoma multiforme (GBM) a local recurrence develops within a short period of time. In this paper the authors describe two patients in whom a second GBM developed after a relatively long time interval at a site remote from the primary tumor. The genetic profiles of the tumors were compared to discriminate between distant recurrence and a second primary tumor. Methods Both patients harboring a supratentorial GBM were treated with surgery and local high-dose radiotherapy. Local control of the disease at the primary tumor site was achieved. Within 2 years, a second GBM developed in both patients, not only outside the previously irradiated target areas but infratentorially in one patient and in the opposite hemisphere in the other. The tumors were examined for the presence of several genetic alterations that are frequently found in GBMs—a loss of heterozygosity at chromosome regions 1p36, 10p15, 19q13, and 22q13, and at the CDKN2A, PTEN, DMBT1, and TP53 gene regions; a TP53 mutation; and EGFR amplification. In the first patient, genetic profiling revealed that the primary tumor had an allelic imbalance for markers in several chromosome regions for which the second tumor displayed a complete loss. In the second patient, genetic profiling demonstrated the presence of genetic changes in the second tumor that were identical with and additional to those found in the primary tumor. Conclusions Based on the similarities between the genetic profiles of the primary and the second tumors in these patients, the authors decided that in each case the second distant GBM was a distant recurrence rather than a second independent primary tumor.

PURPOSE: To establish the alpha/beta ratio of bladder cancer from different radiotherapy schedules reported in the literature and provide guidelines for the design of new treatment schemes.METHODS AND MATERIALS: Ten external beam radiotherapy (EBRT) and five brachytherapy schedules were selected. The biologically effective dose (BED) of each schedule was calculated. Logistic modeling was used to describe the relationship between 3-year local control (LC3y) and BED.RESULTS: The estimated alpha/beta ratio was 13 Gy (95% confidence interval [CI], 2.5-69 Gy) for EBRT and 24 Gy (95% CI, 1.3-460 Gy) for EBRT and brachytherapy combined. There is evidence for an overall dose-response relationship. After an increase in total dose of 10 Gy, the odds of LC3y increase by a factor of 1.44 (95% CI, 1.23-1.70) for EBRT and 1.47 (95% CI, 1.25-1.72) for the data sets of EBRT and brachytherapy combined.CONCLUSION: With the clinical data currently available, a reliable estimation of the alpha/beta ratio for bladder cancer is not feasible. It seems reasonable to use a conventional alpha/beta ratio of 10-15 Gy. Dose escalation could significantly increase local control. There is no evidence to support short overall treatment times or large fraction sizes in radiotherapy for bladder cancer.

PURPOSE: Investigation of cell migration and proliferation of human glioma cell line spheroids (CLS) and evaluation of morphology, apoptosis, and immunohistochemical expression of MIB-1, p53, and p21 of organotypic muticellular spheroids (OMS) following cisplatin (CDDP) and irradiation (RT).MATERIAL AND METHODS: Spheroids of the GaMg glioma cell line and OMS prepared from biopsy tissue of six glioblastoma patients were used. Radiochemosensitvity (5 microg/ml CDDP followed by RT) was determined using migration and proliferation assays on CLS. In OMS, histology and immunohistochemical studies of MIB-1, p53, and p21 expression were examined 24 and 48 h following treatment.RESULTS: Combination treatment led to a migration inhibition of 38% (CDDP 13%; RT 27%) and specific growth delay of 2.6 (CDDP 1.3; RT 2.1) in CLS. Cell cycle analysis after combination treatment showed an accumulation of cells in the G2/M phase. In OMS, apoptosis increased, cell proliferation decreased, and p53/p21 expression increased more pronounced following CDDP+RT. No morphological damage was observed.CONCLUSION: CDDP can lead to enhancement of the RT effect in spheroids of both human glioma cell line spheroids and biopsy spheroids from glioblastoma specimens. The exerted effect is additive rather than synergistic.

PURPOSE: To investigate the pattern and level of cyclooxygenase-2 (COX-2) expression in a series of high grade primary and recurrent glioblastoma multiforme (GBM) and correlation with time to recurrence and patients' survival following therapy. The relationship between COX-2 and epidermal growth factor receptor (EGFR) immunoreactivities was evaluated.MATERIALS AND METHODS: Specimens of 14 primary and 14 recurrent GBMs (eight pairs) following surgery and full course radiation therapy were processed for immunostaining on COX-2 and EGFR. Tumor cell positivity was semi-quantitatively scored. COX-2 scores of the primary tumor and recurrence were correlated with the time to radiological tumor progression and patients' survival.RESULTS: COX-2 positive tumor cells were disseminated throughout the tumor parenchyma. The intensity and pattern of COX-2 expression were heterogeneous, with predominant expression in areas surrounding tumor necrosis. Scoring of COX-2 positivity revealed values between 1 and 80% of the cells. Primary GBMs with COX-2 expression levels between 25% and 70% of the tumor cells showed a shorter time to radiological recurrence than GBMs with CONCLUSIONS: Immunohistochemical expression of COX-2 in GBM showed large variation. Hence, determination of COX-2 expression in tumor specimen for each individual might be relevant for selection of those patients, who could benefit from adjuvant therapy with selective COX-2 inhibitors.

Animal studies show that nervous tissue is sensitive to heat. Although inter- species variations may play a role, the data indicate that the maximum heat dose without obvious complications after localized hyperthermia in regions of the central nervous system ( CNS) lies in the range of 40 - 60 min at 42 - 42.5 degrees C or 10 - 30 min at 43 degrees C. Expression of thermotolerance after a ' conditioning' heat dose was clearly observed in the spinal cord of rodents and the thermotolerance ratio's ( ratio between heat doses with and without conditioning required to obtain a certain defined effect) were high, similar to 2. The thermotolerant state of CNS is shown to protect also against other types of injury as well: pretreatment of rats with hyperthermia protected against spinal cord ischemic injury. During the rather long period required for temperature elevation which is inherent to WBH, some degree of thermotolerance may develop. The correlation between thermotolerance and hsp70 induction in CNS is obvious. Heat, at least if applied shortly after X- rays, enhances the response of nervous tissue to radiation. Data on the combined effects of X- ray irradiation and hyperthermia on rodent spinal cord clearly show that the radiation response can be enhanced with a factor of 1.1 - 1.3. There are no clear experimental data indicating an increase in adverse effects specific to the CNS after localized or whole body hyperthermia as a result of combined treatment with chemotherapy

PURPOSE: To determine the efficacy and safety of a high-dose-rate (HDR) brachytherapy schedule in the treatment of bladder cancer and to investigate the impact of different values of repair half-times and alpha/beta ratios on the design of the HDR schedule.METHODS AND MATERIALS: Between 2000 and 2002, 40 patients with T1G3 and T2 bladder carcinoma were treated with 30 Gy external beam radiotherapy followed by interstitial HDR brachytherapy to a total dose of 32 Gy in 10 sessions of 3.2-Gy fractions in two fractions daily with a 6-h interfraction interval. The local control rate and toxicity were compared with a historical group of 108 patients treated with 30 Gy external beam radiotherapy followed by 40-Gy interstitial low-dose-rate (LDR) brachytherapy. The HDR schedule was designed to be biologically equivalent to the previously used LDR schedule with the linear-quadratic model, including incomplete mono-exponential repair.RESULTS: The local control rate at 2 years was 72% for HDR vs. 88% for LDR brachytherapy (p = 0.04). In the HDR group, 5 of 30 evaluable patients encountered serious late toxicity: 4 patients developed a contracted bladder with inadequate capacity (CONCLUSION: Local control of HDR brachytherapy for bladder cancer was disappointing and late toxicity unexpectedly high. The increase in late toxicity suggested a short repair half-time of 0.5-1 h for late-responding normal bladder tissue, which would not support HDR brachytherapy in the treatment of bladder cancer. The analysis demonstrated that the calculation of equivalent HDR schedules on the basis of the LDR schedules used in clinical practice might be hazardous.

Normal tissue injury may lead to severe, life threatening, late side effects after therapeutic use of irradiation. Neurological complications caused by radiation of the spinal cord are ascribed to progressive, irreversible damage to the vasculature. Hyperbaric oxygen (HBO) is known to induce angiogenesis in irradiated tissue and has been proven to reduce late radiation injury in several normal tissues when applied during the latent period before complications become manifest. In the present study: (1). the prophylactic potential of HBO; (2). optimal timing of HBO therapy after spinal cord irradiation, i.e. during the latent period; and (3). effect of HBO on the re-irradiation tolerance of the spinal cord were investigated. The rat cervical spinal cord was locally X-ray irradiated with ten fractions of 6.5 Gy in 11 days. Five treatment groups (n=10) included: irradiation alone and irradiation followed by 30 HBO treatments (100% oxygen at 240 kPa for 90 min) during latency, with HBO starting either immediately, 5, 10 or 15 weeks after the primary irradiation course. One year after the primary treatment, the same spinal cord volume was re-irradiated with 20 Gy single dose. During life span, the animals were observed on the incidence of myelitis and the duration of the latent period. The actuarial analysis revealed no significant difference in neurological complications free survival between the irradiation alone and the irradiation+HBO treatment groups. A tendency towards radiosensitization was found in the group in which the primary irradiation course was immediately followed by the HBO treatment course. The data show that HBO applied during the latent period of progressively developing irradiation damage to the spinal cord does not increase the re-irradiation tolerance of this tissue.

Experimental animal data give evidence of long-term recovery of the spinal cord after irradiation. By extrapolation of these data, re-irradiation regimens were designed for eight patients who required palliative radiotherapy. As a consequence of re-irradiation, their spinal cords were exposed to cumulative doses exceeding the tolerance dose. Radiobiological and clinical data are presented. Patients and Method: Eight patients were re-irradiated on the cervical (n=1), thoracic (n=5) and lumbar (n=2) spinal cord. The time interval between the initial and re-treatment ranged from 4 months to 12.7 years (median: 2.5 years). (Re-)treatment schemes were designed and analyzed on basis of the biologically effective dose (BED) according to the linear-quadratic model. The repair capacity (α/β ratio) for the cervico-thoracic and lumbar spinal cord was assumed to be 2 Gy and 4 Gy, with a BEDtolerance of 100 Gy and 84 Gy, respectively. Results: The cumulative irradiation dose applied to the spinal cord varied between 125 and 172% of the BEDtolerance. During follow-up, ranging from 33 days to >4.5 years (median: 370 days) none of the patients developed neurological complications. Seven patients died from tumor progression, and one patient is still alive. Conclusion: Long-term recovery of the spinal cord from radiation injury, which has been demonstrated in rodents and primates, may also occur in humans.

Background and Purpose: Indications of the existence of long repair half-times on the order of 2–4 h for late-responding human normal tissues have been obtained from continuous hyperfractionated accelerated radiotherapy (CHART). Recently, these data were used to explain, on the basis of the biologically effective dose (BED), the potential superiority of fractionated high-dose rate (HDR) with large fraction sizes of 5–7 Gy over continuous low-dose rate (LDR) irradiation at 0.5 Gy/h in cervical carcinoma. We investigated the optimal fraction size in HDR brachytherapy and its dependency on treatment choices (overall treatment time, number of HDR fractions, and time interval between fractions) and treatment conditions (reference low-dose rate, tissue repair characteristics). Methods and Materials: Radiobiologic model calculations were performed using the linear-quadratic model for incomplete mono-exponential repair. An irradiation dose of 20 Gy was assumed to be applied either with HDR in 2–12 fractions or continuously with LDR for a range of dose rates. HDR and LDR treatment regimens were compared on the basis of the BED and BED ratio of normal tissue and tumor, assuming repair half-times between 1 h and 4 h. Results: With the assumption that the repair half-time of normal tissue was three times longer than that of the tumor, hypofractionation in HDR relative to LDR could result in relative normal tissue sparing if the optimum fraction size is selected. By dose reduction while keeping the tumor BED constant, absolute normal tissue sparing might therefore be achieved. This optimum HDR fraction size was found to be largely dependent on the LDR dose rate. On the basis of the BED NT/TUM ratio of HDR over LDR, 3 × 6.7 Gy would be the optimal HDR fractionation scheme for replacement of an LDR scheme of 20 Gy in 10–30 h (dose rate 2–0.67 Gy/h), while at a lower dose rate of 0.5 Gy/h, four fractions of 5 Gy would be preferential, still assuming large differences between tumor and normal tissue repair half-times and equal overall treatment time. For the same fraction size, an even larger normal tissue sparing can be obtained by prolongation of the HDR overall treatment time. Conclusion: Radiobiologic model calculations presented here aim to demonstrate that hypofractionation in HDR might have its opportunities for widening the therapeutic window, but definitely has its limits. For each specific combination of the parameters, a theoretical optimal HDR fraction size with regard to relative or absolute normal tissue sparing can be estimated, but because of uncertainty in the biologic parameters, these hypofractionation schemes cannot be generalized for all HDR brachytherapy indications.

Analysis of the feasibility and effect of hyperbaric oxygen treatment (HBO) on cognitive functioning in patients with cognitive disorders after irradiation of the brain. Patients and Method: Seven patients with cognitive impairment after brain irradiation, with an interval of at least 1.5 years after treatment, were treated with 30 sessions of HBO in a phase I–II study. A comprehensive neuropsychological test battery was performed before treatment, at 3 and 6 months thereafter. Patients were randomized into an immediate treatment group and a delayed treatment group. The delayed group had a second neuropsychological test at 3 months without treatment in that period and started HBO thereafter. Results: All eligible patients completed the HBO treatment and the extensive neuropsychological testing. One out of seven patients had a meaningful improvement in neuropsychological functioning. At 3 months there was a small, but not significant benefit in neuropsychological performance for the group with HBO compared to the group without HBO treatment. Six out of seven patients eventually showed improvement after HBO in one to nine (median 2.5) of the 31 tests, although without statistical significance. Conclusion: HBO treatment was feasible and resulted in a meaningful improvement of congitive functioning in one out of seven patients. Overall there was a small but not significant improvement.

AIM: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines.MATERIAL AND METHODS: Fresh brain tumor specimens of six patients were processed to early passage cell cultures. In addition the cell lines D 384 and Gli 6 were used. Cell cultures were irradiated with doses from 2 to 10 Gy. Following irradiation, cell survival was determined by clonogenic assay and survival curves were generated. The surviving fractions after 2 Gy (SF2) and 4 Gy (SF4) were used as radiosensitivity parameters. Genetic analysis included determination of the mutational and loss of heterozygosity (LOH) status of TP 53 (exons 5-8), the LOH 10- and epidermal growth factor receptor gene (EGFR) amplification status.RESULTS: The SF2 and SF4 values ranged from 0.54 to 0.88 (mean: 0.70) and from 0.13 to 0.52 (mean: 0.32), respectively. Genetic alterations were found in the Gli 6 cell line and in two primary cell cultures. The genetic profile of Gli 6 showed LOH but no TP 53 mutation, complete LOH 10 and no EGFR amplification. The VU 15 cell culture showed TP 53 mutation but no LOH 10 or EGFR amplification, while VU 24 showed incomplete LOH 10, EGFR amplification and no TP 53 mutation. In the other four cell cultures and D 384 cell line no genetic alterations were diagnosed. Histopathological classification of glioblastoma multiforme and/or genetic alterations resulted in lower radiosensitivity.CONCLUSION: In this small series of early passage glioma cell cultures low radiosensitivity and alterations in cell regulatory genes were seen. Further testing of biological behavior in larger series of patient-derived material is ongoing.

Glioblastoma multiforme (GBM) has nearly uniformly fatal with a median survival of less than 2 years. While there have not been any novel anti-GBM therapeutics approved for many years, there has been the gradual accumulation of clinical data suggesting that the widely used anti-convulsant agent, valproic acid (VPA) may significantly prolong survival in GBM patients. This pre-clinical study aimed to determine the potential clinical utility of VPA in the treatment of GBM. Primary GBM cells were treated with VPA as a monotherapy and in combination with temozolomide and irradiation. At clinically achievable concentrations, VPA was shown to be effective as a monotherapy agent in the five primary lines tested. VPA was then used as a sensitizing agent to in vitro radiation and showed significant augmentation of in vitro irradiation therapy. In addition, when VPA, radiation and temozolomide were combined an additive, rather than synergistic effect was noted. Gene expression profiling demonstrated close clustering of triple treated cells with VPA mono-treated cells while untreated cells clustered closer with TMZ-irradiation dual treated cells. These microarray data suggest a dominant role of VPA at the gene expression level when combining these different treatment options. Moreover, in an in vivo tumor transplantation model, we were able to demonstrate an increase in animal survival when cells were pre-treated with irradiation-VPA and when triple treated. These findings provide a significant rationale for the investigation of VPA in the treatment of GBM patients. Refereed/Peer-reviewed

Purpose: Hypoxic tumor cells are an important factor of radioresistance. Hyperbaric oxygen (HBO) and normobaric carbogen (95% oxygen, 5% carbon dioxide) increase the oxygen delivery to tumors. This study was performed to explore changes of tumor oxygenation during a course of fractionated irradiation and to determine the effectiveness of normobaric carbogen and HBO during the final phase of the radiation treatment. Methods and Materials: Experiments were performed on the rhabdomyosarcoma R1H growing on WAG/Rij rats. After 20 X-ray fractions of 2 Gy within 4 weeks, oxygen partial pressure (pO 2 ) was measured using the Eppendorf oxygen electrode under ambient conditions, with normobaric carbogen or HBO at a pressure of 240 kPa. Following the 4-week radiation course, a top-up dose of 10–50 Gy was applied in 2–10 fractions of 5 Gy with or without hyperoxygenation. Results: HBO but not carbogen significantly increased the median pO 2 in irradiated tumors. The radiation doses to control 50% of tumors were 38.0 Gy, 29.5 Gy, and 25.0 Gy for air, carbogen, and HBO, respectively. Both high oxygen content gas inspirations led to significantly improved tumor responses with oxygen enhancement ratios (OERs) of 1.3 for normobaric carbogen and 1.5 for HBO (air vs. carbogen: p = 0.044; air vs. HBO: p = 0.02; carbogen vs. HBO: p = 0.048). Conclusion: Both normobaric carbogen and HBO significantly improved the radiation response of R1H tumors. HBO appeared to be more effective than normobaric carbogen, both with regard to tumor oxygenation and response to irradiation.

PURPOSE: Investigation of the in vitro cytotoxic effect of X-rays, either alone or combined with cisplatin on early passage cell cultures derived from human glioblastoma multiforme biopsy tissue.MATERIALS AND METHODS: Fresh tumour specimens from four patients were processed to cell cultures. The U373 glioma cell line was used as a reference. Early passage cell cultures were X-irradiated (0-8 Gy) either alone or in combination with cisplatin (0.5-1 microgram/ml). Cell survival was determined by either clonogenic assay or the colorimetric MTT assay. Survival curves were generated and mathematically analysed using the linear quadratic model, to obtain the radiosensitivity parameters alpha, beta, and SF2, i.e., the Surviving Fraction after 2 Gy.RESULTS: Two patient-derived glioma cell cultures and the U373 cell line showed rather high SF2 values of 0.61-0.72 in the clonogenic assay, indicating relative high radiation resistance. Cisplatin alone (1 microgram/ml) reduced cell survival by 10-30% (n = 4). When combined with irradiation, a clear additive cytotoxic effect of cisplatin was demonstrated by the unaltered value of the alpha-parameter for reproductive cell death.CONCLUSION: Cisplatin exerted an additive rather than radiosensitising cytotoxic effect in uncharacterised patient derived glioma cell cultures.

It is known that the pleiotropic cytokine transforming growth factor beta (TGF-beta) has a regulatory role in the process of tissue repair and remodelling following injury. As reports on these molecules in multiple sclerosis (MS) lesion with different lesional activity are rare, we studied the cellular localization of TGF-beta1, -beta2, and -beta3 isoforms, and TGF-beta receptor type I (TGF-betaR-I) and TGF-betaR-II expression by immunohistochemistry on postmortem brain tissue from MS and normal control cases. To validate the TGF-beta staining results we demonstrated that cultured human adult astrocytes that produce biological active TGF-beta2, and to a lesser extent TGF-beta1, were immunoreactive for all 3 TGF-beta isoforms. Moreover, at mRNA level TGF-beta1 was detected in MS and normal control brain tissue. In normal control brain tissue, TGF-beta isoforms were expressed in ramified microglia and TGF-beta2, and -beta3 on neuronal cells in the gray matter TGF-betaR-I and TGF-betaR-II expression was found on endothelial cells, astrocytes, microglia, and neurons. In active demyelinating MS lesions a strong to intense immunoreactivity was detected for all 3 TGF-beta isoforms in perivascular and parenchymal (foamy) macrophages and in hypertrophic astrocytes. Strong immunoreactivity for TGF-betaR-I and TGF-betaR-II was found on macrophages in both parenchymal and perivascular areas and on hypertrophic astrocytes and endothelial cells in active demyelinating MS lesions. In chronic active and inactive MS lesions, all 3 TGF-beta isoforms and their receptors were strongly expressed in hypertrophic astrocytes. Our findings strongly suggest that the expression of the various TGF-beta isoforms and their receptor types found in MS lesions with different cellular activity participate in reactive processes leading to the formation of chronic MS lesions.

PURPOSE: Investigation of normal tissue sparing in pulsed brachytherapy (PB) relative to continuous low-dose rate irradiation (CLDR) by adjusting pulse frequency based on tissue repair characteristics.METHOD: Using the linear quadratic model, the relative effectiveness (RE) of a 20 Gy boost was calculated for tissue with an alpha/beta ratio ranging from 2 to 10 Gy and a half-time of sublethal damage repair between 0.1 and 3 h. The boost dose was considered to be delivered either in a number of pulses varying from 2 to 25, or continuously at a dose rate of 0.50, 0.80, or 1.20 Gy/h.RESULTS: The RE of 20 Gy was found to be identical for PB in 25 pulses of 0.80 Gy each h and CLDR delivered at 0.80 Gy/h for any alpha/beta value and for a repair half-time > 0.75 h. When normal tissue repair half-times are assumed to be longer than tumor repair half-times, normal tissue sparing can be obtained, within the restriction of a fixed overall treatment time, with higher dose per pulse and longer period time (time elapsed between start of pulse n and start of pulse n + 1). An optimum relative normal tissue sparing larger than 10% was found with 4 pulses of 5 Gy every 8 h. Hence, a therapeutic gain might be obtained when changing from CLDR to PB by adjusting the physical dose in such a way that the biological dose on the tumor is maintained. The normal tissue-sparing phenomenon can be explained by an increase in RE with longer period time for tissue with high alpha/beta ratio and fast or intermediate repair half-time, and the RE for tissue with low alpha/beta ratio and long repair half-time remains almost constant.CONCLUSION: Within the benchmark of the LQ model, advantage in normal tissue-sparing is expected when matching the pulse frequency to the repair kinetics of the normal tissue exposed. A period time longer than 1 h may lead to a reduction of late normal tissue complications. This theoretical advantage emphasizes the need for better knowledge of human tissue-repair kinetics.

Radionecrosis (RN) in children treated for brain tumors represents a potentially severe long-term complication. Its diagnosis is challenging, since magnetic resonance imaging (MRI) cannot clearly discriminate between RN and tumor recurrence. A retrospective single-center study was undertaken to describe the incidence and clinical course of RN in a cohort of 107 children treated with external radiotherapy (RT) for various brain tumors between 1992 and 2012. During a median follow-up of 4.6 years (range 0.29-20.1 years), RN was implied by suspicious MRI findings in in 5 children (4.7 %), 5-131 months after RT. Suspicion was confirmed histologically (1 patient) or substantiated by FDG positron-emission tomography (FDG-PET, 2 patients) or by FDG-PET and MR spectroscopy (1 patient). Before developing RN, all 5 patients had received cytotoxic chemotherapy in addition to RT. In addition to standard treatment protocols, 2 patients had received further chemotherapy for progression or relapse. Median radiation dose expressed as the biologically equivalent total dose applied in 2 Gy fractions (EQD2) was 51.7 Gy (range 51.0-60.0 Gy). At RN onset, 4 children presented with neurological symptoms. Treatment of RN included resection (n = 1), corticosteroids (n = 2) and a combination of corticosteroids, hyperbaric oxygen (HBO) and bevacizumab (n = 1). One patient with asymptomatic RN was not treated. Complete radiological regression of the lesions was observed in all patients. Clinical symptoms normalized in 3 patients, whereas 2 developed permanent severe neurological deficits. RN represents a severe long-term treatment complication in children with brain tumors. The spectrum of clinical presentation is wide; ranging from asymptomatic lesions to progressive neurological deterioration. FDG-PET and MR spectroscopy may be useful for distinguishing between RN and tumor recurrence. Treatment options in patients with symptomatic RN include conservative management (steroids, HBO, bevacizumab) and surgical resection.

Investigation of clonogenic cell survival and cell proliferation following single dose and fractionated delivery of high dose rate flattening filter free (FFF) irradiation compared to conventional dose rates.The human astrocytoma D384, glioma T98 and lung carcinoma SW1573 cell lines were irradiated using either a single dose (0-12 Gy) or a fractionated protocol of 5 daily fractions of 2 Gy (D384) or 3 Gy (SW1573). Cells were irradiated inside a phantom using fixed gantry beams of a linear accelerator. A sliding window technique created homogeneous dose distributions over the surface of the cell cultures. Irradiations using standard beams (6 MV, 600 MU/min.) and high dose rate FFF beams (10 MV, 2400 MU/min.) were compared. Cell survival was determined by clonogenic assay. In the fractionated irradiation set-up, the number of clonogenic cells was estimated by including tumor cell proliferation during the overall treatment time in the analysis.All cell lines showed equal cell survival following irradiation using either the FFF beams or conventional flattened (FF) beams. This was observed after single dose exposure (0-12 Gy) as well as after fractionated irradiation (p = 0.08 for D384 and 0.20 for SW1373 cell lines).FFF irradiation with a dose rate of 2400 MU/min and four times higher dose per pulse compared to irradiation with FF beams did not change cell survival for three human cancer cell lines up to a fraction dose of 12 Gy compared to irradiation using FF beams.

The linear-quadratic model (LQ model) provides a biologically plausible and experimentally established method to quantitatively describe the dose-response to irradiation in terms of clonogenic survival. In the basic LQ formula, the clonogenic surviving fraction Sd/S₀ following a radiation dose d (Gy) is described by an inverse exponential approximation: Sd/S₀ = e-(αd+βd²), wherein α and β are experimentally derived parameters for the linear and quadratic terms, respectively. Radiation is often combined with other agents to achieve radiosensitisation. In this study, we reviewed radiation enhancement ratios of hyperthermia (HT), halogenated pyrimidines (HPs), various cytostatic drugs and poly(ADP-ribose) polymerase‑1 (PARP1) inhibitors expressed in the parameters α and β derived from cell survival curves of various mammalian cell cultures. A significant change in the α/β ratio is of direct clinical interest for the selection of optimal fractionation schedules in radiation oncology, influencing the dose per fraction, dose fractionation and dose rate in combined treatments. The α/β ratio may increase by a mutually independent increase of α or decrease of β. The results demonstrated that the different agents increased the values of both α and β. However, depending on culture conditions, both parameters can also be separately influenced. Moreover, it appeared that radiosensitisation was more effective in radioresistant cell lines than in radiosensitive cell lines. Furthermore, radiosensitisation is also dependent on the cell cycle stage, such as the plateau or exponentially growing phase, as well as on post-treatment plating conditions. The LQ model provides a useful tool in the quantification of the effects of radiosensitising agents. These insights will help optimize fractionation schedules in multimodality treatments.

Temozolomide (TMZ) is given in addition to radiotherapy in glioma patients, but its interaction with the commonly prescribed antiepileptic drug valproic acid (VPA) is largely unknown. Induction of DNA demethylation by VPA could potentially induce expression of the O-6-methylguanine-DNA-methyltransferase (MGMT) protein, causing resistance to TMZ and thereby antagonizing its effect. Therefore, this study investigates the interaction between VPA, TMZ, and gamma-radiation. Two glioma cell lines were used that differ in TMZ sensitivity caused by the absence (D384) or presence (T98) of the MGMT protein. VPA was administered before (24/48 h) or after (24 h) single doses of gamma-radiation; or, after 24 h, VPA treatment was accompanied by a single dose of TMZ for another 24 h. For trimodal treatment the combination of VPA and TMZ was followed by single doses of gamma-radiation. In both cell lines VPA caused enhancement of the radiation response after preincubation (DMF0.2 1.4 and 1.5) but not after postirradiation (DMF0.2 1.1 and 1.0). The combination of VPA and TMZ caused enhanced cytotoxicity (DMF0.2 1.7) in both the TMZ-sensitive cell line (D384) and the TMZ-resistant cell line (T98). The combination of VPA and TMZ caused a significant radiation enhancement (DMF0.2 1.9 and 1.6) that was slightly more effective than that of VPA alone. VPA does not antagonize the cytotoxic effects of TMZ. Preincubation with VPA enhances the effect of both gamma-radiation and TMZ, in both a TMZ-sensitive and a TMZ-resistant human glioma cell line. VPA combined with TMZ may lead to further enhancement of the radiation response

Thirty-three patients with an inoperable NSCLC were treated with a dose of 60 Gy/20 fractions/25 days, using a concomitant boost technique. A dose of 40 Gy/2 Gy/25 days was given to the tumor area and a part (15 patients) or the whole (18 patients) mediastinum. During each session a simultaneous boost to the tumor of 1 Gy was administered. Moderate acute oesophageal toxicity was observed in 7/33 patients (22%). One out of 33 patients developed serious late oesophageal toxicity. A correlation between the oesophageal toxicity, absorbed oesophageal dose of irradiation and length of the elective field was observed. Five out of 33 patients developed subacute radiation pneumonitis grade 2 or 3. In selected patients with inoperable NSCLC radiotherapy, with a dose of 60 Gy/20 fractions/25 days, using a concomitant technique is feasible.

Background:The DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins.Methods:Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing.Results:The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%).Conclusion:The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell lines.

Hyperthermia was applied in the region of the vertebral column from the second to the fifth lumbar vertebra using a ring-shaped 434 MHz microwave radiator. In all experiments temperatures were measured at a 'reference' thermocouple which was placed against the fourth lumbar vertebra. After 60 min of heat treatment at 'reference' temperatures of 43.0 degrees C, 44.0 degrees C and 45.0 degrees C (+/- 0.05 degrees C) the average maximal temperature inside the vertebral canal were 42.6 degrees C, 43.0 degrees C and 43.8 degrees C (+/- 0.3 degrees C), respectively. At all 'reference' temperatures the maximal core temperature of the animal did not exceed 40.5 +/- 0.3 degrees C after 60 min of heat treatment. Dorsal skin and muscle temperatures in the treatment area reached 'reference' temperature, and transient skin and muscle necrosis was observed after treatment for 1 h at 'reference' temperatures at 44 degrees C and 45 degrees C. Temperatures in the peritoneal cavity approximately 1 mm ventrally of the vertebral column rose to 41.8 degrees C after 60 min at reference 43.0 degrees C. Treatment at spinal cord temperature 42.6 degrees C for 60 min did not induce any significant neurological effects. Motoric dysfunction of the hind legs, such as difficulties with walking, was observed after 60 min treatment at spinal cord temperatures of 43.0 degrees C or 43.8 degrees C. In addition, 24 h after treatment at 43.8 degrees C for 60 min loss of tail tonus was observed, as well as loss of sensory function in the hind limbs. Recovery from the neurological disorders, except for the loss of tail tonus, occurred within 2 weeks after treatment. Histopathological examination revealed necrosis in the central areas of the spinal cord at 3 days and complete necrosis at 7 days after treatment at 43.8 degrees C for 60 min.

The cervical region of the rat, including the spinal cord (cervical 5-thoracic 2) was irradiated with single doses of 15-32 Gy 250 kV X-rays. Hyperthermia, at temperatures of 42-, 43- and 44 +/- 0.1 degrees C for 30 min was applied to the cervical vertebral column and immediate adjacent tissues for 5-10 min or 7 h after X-irradiation. Over a period of 18-21 months, animals were followed up to monitor neurological complications occurring as a result of damage to the spinal cord (Sminia et al. 1991). We also noted the development of neoplasms either inside or outside the cervical region. The data on tumour incidence were analysed retrospectively using the actuarial method. Although hyperthermia alone was not carcinogenic, it led to a significant increase of radiation-induced tumours. This increase of radiation carcinogenesis was observed both with hyperthermia applied 5-10 min after X-rays and with an interval of 7 h between X-rays and heat. Cancer induction was highest after the lower radiation doses (16 Gy) combined with high heat doses (30 min 44 degrees C). The latent period for induction of tumours by X-rays was 472 +/- 19 days (mean +/- SEM; n = 24). Latency was significantly shortened by hyperthermia to 404 +/- 34 days (n = 22) if applied 5-10 min after X-rays and to 348 +/- 6 days (n = 33) with an interval of 7 h. Histology revealed that 86% (38/44) of the examined tumours found inside the volume treated with hyperthermia and irradiation were sarcomas. The percentage of animals with a tumour outside the treated volume was almost the same for all treatment groups. Most of these tumours were of the mammary gland type.

Screening of therapeutics relies on representative cancer models. The representation of human glioblastoma by in vitro cell culture models is questionable. We obtained genomic profiles by array comparative genomic hybridization of both short- and long-term primary cell and spheroid cultures, derived from seven glioblastomas and one anaplastic oligodendroglioma. Chromosomal copy numbers were compared between cell cultures and spheroids and related to the parental gliomas using unsupervised hierarchical clustering and correlation coefficient. In seven out of eight short-term cell cultures, the genomic profiles clustered further apart from their parental tumors than spheroid cultures. In four out of eight samples, the genetic changes in cell culture were substantial. The average correlation coefficient between parental tumors and spheroid profiles was 0.89 (range: 0.79-0.97), whereas that between parental tumors and cell cultures was 0.62 (range: 0.10-0.96). In two out of three long-term cell cultures progressive genetic changes had developed, whereas the spheroid cultures were genetically stable. It is concluded that genomic profiles of primary cell cultures from glioblastoma are frequently deviant from parental tumor profiles, whereas spheroids are genetically more representative of the glioblastoma. This implies that glioma cell culture data have to be handled with the highest caution.

PURPOSE: To investigate the radiosensitizing potential of temozolomide (TMZ) for human glioblastoma multiforme (GBM) cell lines using single-dose and fractionated gamma-irradiation.METHODS AND MATERIALS: Three genetically characterized human GBM cell lines (AMC-3046, VU-109, and VU-122) were exposed to various single (0-6 Gy) and daily fractionated doses (2 Gy per fraction) of gamma-irradiation. Repeated TMZ doses were given before and concurrent with irradiation treatment. Immediately plated clonogenic cell-survival curves were determined for both the single-dose and the fractionated irradiation experiments. To establish the net effect of clonogenic cell survival and cell proliferation, growth curves were determined, expressed as the number of surviving cells.RESULTS: All three cell lines showed MGMT promoter methylation, lacked MGMT protein expression, and were sensitive to TMZ. The isotoxic TMZ concentrations used were in a clinically feasible range of 10 micromol/L (AMC-3046), 3 micromol/L (VU-109), and 2.5 micromol/L (VU-122). Temozolomide was able to radiosensitize two cell lines (AMC 3046 and VU-122) using single-dose irradiation. A reduction in the number of surviving cells after treatment with the combination of TMZ and fractionated irradiation was seen in all three cell lines, but only AMC 3046 showed a radiosensitizing effect.CONCLUSIONS: This study on TMZ-sensitive GBM cell lines shows that TMZ can act as a radiosensitizer and is at least additive to gamma-irradiation. Enhancement of the radiation response by TMZ seems to be independent of the epigenetically silenced MGMT gene.