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Background: We aimed to investigate the prognostic value of tumour-infiltrating lymphocytes' (TILs) expression in pretreatment specimens from patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT)., Methods: The prevalence of CD3+, CD8+, CD4+ and FOXP3+ TILs was assessed using immunohistochemistry in tumour tissue obtained from 101 patients before CRT and was correlated with clinicopathological characteristics as well as local failure-free- (LFFS), distant metastases free- (DMFS), progression-free (PFS) and overall survival (OS). Survival curves were measured using the Kaplan-Meier method, and differences in survival between the groups were estimated using the log-rank test. Prognostic effects of TIL subset density were determined using the Cox regression analysis., Results: With a mean follow-up of 25 months (range, 2.3-63 months), OS at 2 years was 57.4% for the entire cohort. Patients with high immunohistochemical CD3 and CD8 expression had significantly increased OS (P=0.024 and P=0.028), PFS (P=0.044 and P=0.047) and DMFS (P=0.021 and P=0.026) but not LFFS (P=0.90 and P=0.104) in multivariate analysis that included predictive clinicopathologic factors, such as age, sex, T-stage, N-stage, tumour grading and localisation. Neither CD4 nor FOXP3 expression showed significance for the clinical outcome. The lower N-stage was associated with improved OS in the multivariate analysis (P=0.049)., Conclusion: The positive correlation between a high number of infiltrating CD3+ and CD8+ cells and clinical outcome indicates that TILs may have a beneficial role in HNSCC patients and may serve as a biomarker to identify patients likely to benefit from definitive CRT.

Tumor-infiltrating lymphocytes (TILs) play an important role in the response of neoplastic lesions to therapy. We have recently shown that a robust tumor infiltration by CD3 + and CD8 + T cells correlates with improved disease outcome upon definitive chemoradiotherapy in patients with head and neck cancer, in a manner that is influenced by tumor compartment and human papilloma virus status. Our data highlight the prognostic and therapeutic relevance of TILs in head and neck cancer.

Purpose: We have previously shown that the addition of cetuximab to chemoradiation therapy failed to improve complete response rates (pCR) in rectal cancer. Here we report the long-term results of the cetuximab added to preoperative radiation therapy with capecitabine and oxaliplatin (CET-CAPOX-RT) phase 1/2 study that evaluated preoperative chemoradiation with cetuximab, capecitabine, and oxaliplatin in patients with rectal cancer., Methods and Materials: The median follow-up was 63 months (range, 5-73 months). Sixty patients were enrolled; 3 patients were excluded due to protocol violation, and 4 died before surgery. Total mesorectal excision was performed in 53 patients, in 85% (n=45) with curative intention (M0-status). Secondary end points including overall survival (OS) disease-free survival (DFS) and cancer-specific survival (CSS) were calculated. The prognostic value of KRAS mutation status was also assessed., Results: Histopathological examination confirmed ypUICC stages 0 (n=4; pCR), I (n=17), II (n=10), III (n=14), and IV (n=8). For patients who underwent surgery (n=53), OS at 1, 3, and 5 years was 88.7%, 83%, and 75.5%, respectively, whereas CSS rates were 94.1%, 88.1%, and 78.1%, respectively. In the 45 patients who were treated with curative intent (M0), the OS rates at 1, 3, and 5 years were 91.1%, 88.9%, and 86.7%, respectively; whereas CSS rates were 97.6%, 95.2%, and 90.3%, respectively; and DFS rates were 90.7%, 88.3%, and 88.3%, respectively. We did not find any locoregional failure in patients with M0-status (n=45). Chronic toxicity was rare. KRAS mutations, as detected in 33.3%, showed no correlation with the clinicopathological parameters nor significance for either OS (P=.112), CSS (P=.264), or DFS (P=.565)., Conclusions: Taken together, chemoradiation therapy combined with cetuximab is safe, feasible, and offers excellent survival rates. KRAS mutation status was not a predictive factor. Importantly, lack of improvement in pCR rate did not translate to poor survival in our clinical trial., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This retrospective study aimed to investigate long-term outcome in patients with arteriovenous malformations (AVM) treated with stereotactic radiosurgery (SRS). Between 1998 and 2008, 164 patients with AVM received SRS. Median age was 36 years (range 7-69 years). Before SRS, 39 % of the patients experienced haemorrhage and 27 % suffered from epileptic seizures, whereas 43 % received previously embolization, 7.9 % neurosurgery and 1.8 % proton radiotherapy. Primary SRS was applied in 51.2 % of the patients. Median single dose was 19 Gy (80 % isodose; range 18-20 Gy) and median target volume was 4 cc (range 0.1-24.4). Median follow-up was 93 months (range 12-140). Complete obliteration (CO) was observed in 100 (61 %) patients at a median time of 29 months (range 6.1-88.5). The 3 and 5-year CO rates were 61 and 88 %, respectively. In multivariate analysis, radiation dose ≥ 19 Gy (p = 0.044) and target volume <4 cc (p = 0.015) were associated with significantly higher rates of CO. Intracranial haemorrhage was seen in nine patients (5.5 %) after SRS, whereas three patients (1.8 %) died as a consequence of bleeding. The annual bleeding risk was 1.3 % after 1 year and 1.3 % after 2 years, respectively. In multivariate analysis, only target volume >4 cm(3) (p = 0.031) and Spetzler-Martin grade III-V (p = 0.046) retained significance for increased risk of intracranial bleeding. After SRS an improvement in epileptic episodes, headaches and motor-sensory deficits was found in 8.5, 14 and 15 % of patients, respectively. Our long-term follow-up data show that SRS is an effective treatment option in AVM with low toxicity and bleeding risk, depending on AVM size and Spetzler-Martin grade. An improvement of neurologic symptoms is achievable.

Background: To examine whether nuclear NF-κB expression correlates with outcome in patients with head and neck squamous cell carcinoma (HNSCC) treated with primary chemoradiation therapy (CRT)., Methods and Materials: Between 2007 and 2010, 101 patients with locally advanced primary HNSCC were treated with definitive simultaneous CRT. Pretreatment biopsy specimens were analyzed for NF-κB p65 (RelA) nuclear immunoreactivity. A sample was assigned to be positive with more than 5% positive nuclear expression. The predictive relevance of NF-κB and clinicopathologic factors for overall survival (OS), progression-free survival (PFS), local progression-free survival (LPFS), and metastasis-free survival (DMFS) was examined by univariate and multivariate analysis., Results: No significant differences between the groups were observed with regard to age, sex, total radiation dose, fractionation mode, total chemotherapy applied, T stage or grading. Patients with p65 nuclear positive biopsy specimens showed significantly a higher rate of lymph node metastasis (cN2c or cN3 status, P=.034). Within a mean follow-up time of 25 months (range, 2.33-62.96 months) OS, PFS, and DMFS were significantly poorer in the p65 nuclear positive group (P=.008, P=.027, and P=.008, respectively). These correlations remained significant in multivariate analysis., Conclusion: NF-κB/p65 nuclear expression is associated with increased lymphatic and hematogenous tumor dissemination and decreased survival in HNSCC patients treated with primary CRT. Our results may foster further investigation of a predictive relevance of NF-κB/p65 and its role as a suitable target for a molecular-based targeted therapy in HNSCC cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background and Purpose: Contouring of target volumes varies significantly in radiotherapy of pancreatic ductal adenocarcinoma (PDAC). There is a lack of consensus as to whether elective lymph nodes (eLN's) should be included or not in the planning target volume (PTV). In the present study we analyzed the dosimetric coverage of the eLN's and organs at risk (OAR) by comparing four different contouring guidelines., Methods and Materials: PTVs were delineated with (Oxford and RTOG guidelines) or without (Michigan and SCALOP guidelines) including the eLNs in eleven patients with PDAC. eLNs included the peripancreatic, paraaortic, paracaval, celiac trunk, superior mesenteric and portal vein clinical target volumes (CTVs). A 3D-CRT plan (50.40 Gy in 28 fractions) was performed to analyze and compare the dosimetric coverage of all eLNs and OAR between the 4 contouring guidelines., Results: The size of Oxford and RTOG PTVs was comparable and significantly larger than the SCALOP and Michigan PTVs. Interestingly the eLNs received a significant amount of incidental dose irradiation by PTV-based plans that only aimed to treat the tumor without the eLNs. The dosimetric coverage of eLN presented a large variability according to the respective contouring methods. The difference in the size of the 4 PTVs was reflected to the dose distribution at the OAR., Conclusions: Our study provides important information regarding the impact of different contouring guidelines on the dose distribution to the eLNs and the OAR in patients with locally advanced PDAC treated with radiotherapy., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Stereotactic radiotherapy (SRT) is well-established in the treatment of meningiomas offering high local control with low toxicity. However, the impact of SRT on quality of life (QoL) of patients remains largely unknown. This work aimed to prospectively evaluate QoL (longitudinal analysis) during and after SRT of meningiomas. We performed a single center, one-armed, prospective non-randomized study to assess QoL before and at the end of SRT (median fraction dose: 1.8 Gy; median cumulative dose: 54.0 Gy) and furthermore biannually until 24 months after SRT with the "medical outcome study short form 36". This questionnaire evaluates 8 health parameters summarized in "physical component scale" (PCS) and "mental component scale" (MCS). Between 2005 and 2007, 67 patients were enrolled and treated with SRT. 42/52 patients underwent previous operations and 10/52 primary SRT. Complete follow-up data were available from 44 patients. Compared to the german normal population (GNP) a general decrease in the mean values of all parameters was observed. After SRT mean values still declined and 12 months after SRT all parameters normalized towards their initial values. The cohort (previous operations) had better values for MCS (p = 0.004). The cohort (primary SRT) had worse values for PCS that increased asymptotically 6 months after SRT to values of cohort (previous operations) (p = 0.054). Gender, age and tumor related symptoms did not affect QoL according to MCS and PCS (p > 0.05). Local control was 98 %. Treatment was well tolerated and no severe side effects were observed. Patients with meningiomas have an impaired QoL compared to GNP. The QoL assessment after SRT revealed three phases: "depressive phase", "recovery phase" and "normalization phase". Patients treated with primary SRT developed a stable increase of the mean values for PCS. Gender, age, applied dose, symptomatology did not affect QoL.

Background and Purpose: Target volume definitions for radiotherapy in pancreatic ductal adenocarcinoma (PDAC) vary substantially. Some groups aim to treat the primary tumor only, whereas others include elective lymph nodes (eLNs). eLNs close to the primary tumor are often included unintentionally within the treatment volume, depending on the respective treatment philosophies. We aimed to measure the percentages of anatomical coverage of eLNs by comparing four different contouring guidelines., Patients and Methods: Planning target volumes (PTVs) were contoured using planning computed tomography (CT) scans of 11 patients with PDAC based on the Oxford, RTOG (Radiation Therapy Oncology Group), Michigan, and SCALOP (Selective Chemoradiation in Advanced Localised Pancreatic Cancer trial) guidelines. Clinical target volumes (CTVs) included the peripancreatic, para-aortic, paracaval, celiac trunk, superior mesenteric, and portal vein lymph node areas. Volumetric comparisons of the coverage of all eLN regions were conducted to illustrate the differences between the four contouring strategies., Results: The PTV sizes of the RTOG and Oxford guidelines were comparable. The SCALOP and Michigan PTV sizes were similar to each other and significantly smaller than the RTOG and Oxford PTVs. A large variability of eLN coverage was found for the various subregions according to the respective contouring strategies., Conclusion: This is the first study to directly compare the percentage of anatomical coverage of eLNs according to four PTVs in the same patient cohort. Potential practical consequences are discussed in detail.

Despite recent improvements in surgery and radiotherapy, and refinements of systemic treatment options, including incorporation of targeted agents, long-term survival remains poor for patients with esophageal cancer. While surgical resection alone constitutes the standard approach for early-stage disease (stage I), multimodality therapy, including perioperative chemotherapy and neoadjuvant or definitive chemoradiotherapy (CRT), are internationally accepted treatment options for patients with locally advanced disease. In lower esophageal and esophagogastric junction adenocarcinomas, data from large, randomized phase III trials and meta-analyses support the use of both perioperative chemotherapy alone or neoadjuvant concurrent CRT. In patients with locally advanced squamous cell carcinoma (SCC) of the esophagus, neoadjuvant CRT but not neoadjuvant chemotherapy alone is the preferred treatment approach. Definitive CRT without surgery has also emerged as a useful option for the treatment of resectable SCC of the esophagus, avoiding potential surgical morbidity and mortality, with salvage surgery reserved for those with persistent disease. Functional imaging modalities, such as PET-CT, may create new opportunities for a more adequate therapy response assessment and patient selection. Patients with SCC that show clinical response by PET-CT are considered to have a more favorable outcome, regardless of whether surgery will be performed or not. In nonresponding patients, salvage surgery improves survival, especially if complete resection is achieved. Recent technological advances in radiotherapy, such as intensity-modulated radiotherapy, image guided-radiotherapy and PET-CT-based radiotherapy planning, may further improve the therapeutic ratio of CRT. Moreover, the traditional backbone of CRT, platinum plus fluorouracil, may be supplanted by more modern and easier-to-administer regimens incorporating taxanes, irinotecan and targeted agents., (Copyright © 2013 S. Karger AG, Basel.)

Brain metastases (BM) occur in 20% to 40% of patients with cancer and result in significant morbidity and poor survival. The main therapeutic options include surgery, whole brain radiotherapy, stereotactic radiosurgery and chemotherapy. Although significant progress has been made in diagnostic and therapeutic methods, the prognosis in these patients remains poor. Furthermore, the poor penetrability of chemotherapy agents through the blood brain barrier (BBB) continues to pose a challenge in the management of this disease. Preclinical evidence suggests that new targeted treatments can improve local tumor control but our clinical experience with these agents remains limited. In addition, several clinical studies with these novel agents have produced disappointing results. This review will examine the knowledge of targeted therapies in BM. The preclinical and clinical evidence of their use in BM induced by breast cancer, non-small cell lung cancer and melanoma will be presented. In addition, we will discuss the role of antiangiogenic and radiosensitising agents in the treatment of BM and the current strategies available to increase BBB permeability. A better understanding of the mechanism of action of these agents will help us to identify the best targets for testing in future clinical studies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.

Tumor cells exploit their microenvironment by growth factors and cytokines such as vascular endothelial growth factor (VEGF) to stimulate abnormal vessel formation that is leaky and tortuous, causing irregular blood flow. The combination of poor perfusion, raised interstitial fluid pressure and areas of vascular collapse leads to hypoxia within tumor. The latter activates factors such as hypoxia inducible factor 1 (HIF-1) that serve to make cancer cells more aggressive and also markedly influences the response of malignant tumors to conventional irradiation and chemotherapy. Accumulating data now suggest that blockade of oncogenic signaling, for example by PI3K/Akt/mTOR inhibitors, might consist a promising strategy since these agents do not only possess antitumor effects but can also alter tumor vasculature and oxygenation to improve the response to radiation and chemotherapy. In many cases, these changes are related to downregulation of HIF-1α and VEGF. Here, we review the pathophysiology of tumor microenvironment (TME) and how it adversely affects cancer treatment. The complex interaction of tumor vasculature and radiotherapy is examined together the preclinical evidence supporting a proinvasive/metastatic role for ionising radiation. We will discuss the expanding role of oncogenic signaling, especially PI3K/Akt/mTOR, on tumor angiogenesis. Special emphasis will be paid to the potential of different oncogenic pathways blockade and other indirect antivascular strategies to alter the TME for the benefit of cancer treatment, as an alternative to the classical angiogenetic treatment.

Aims and Background: We investigated the impact of human epidermal growth factor receptor 2 (HER2) and prognostic factors in the outcome of patients with breast cancer that developed brain metastases., Methods: . The data from 94 patients who received multidisciplinary therapy from 2001 to 2007 were retrospectively reviewed. Patients were assigned according to their HER2 status, and overall survival and time to brain metastases recurrence/progression were evaluated. The prognostic value of age, presence of extracerebral metastases, recursive partitioning analysis class, hormone therapy, systemic therapy and trastuzumab was assessed., Results: The median overall survival and time to brain disease progression were 7.1 and 6.5 months, respectively. HER2 positivity (P = 0.006), treatment with trastuzumab (P = 0.025), chemotherapy (P = 0.011) and recursive partitioning analysis class I-II (P <0.001) were associated with prolonged survival on univariate analysis. On multivariate analysis, only recursive partitioning analysis class I-II (P <0.001) and triple-negative disease (P = 0.04) remained significant for overall survival, whereas time to brain metastases progression was only associated with recursive partitioning analysis class I-II (P = 0.001). The time from the diagnosis of primary disease to brain metastasis was slightly shorter in the HER2+ patients than in HER2- patients (36 vs 39 months). Intensified local treatment of brain metastasis incorporating whole-brain radiotherapy and/or radiosurgery and neurosurgery did not affect survival. Patients with triple-negative disease presented a significantly lower survival than the rest of the cohort (4 vs 8 months; P = 0.012)., Conclusions: Recursive partitioning analysis class I-II was found to be the strongest independent predictive factor. Treatment with trastuzumab in HER2+ patients appeared to improve overall survival, probably due to the better control of systemic metastatic disease, but did not maintain significance in multivariate analysis. The dismal prognosis of patients with triple-negative breast cancer highlights the need to develop novel therapies to improve the poor survival.

DNA damaging agents such as radiotherapy and gemcitabine are frequently used for the treatment of pancreatic cancer. However, these treatments typically provide only modest benefit. Improving the low survival rate for pancreatic cancer patients therefore remains a major challenge in oncology. Inhibition of the key DNA damage response kinase ATR has been suggested as an attractive approach for sensitization of tumor cells to DNA damaging agents, but specific ATR inhibitors have remained elusive. Here we investigated the sensitization potential of the first highly selective and potent ATR inhibitor, VE-821, in vitro. VE-821 inhibited radiation- and gemcitabine-induced phosphorylation of Chk1, confirming inhibition of ATR signaling. Consistently, VE-821 significantly enhanced the sensitivity of PSN-1, MiaPaCa-2 and primary PancM pancreatic cancer cells to radiation and gemcitabine under both normoxic and hypoxic conditions. ATR inhibition by VE-821 led to inhibition of radiation-induced G 2/M arrest in cancer cells. Reduced cancer cell radiosurvival following treatment with VE-821 was also accompanied by increased DNA damage and inhibition of homologous recombination repair, as evidenced by persistence of γH2AX and 53BP1 foci and inhibition of Rad51 foci, respectively. These findings support ATR inhibition as a novel approach to improve the efficacy and therapeutic index of standard cancer treatments across a large proportion of pancreatic cancer patients.

We retrospectively evaluated and compared the efficacy and the toxicity profile of stereotactic radiosurgery (SRS) and fractionated stereotactic radiotherapy (FSRT) for the treatment of patients with brain metastases (BM). Between 2000 and 2009, 260 patients with 1-3 BM were treated using either SRS (median dose 20 Gy; n = 138) or two different FSRT dose concepts: 7 × 5 Gy (n = 61) or 10 × 4 Gy (n = 61). The median survival for SRS, 7 × 5 Gy and 10 × 4 Gy was 8, 7 and 10 months (p = 0.575), respectively, and the overall survival (OS) was 9 months. Follow-up imaging data were available in 214 of the 260 patients. The 1-year local progression-free survival (LPFS) was 73, 75 and 71 %, respectively (p = 0.191). After a mean follow-up of 28 months (range: 2.1-77 months), the rate of complete remission, partial remission, stable disease and progressive disease were 29, 40, 21 and 10 %, respectively. On multivariate analysis, RPA class I was associated with better OS and regional progression-free survival (both p < 0.001). SRS was associated with a higher toxicity rate (grade I-III) compared to the 7 × 5 Gy and 10 × 4 Gy groups (14 vs. 6 vs. 2 %, respectively; p = 0.01). Although FSRT was used for large lesions and/or lesions near critical structures, the LPFS was comparable to SRS. Importantly, FSRT presented low toxicity and appears to be an effective and safe treatment for BM not amenable to SRS. The 10 × 4 Gy fractionation scheme warrants further investigation due to its efficacy and safe toxicity profile.

Background: The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition., Materials and Methods: We investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual γH2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well., Results: Both compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of γH2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor., Conclusions: The results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy.

The aberrant vascular architecture of solid tumors results in hypoxia that limits the efficacy of radiotherapy. Vascular normalization using antiangiogenic agents has been proposed as a means to improve radiation therapy by enhancing tumor oxygenation, but only short-lived effects for this strategy have been reported so far. Here, we show that NVP-BEZ235, a dual inhibitor of phosphoinositide-3-kinase (PI3K) and mTOR, can improve tumor oxygenation and vascular structure over a prolonged period that achieves the aim of effective vascular normalization. Because PI3K inhibition can radiosensitize tumor cells themselves, our experimental design explicitly distinguished effects on the blood vasculature versus tumor cells. Drug administration coincident with radiation enhanced the delay in tumor growth without changing tumor oxygenation, establishing that radiosensitization is a component of the response. However, the enhanced growth delay was substantially greater after induction of vascular normalization, meaning that this treatment enhanced the tumoral radioresponse. Importantly, changes in vascular morphology persisted throughout the entire course of the experiment. Our findings indicated that targeting the PI3K/mTOR pathway can modulate the tumor microenvironment to induce a prolonged normalization of blood vessels. The substantial therapeutic gain observed after combination of NVP-BEZ235 with irradiation has conceptual implications for cancer therapy and could be of broad translational importance., (©2011 AACR.)

Background: The microenvironment plays an important role in regulating tumor response to radiotherapy. Ionizing radiation can disrupt tumor vasculature, and Notch pathway inhibition can interfere with functional angiogenesis. We explored the potential cooperativity between Notch inhibition and ionizing radiation in delaying tumor growth., Methods: Human colorectal carcinoma LS174T cells, which express the Notch ligand delta-like ligand 4 (DLL4), and human head and neck cancer FaDu cells, which do not, were grown as subcutaneous xenografts in nude mice. The mice were treated with dibenzazepine (DBZ), a γ-secretase inhibitor that blocks all Notch signaling, or a DLL4-specific blocking monoclonal antibody, alone or in combination with ionizing radiation (n = 5-10 mice per group), and response was assessed by tumor growth delay. Microbubble contrast Doppler ultrasound was used to measure tumor blood flow. Tumor Notch activity was monitored by in vivo bioluminescence from a Notch luciferase reporter. Vessel density was assessed using Chalkley vessel counting. All statistical tests were two-sided., Results: In LS174T xenografts, the average time for tumor volumes to reach four times the starting volume was longer for mice treated with the DLL4 monoclonal antibody than for mice treated with DBZ (16.4 vs 9.5 days, difference = 6.9 days, 95% confidence interval [CI] = 3.7 to 10.1 days, P < .001). Both Notch inhibitors suppressed tumor Notch activity within 24 hours of administration compared with vehicle (change in luciferase activity, vehicle vs DBZ: 103% vs 28%, difference = 75%, 95% CI = 39% to 109%, P = .002; vehicle vs DLL4 antibody: 172% vs 26%, difference = 146%, 95% CI = 86% to 205%, P < .001). Administration of the DLL4 antibody or DBZ after ionizing radiation resulted in a supra-additive growth delay compared with vehicle (vehicle vs DLL4 antibody + ionizing radiation: 6.8 vs 44.3 days, difference = 37.5 days, 95% CI = 32 to 43 days, P < .001; vehicle vs DBZ + ionizing radiation: 7.1 vs 24.4 days, difference = 17.3 days, 95% CI = 15.9 to 18.6 days, P < .001). Treatment of mice with the DLL4 antibody alone or in combination with ionizing radiation increased tumor vessel density but reduced tumor blood flow. Combination therapy with DLL4 antibody and ionizing radiation resulted in extensive tumor necrosis in LS174T xenografts and enhanced tumor growth delay in FaDu xenografts., Conclusion: The combination of specific DLL4-Notch blockade and ionizing radiation impairs tumor growth by promoting nonfunctional tumor angiogenesis and extensive tumor necrosis, independent of tumor DLL4 expression.

Background: We investigated the role of radiotherapy, including whole brain radiotherapy and stereotactic radiosurgery (SRS), and prognostic factors in patients with colorectal cancer (CRC) who developed brain metastases., Patients and Methods: The data of 78 patients who received multidisciplinary treatment from 1996 to 2007 were reviewed. Overall survival (OS), intracerebral control (ICC), and local control (LC) were retrospectively analyzed. Six potential prognostic factors were evaluated: age, gender, number of brain metastases, extracerebral metastases, recursive partitioning analysis (RPA) class, and interval from tumor diagnosis to radiotherapy., Results: The median OS and ICC for the entire cohort were 8 and 6 months, respectively. Surgical resection-incorporating treatment resulted in significant improvement in OS (P = .036). On multivariate analysis, OS and ICC were significantly correlated with lack of extracerebral metastases (P = .024 and P = .041, respectively), lower number of lesions (P < .001 and P = .007, respectively) and interval from primary CRC diagnosis (P < .001 and .005, respectively) whereas RPA class I-II demonstrated significance only for OS (P = .045). SRS-incorporating therapy revealed a 1-year LC probability of 85%. No association between LC and any of the potential prognostic factors was observed., Conclusion: Our data indicate that surgery can prolong survival in CRC patients with brain metastases. SRS-incorporating treatment provides excellent LC rates and should be considered for patients with 1-3 lesions. The strong association between survival and the prognostic factors identified in this study highlights a patient subset that may potentially benefit from new, more aggressive therapies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: To compare the accuracy of the robot-assisted needle positioning with that of the conventional template-guided method with the help of a prostate model in high dose rate (HDR) brachytherapy., Materials and Methods: A prostate model of fresh porcine abdomen and special polyvinylchloride (PVC) sheets was developed. To verify the model, deviations from 311 needle placements of real prostate implants were analyzed. Second, the accuracy of the template-guided positioning versus robot-assisted positioning was measured with 20 needle insertions in the model. For robot-assisted positioning, different velocities (2.7, 5.4, 9.8 mm/s) of needle insertion were investigated., Results: The average needle positioning accuracies of manual template guidance on the model closely resembled those of real patients (approximately 3 mm). The average needle positioning accuracy for the robot-assisted method on the prostate model was 1.8 ± 0.6 mm, at a velocity of 2.7 mm/s and, in comparison to the template-guided method (2.7 ± 0.7 mm), was statistically more precise (p < 0.001). At higher robotic velocities, the measured needle positioning accuracy showed no significant difference from that of the manual insertion procedure., Conclusion: By employing a prostate model, we showed for the first time that robot-assisted needle placement for HDR brachy-therapy is significantly more precise than the conventional method at a velocity of 2.7 mm/s. The robot-assisted needle positioning technique improves the degree of freedom by providing additional oblique insertion channels and could be potentially exploited not only for LDR but also for HDR brachytherapy.

Purpose: The Wnt pathway plays an important role in embryonic development, and defects in this pathway have been implicated in the tumorigenesis. The Dickkopf 3 (DKK3) is a putative Wnt signaling inhibitor that is frequently inactivated in human cancers. However, the expression of DKK3 in ovarian cancer remains unknown., Methods: We investigated the expression of DKK3 in silico using the Digital Differential Display. DKK3 mRNA expression was also analyzed by real-time RT-PCR in ovarian carcinomas and normal ovarian tissues. DKK3 protein expression was determined by immunohistochemistry in the same ovarian carcinomas and normal ovarian tissues., Results: A significantly reduced expression of DKK3 (P < 0.05) was found after comparison of normal ovary- and tumor-derived libraries in the Cancer Genome Anatomy Project (CGAP). DKK3 mRNA expression was reduced in 63% (35 of 56) of tumors compared with normal ovarian samples (P < 0.02). Analysis of 13 matched pairs of ovarian carcinomas and adjacent normal tissues showed significant transcriptional downregulation of DKK3 (>twofold) in 9 paired carcinomas (69%). Loss or weak membranous expression of DKK3 protein was observed in 66% of ovarian cancers (37 of 56) including all tumors with low transcriptional level of DKK3 gene analyzed by real-time PCR., Conclusions: To our best knowledge, this is the first time to demonstrate altered expression of DKK3 in ovarian cancer. The latter could be a relevant mechanism for the activation of the Wnt pathway in the carcinogenesis of ovarian cancer but additional studies are required to elucidate the function of DKK3 silencing in ovarian carcinogenesis.

The aim of this study was to investigate the difference in gene expression profiles of human lung adenocarcinoma cells and identify genes whose expression is altered by heavy ions but not X-rays. The lung adenocarcinoma cell line A549 was irradiated with carbon ion beams and X-rays using biologically equivalent doses (2 Gy and 6 Gy, respectively). The transcriptional profiling was determined with a high density cDNA microarray containing 11.800 genes, and genetic network and gene ontology analysis was performed. The changes in selected genes involved were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The microarray analysis identified 49 mapped, network-eligible genes, the expression level of which was altered by carbon ions but not by X-rays. From these, 29 were upregulated while 20 genes were downregulated 4 h post-irradiation with carbon ions in A549 cells, as compared to the control. Among these, three genes (CCND2, RARG and E2F5) were involved in the aryl hydrocarbon receptor signalling and G1/S cell cycle checkpoint pathways. The microarray data were corroborated by qRT-PCR analysis of the selected genes (p<0.05). Our findings provide information on the genetic signature of carbon ions in human lung adenocarcinoma cells and add to the understanding of the complicated molecular response to carbon ion irradiation.

Purpose: To develop a new semiautomatic method to improve target delineation in head-and-neck cancer., Methods and Materials: We implemented an atlas-based software program using fourteen anatomic landmarks as well as the most superior and inferior computerd tomography slices for automatic target delineation, using an advanced laryngeal carcinoma as an example. Registration was made by an affine transformation. Evaluation was performed with manually drawn contours for comparison. Three physicians sampled and further applied a target volume atlas to ten other computer tomography data sets. In addition, a rapid three-dimensional (3D) correction program was developed., Results: The mean time to the first semiautomatic target delineation proposal was 2.7 minutes. Manual contouring required 20.2 minutes per target, whereas semiautomatic target volume definition with the rapid 3D correction was completed in only 9.7 minutes. The net calculation time for image registration of the target volume atlas was negligible (approximately 0.6 seconds). Our method depicted a sufficient adaptation of the target volume atlas on the new data sets, with a mean similarity index of 77.2%. The similarity index increased up to 85% after 3D correction performed by the physicians., Conclusions: We have developed a new, feasible method for semiautomatic contouring that saves a significant amount (51.8%) of target delineation time for head-and-neck cancer patients. This approach uses a target volume atlas and a landmark model. The software was evaluated by means of laryngeal cancer but has important implications for various tumor types whereby target volumes remain constant in form and do not move with respiration., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: EphA2 tyrosine kinase plays an important role in tumor angiogenesis, but whether targeting this pathway can affect response to ionizing radiation (IR) remains unknown., Methods: We investigated, using a soluble EphA2-Fc chimera, whether EphA2 inhibition could sensitize A549 and MCF-7 tumor cells, as well as human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (HDMEC), to IR., Results: EphA2-Fc resulted in a greater response of endothelial cells (EC) to IR than either treatment alone. EphA2-Fc significantly increased apoptosis and decreased clonogenic survival, tube formation and migration in irradiated EC after stimulation with vascular endothelial growth factor (VEGF), without an affecting their proliferation. No difference in proliferation or survival was found in A549 and MCF-7 tumor cells. In a co-culture model, EphA2-Fc inhibited an irradiated A549 cell-induced increase in EC migration. VEGF supplementation, as well as condiotioned medium from irradiated A549 cells, phosphorylated EphA2 in EC. The latter was abrogated by EphA2-Fc., Conclusions: EC were most sensitive to a combination of EphA2 inhibition and radiotherapy. The induction of paracrine growth factors and activation of EphA2 in EC suggest a protective mechanism that tumors probably use to attenuate IR-induced antivascular effects. Our data justify further investigation to explore targeting EphA2 in tumor radiosensitivity in vivo., (© Tianjin Lung Cancer Institute and Blackwell Publishing Asia Pty. Ltd.)

Purpose: To evaluate the role of radiotherapy (RT) and prognostic factors in 62 patients with brain metastases from transitional cell carcinoma (TCC) of the urinary bladder., Patients and Methods: 62 patients received either RT (n = 49), including whole-brain radiotherapy (WBRT) and/or stereotactic radiosurgery (SRS), or surgery (OP) combined with WBRT (n = 13). Overall survival (OS), intracerebral control (ICC) and local control (LC) were retrospectively analyzed. Six potential prognostic factors were assessed: age, gender, number of brain metastases, extracerebral metastases, recursive partitioning analysis (RPA) class, and interval from tumor diagnosis to RT., Results: Median OS and ICC for the entire cohort were 9 and 7 months. No significant difference between RT and OP + RT was found for OS (p = 0.696) and ICC (p = 0.996). On multivariate analysis, improved OS was associated with lack of extracerebral metastases (p < 0.001) and RPA class (p < 0.001), and ICC with the latter (p < 0.001). SRS-incorporating RT resulted in 1-, 2-, and 3-year LC probability of 78%, 66%, and 51%. No association between LC and any of the potential prognostic factors was observed. The results of the subgroup RPA class analyses were similar to the entire cohort., Conclusion: Patient outcome for the RT-alone arm was not significantly different from OP + RT. SRS-incorporating treatment offers excellent LC rates. RPA class and the presence of extracerebral metastases demonstrated a significant prognostic role for survival. The latter should be used as stratification factors in randomized trials and can help define the cohort of patients that may benefit from more aggressive therapies.

Background: Siah proteins play an important role in cancer progression. We evaluated the effect of Siah1, its splice variants Siah1L and the Siah1 mutant with the RING finger deleted (Siah1DeltaR) on radiosensitization of human breast cancer cells., Methods: The status of Siah1 and Siah1L was analysed in five breast cancer cell lines. To establish stable cells, SKBR3 cells were transfected with Siah1, Siah-1L and Siah1DeltaR. Siah1 function was suppressed by siRNA in MCF-7 cells. The impact of Siah1 overexpression and silencing on apoptosis, proliferation, survival, invasion ability and DNA repair was assessed in SKBR3 and MCF-7 cells, also in regards to radiation., Results: Siah1 and Siah1L mRNA expression was absent in four of five breast cancer cells lines analysed. Overexpression of Siah1 and Siah1L enhanced radiation-induced apoptosis in stable transfected SKBR3 cells, while Siah1DeltaR failed to show this effect. In addition, Siah1 and Siah1L significantly reduced cell clonogenic survival and proliferation. Siah1L sensitization enhancement ratio values were over 1.5 and 4.0 for clonogenic survival and proliferation, respectively, pointing to a highly cooperative and potentially synergistic fashion with radiation. Siah1 or Siah1L significantly reduced invasion ability of SKBR3 and suppressed Tcf/Lef factor activity. Importantly, Siah1 siRNA demonstrated opposite effects in MCF-7 cells. Siah1 and Siah1L overexpression resulted in inhibition of DNA repair as inferred by increased levels of DNA double-strand breaks in irradiated SKBR3 cells., Conclusion: Our results reveal for the first time how overexpression of Siah1L and Siah1 can determine radiosensitivity of breast cancer cells. These findings suggest that development of drugs augmenting Siah1 and Siah1L activity could be a novel approach in improving tumor cell kill.

Purpose: Hypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia and perfusion in A549 human lung adenocarcinoma xenografts., Materials and Methods: Immunohistological analysis of two exogenously added chemical hypoxic markers, pimonidazole and CCI-103F, and of the endogenous marker Glut-1 was performed time dependently after IR. Tumor vessels and apoptosis were analyzed using CD31 and caspase-3 antibodies. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and fluorescent beads (Hoechst 33342) were used to monitor vascular perfusion., Results: CCI-103F signals measuring the fraction of hypoxic areas after IR were significantly decreased by approximately 50% when compared with pimonidazole signals, representing the fraction of hypoxic areas from the same tumors before IR. Interestingly, Glut-1 signals were significantly decreased at early time point (6.5 h) after IR returning to the initial levels at 30.5 h. Vascular density showed no difference between irradiated and control groups, whereas apoptosis was significantly induced at 10.5 h post-IR. DCE-MRI indicated increased perfusion 1 h post-IR., Conclusions: The discrepancy between the hypoxic fractions of CCI-103F and Glut-1 forces us to consider the possibility that both markers reflect different metabolic alterations of tumor microenvironment. The reliability of endogenous markers such as Glut-1 to measure reoxygenation in irradiated tumors needs further consideration. Monitoring tumor microvascular response to IR by DCE-MRI and measuring tumor volume alterations should be encouraged., (Copyright 2010 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the role of stereotactic radiosurgery (SRS) and whole-brain radiotherapy (WBRT) for the treatment of brain metastases in patients with renal cell cancer (RCC)., Patients and Methods: 88 patients were treated with either SRS (n = 51) or SRS + WBRT (n = 17) for one to three lesions, or with WBRT (n = 20) for more than three brain metastases. Overall survival (OS), intracerebral control (IC) and local control (LC) were retrospectively analyzed. Six potential prognostic factors were assessed: age, gender, number of brain metastases, extracerebral metastases, recursive partitioning analysis (RPA) class, and interval from tumor diagnosis to irradiation., Results: The median times for OS, IC, and LC from the time of diagnosis were 11, 9, and 10 months. The median OS times for SRS, SRS + WBRT, and WBRT were 12, 16, and 2 months. Addition of WBRT to the SRS improved IC (p = 0.032) but not OS (p = 0.703). On multivariate analyses, improved OS was associated with the absence of extracerebral metastases (p < 0.001) and RPA class (p = 0.04), and IC with treatment (p = 0.019). SRS provided a 1-year, 2-year, and 3-year LC probability of 81%, 78%, and 55%, respectively. No association between LC and any of the potential prognostic factors was observed. The results of the subgroup analyses, regarding treatment modality, were similar to the entire cohort, particularly for RPA class I patients., Conclusion: Addition of WBRT to SRS offers better IC and should be considered for RCC patients with one to three brain metastases, especially in RPA class I group. SRS offers excellent LC rates, while WBRT should be reserved for patients with multiple metastases and poor prognosis.

Background: Alterations of mitochondrial DNA (mtDNA) have been implicated in carcinogenesis. We developed an accurate multiplex quantitative real-time PCR for synchronized determination of mtDNA and nuclear DNA (nDNA). We sought to investigate whether mtDNA content in the peripheral blood of breast cancer patients is associated with clinical and pathological parameters., Methods: Peripheral blood samples were collected from 60 patients with breast cancer and 51 age-matched healthy individuals as control. DNA was extracted from peripheral blood for the quantification of mtDNA and nDNA, using a one-step multiplex real-time PCR. A FAM labeled MGB probe and primers were used to amplify the mtDNA sequence of the ATP 8 gene, and a VIC labeled MGB probe and primers were employed to amplify the glyceraldehyde-3-phosphate-dehydrogenase gene. mtDNA content was correlated with tumor stage, menstruation status, and age of patients as well as lymph node status and the expression of estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu protein., Results: The content of mtDNA in stage I breast cancer patients was significantly lower than in other stages (overall P = 0.023). Reduced mtDNA was found often in post menopausal cancer group (P = 0.024). No difference in mtDNA content, in regards to age (p = 0.564), lymph node involvement (p = 0.673), ER (p = 0.877), PR (p = 0.763), and Her-2/neu expression (p = 0.335), was observed., Conclusion: Early detection of breast cancer has proved difficult and current detection methods are inadequate. In the present study, decreased mtDNA content in the peripheral blood of patients with breast cancer was strongly associated with stage I. The use of mtDNA may have diagnostic value and further studies are required to validate it as a potential biomarker for early detection of breast cancer.

High-energy protons and carbon ions exhibit an inverse dose profile allowing for increased energy deposition with penetration depth. Additionally, heavier ions like carbon beams have the advantage of a markedly increased biological effectiveness characterized by enhanced ionization density in the individual tracks of the heavy particles, where DNA damage becomes clustered and therefore more difficult to repair, but is restricted to the end of their range. These superior biophysical and biological profiles of particle beams over conventional radiotherapy permit more precise dose localization and make them highly attractive for treating anatomically complex and radioresistant malignant tumors but without increasing the severe side effects in the normal tissue. More than half a century since Wilson proposed their use in cancer therapy, the effects of particle beams have been extensively investigated and the biological complexity of particle beam irradiation begins to unfold itself. The goal of this review is to provide an as comprehensive and up-to-date summary as possible of the different radiobiological aspects of particle beams for effective application in cancer treatment.

Background and Purpose: Recurrent malignant gliomas have a very poor prognosis. This trial aimed to evaluate the benefits of reirradiation in case of recurrent glioblastoma multiforme (GBM) using hypofractionated stereotactic radiotherapy (hFSRT) after primary high-dose percutaneous irradiation., Patients and Methods: Between 1998 and 2008, 53 patients with recurrent GBM were treated by hFSRT based on CT and MR imaging. At the time of recurrence, a median total dose of 30 Gy (20-60 Gy) was delivered in median fractions of 3 Gy/day (2-5Gy)., Results: The reirradiation was well tolerated (no acute or late toxicity > grade 2), despite the relatively large median tumor volume (35.01 ml). Karnofsky Performance Score was the strongest predictor for survival after reirradiation (p = 0.0159). Tumor volume (p = 0.4690), patient age (p = 0.4301), second operation (p = 0.6930), and chemotherapy (p = 0.1466) at the time of reirradiation did not affect survival. After hFSRT, the median survival was 9 months, and the 1-year progression-free survival (PFS) amounted to 22%.The median overall survival from initial diagnosis was 27 months. 1-year survival from first diagnosis was 83%, 2-year survival 45%. The median time to progression from the end of initial irradiation to recurrence was 12 months. 1-year PFS before reirradiation was 40%., Conclusion: hFSRT as a secondary treatment of recurrent GBM is a feasible and effective treatment option. Only minor side effects were observed with prolonged life expectancy of 9 months.

The metastatic spread of tumor cells to distant sites represents the major cause of cancer-related deaths. Cancer metastasis involves a series of complex interactions between tumor cells and microenvironment that influence its biological effectiveness and facilitate tumor cell arrest to distant organs. More than a century since Paget developed the theory of seed and soil, the enigma of tissue specificity observed in metastatic colonization of tumor cells begins to unfold itself. The advent of new technologies has led to the discovery of novel molecules and pathways that confer metastasis-associated properties to the cancer cells, mediating organ specificity and unique genetic signatures have been developed using microarray studies. Future clinical studies and new antimetastatic compounds aiming to improve survival of patients with metastasis will most probably be based on these signatures. This review summarizes the plethora of old and new molecules that are strongly correlated with organ-specific metastases and which provide now an identity to the theory of seed and soil.