[en] Background: EGFR (epidermal growth factor receptor) and VEGF (vascular endothelial growth factor) inhibitors confer clinical benefit in metastatic colorectal cancer when combined with chemotherapy. An emerging strategy to improve outcomes in rectal cancer is to integrate biologically active, targeted agents as triple therapy into chemoradiation protocols. Material and methods: cetuximab and bevacizumab have now been incorporated into phase I-II studies of preoperative chemoradiation therapy (CRT) for rectal cancer. The rationale of these combinations, early efficacy and toxicity data, and possible molecular predictors for tumor response are reviewed. Computerized bibliographic searches of Pubmed were supplemented with hand searches of reference lists and abstracts of ASCO and ASTRO meetings. Results: the combination of cetuximab and CRT can be safely applied without dose compromises of the respective treatment components. Disappointingly low rates of pathologic complete remission have been noted in several phase II studies. The K-ras mutation status and the gene copy number of EGFR may predict tumor response. The toxicity pattern (radiation-induced enteritis, perforations) and surgical complications (wound healing, fistula, bleeding) observed in at least some of the clinical studies with bevacizumab and CRT warrant further investigations. Conclusion: longer follow-up (and, finally, randomized trials) is needed to draw any firm conclusions with respect to local and distant failure rates, and toxicity associated with these novel treatment approaches. (orig.)

[en] Background: Tumor shrinkage by preoperative radiochemotherapy (RCT) can markedly improve surgery in locally advanced (T4) rectal cancer with clear resection margins and may enable sphincter preservation in low-lying tumors. However, tumor response varies considerably, even among tumors treated according to the same protocol. If one is able to identify patients with highly radioresponsive tumors at the time of diagnosis, a selective and individualized policy of preoperative RCT might be pursued. Methods: The apoptotic index (AI), Ki-67, p53, and bcl-2 were evaluated by immunohistochemistry on pretreatment biopsies from 44 patients treated uniformly according to a prospective neoadjuvant RCT protocol (CAO/AIO/ARO-94). Treatment response was assessed histopathologically in the resected surgical specimen, using a five-point grading system. Expression of each marker was correlated with tumor response and relapse-free survival after curative surgery. Results: Tumors with complete (n=3) or good (n=28) response to RCT showed significantly higher pretreatment levels of apoptosis (mean AI: 2.06%) than tumors with moderate (n=7), minimal (n=5), or no regression (n=1) from RCT (AI: 1.44%, p=0.003). The AI was significantly related to Ki-67 (p=0.05), but not to p53 and bcl-2 status. Tumor regression and AI best predicted relapse-free survival after combined modality treatment and curative surgery. Conclusion: Spontaneous apoptosis in rectal cancer may serve as an important predictor of tumor regression from RCT in rectal cancer and as a significant prognosticator of relapse-free survival. Thus, this molecular marker may finally help to tailor therapy with regard to (neo-) adjuvant treatment of rectal cancer

[en] Purpose: To assess the prognostic value of biologic (p53, Ki-67) and clinical factors in squamous cell carcinoma of the oropharynx after radical surgery and postoperative radiotherapy (RT). Methods and Materials: Between 1985 and 1995, a total of 102 patients with 104 tumor sites were entered onto the study. Fifty-five primary tumors (53%) involved the tonsils, 26 (25%) the soft palate, and 23 (22%) the base of the tongue. Median age was 53 years (range 36-80 years). The clinical T- and N-categories (UICC 1997) were: T1 (30), T2 (47), T3 (22), T4 (5), N0 (33), N1 (28), N2 (42), and N3 (1). Histologically-clear margins were achieved in all patients by initial surgery. Postoperative RT to the primary and regional lymphatics was given, to a total of 60 Gy in 6 weeks, and single daily fractions of 2 Gy. The expression of the nuclear p53- and Ki-67-labeling index (LI) was investigated by immunostaining using the monoclonal antibodies DO-7 and MIB 1. The nuclear p53-intensity (p53-I) was graded into 4 categories (0/+/++/+++) by densitometry. Median follow-up was 43 months (range 14-132 months). Results: Cancer-specific survival, disease-free survival, and locoregional tumor control rates were 74%, 69%, and 75%, respectively, at 5 years. Significant prognostic factors for disease-free survival were: T-category (T1/2: 77% vs. T3/4: 53%, p = 0.02), tumor site (tonsils: 79% vs. soft palate: 70% vs. base of tongue: 45%, p = 0.05), duration of RT (≤ 46 days: 80% vs. > 46 days: 60%, p = 0.04), Ki-67 LI (≤ 20%: 84% vs. > 20%: 49%, p = 0.006) and p53-I (0/+: 56% vs. ++/+++: 79%, p = 0.008). A significant prognostic impact on locoregional control was noted for the duration of RT (≤ 46 days: 86% vs. > 46 days: 68%, p = 0.01), tumor site (tonsils: 88% vs. soft palate: 67% vs. base of tongue: 51%, p = 0.02), Ki-67 LI (≤ 20% LI: 87% vs. > 20% LI: 56%, p = 0.018), and the p53-I (0/+: 58% vs. ++/+++: 88%, p = 0.0006). On multivariate analysis, the p53 nuclear intensity (p = 0.002) and the Ki-67 index (p = 0.01) remained the only significant factors for locoregional control. Conclusion: Ki-67 labeling index above 20% and a weak p53 nuclear intensity (0/+) are both able to identify patients with squamous cell carcinoma of the oropharynx being at high risk for local recurrence after surgery and postoperative RT. Consequently, in this subgroup an intensification of treatment may be contemplated in prospective trials

[en] Purpose: Several groups have reported the value of bladder preservation by a combined treatment protocol, including transurethral resection (TUR-B) and radiochemotherapy (RCT). As more experience is acquired with organ-sparing treatment, patient selection should be optimized. The purpose of this study was to investigate the role of several biologic markers that may predict response to RCT in muscle-invasive bladder carcinoma. Methods and Materials: The apoptotic index (AI), Ki-67, p53, and bcl-2 were evaluated by immunohistochemistry on pretreatment biopsies from 70 patients treated for invasive bladder cancer by TUR-B and RCT. Expression of each marker was correlated with initial response, local control, and cancer-specific survival with preserved bladder. An exploratory multivariate analysis was also performed that included clinical and immunohistochemical variables. Results: A high AI (> median = 1.6%) and a high Ki-67 index (> median = 8.8%), but not the p53- and bcl-2 expression, were significantly related to initial complete response (CR) and local control with preserved bladder after 5 years. When the AI and Ki-67 expression were considered simultaneously, the association with initial CR (p < 0.001), local control (p = 0.0002), and cancer-specific survival with preserved bladder (p = 0.008) was highly significant. In an exploratory multivariate analysis (final model), only AI, Ki-67, and the combined AI/Ki-67 variable retained significance for local control with preserved bladder at 5 years. Conclusion: Patients with a high spontaneous AI and a high pretreatment Ki-67 index should be considered preferentially for treatment with RCT, whereas tumors with low proliferation and low levels of apoptosis are less likely to respond to RCT

[en] Purpose: Caveolin-1 is a protein marker for caveolae organelles and has an essential impact on cellular signal transduction pathways (e.g., receptor tyrosine kinases, adhesion molecules, and G-protein-coupled receptors). In the present study, we investigated the expression of caveolin-1 in patients with rectal adenocarcinoma and correlated its expression pattern with the risk for disease recurrences after preoperative chemoradiation therapy (CRT) and surgical resection. Methods and Materials: Caveolin-1 mRNA and protein expression were evaluated by Affymetrix microarray analysis (n = 20) and immunohistochemistry (n = 44) on pretreatment biopsy samples of patients with locally advanced adenocarcinoma of the rectum, and were correlated with clinical and histopathologic characteristics as well as with 5-year rates of local failure and overall survival. Results: A significantly decreased median caveolin-1 intracellular mRNA level was observed in tumor biopsy samples as compared with noncancerous mucosa. Individual mRNA levels and immunohistologic staining, however, revealed an overexpression in 7 of 20 patients (35%) and 17 of 44 patients (38.6%), respectively. Based on immunohistochemical evaluation, local control rates at 5 years for patients with tumors showing low caveolin-1 expression were significantly better than for patients with high caveolin-1-expressing carcinoma cells (p = 0.05; 92%, 95% confidence interval [95% CI] = 82-102% vs. 72%, 95% CI = 49-84%). A low caveolin-1 protein expression was also significantly related to an increased overall survival rate (p = 0.05; 45%, 95% CI 16-60% vs. 82%, 95% CI = 67-97%). Conclusion: Caveolin-1 may provide a novel prognostic marker for local control and survival after preoperative CRT and surgical resection in rectal cancer

[en] Background and purpose: Novel strategies to overcome an irradiation resistant phenotype may help to increase therapeutic efficacy in glioblastoma multiforme. The present study aimed to elucidate radiation sensitizing properties of artesunate, a semi synthetic derivate of artemisinin and to assess factors involved in this effect. Materials and methods: LN229 and U87MG cells were treated with various concentrations of artesunate and radiation response was determined by a colony forming assay. Cell numbers, apoptosis induction, cell cycle distribution, and DNA repair following combined modality treatment were monitored by MTT-, caspase 3/7 assay, cytofluorometry, and γ-H2AX foci formation. Expression of survivin, survivin–GFP fusion protein, XIAP, cellular (c)IAP1 and cIAP2 was monitored by Western immunoblotting. Results: Treatment of glioma cells with artesunate and irradiation resulted in an increased apoptotic fraction, pronounced G2/M arrest and increased DNA damage as demonstrated by an elevated amount of γ-H2AX foci/nucleus. Incubation with artesunate lowers survivin expression in a time and dose-dependent manner, whereas expression of XIAP, cIAP1 and cIAP2 was not affected. In clonogenic assays, treatment with artesunate revealed a significantly reduced surviving fraction, whereas stable over expression of a survivin–GFP protein reversed artesunate-mediated radiosensitization. Conclusion: Artesunate selectively down regulates survivin that contributes to a radio-sensitization of glioma cells by an increased induction of apoptosis, cell cycle arrest, and a hampered DNA damage response.

[en] A discontinuous dose response relationship is a major characteristic of the anti-inflammatory effects of low-dose X-irradiation therapy. Although recent data indicate an involvement of a variety of molecular mechanisms in these characteristics, the impact of reactive oxygen species (ROS) production to give rise or contribute to these phenomena in endothelial cells (EC) remains elusive. HUVEC derived immortalized EA.hy926 cells were stimulated by tumor necrosis factor-α (TNF-α, 20 ng/ml) 4 h before irradiation with doses ranging from 0.3 to 1 Gy. To analyse DNA repair capacity, phospho-histone H2AX foci were assayed at 1 h, 4 h and 24 h after irradiation. ROS production and superoxide dismutase (SOD) activity were analysed by fluorometric 2′,7′-dichlorodihydrofluorescein-diacetate (H2DCFDA) and colorimetric assays. A functional impact of ROS on γH2AX production was analysed by treatment with the scavenger N-acetyl-L-cysteine (NAC). Irrespective of stimulation by TNF-α, EA.hy926 cells revealed a linear dose response characteristic of γH2AX foci detection at 1 h and 4 h after irradiation. By contrast, we observed a discontinuity in residual γH2AX foci detection at 24 h after irradiation with locally elevated values following a 0.5 Gy exposure that was abolished by inhibition of ROS by NAC. Moreover, SOD protein expression was significantly decreased at doses of 0.5 Gy and 0.7 Gy concomitant with a reduced SOD activity. These data implicate a non-linear regulation of ROS production and SOD activity in EA.hy926 EC following irradiation with doses < 1 Gy that may contribute to a discontinuous dose-response relationship of residual γH2AX foci detection

[en] Purpose: Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. Methods and Materials: SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, γH2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. Results: In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G₂/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. Conclusion: Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer

[en] Background and purpose: Gliomas display prime examples of ionizing radiation (IR) resistant tumors. The IAP Survivin is reported to be critically involved in radiation resistance by anti-apoptotic and by caspase-independent mechanisms. The present study aimed to elucidate an interrelationship between Survivin’s cellular localization and DNA damage repair in glioma cells. Material and methods: Cellular distribution and nuclear complex formation were assayed by immunoblotting, immunofluorescence staining and co-immunoprecipitation of Survivin bound proteins in LN229 glioblastoma cells. Apoptosis induction, survival and DNA repair following IR were assayed by means of caspase3/7 activity, clonogenic assay, γ-H2AX/53BP1 foci formation, single cell gel electrophoresis assay, and DNA-PKcs kinase assay in the presence of Survivin siRNA or over expression of Survivin-GFP. Results: Following irradiation, we observed a nuclear accumulation and a direct interrelationship between Survivin, MDC1, γ-H2AX, 53BP1 and DNA-PKcs, which was confirmed by immunofluorescence co-localization. Survivin downregulation by siRNA resulted in an increased apoptotic fraction, decreased clonogenic survival and increased DNA-damage, as demonstrated by higher amount of DNA breaks and an increased amount of γ-H2AX/53BP1 foci post irradiation. Furthermore, we detected in Survivin-depleted LN229 cells a hampered S2056 (auto)phosphorylation and a significantly decreased DNA-PKcs kinase activity. Conclusion: Nuclear accumulation of Survivin and interaction with components of the DNA-double-strand break (DSB) repair machinery indicates Survivin to regulate DSB damage repair that leads to a significant improvement of survival of LN229 glioblastoma cells.

[en] Purpose: Therapy with low doses of ionising radiation (X-rays) exerts anti-inflammatory effects. Little is known about whether and how low doses of X-ray treatment modulate the inflammatory phenotype of macrophages, especially the secretion of Interleukin-1beta (IL-1β). Materials and methods: Macrophages were differentiated from human THP-1 monocytes, activated with lipopolysaccharide (LPS), treated with distinct low doses of X-rays, and co-activated with monosodium urate crystals (MSU) to induce inflammasome activation. Secretion of IL-1β was analysed by an enzyme-linked immunosorbent assay (ELISA) and Western blot. Furthermore, we analysed the intracellular amounts of the serine/threonine protein kinase B (named: Akt), mitogen-activated protein kinase p38 (p38), the v-rel reticuloendotheliosis viral oncogene homolog A (RelA), and pro- and cleaved IL-1β. Results: Low dose X-rays led to decreased secretion of active IL-1β in a manner discontinuous with dose which was most pronounced after 0.5 or 0.7 Gy. Passive release of lactate dehydrogenase (LDH) was not influenced by X-rays. The decreased secretion of IL-1β correlated with reduced translocation of RelA, being part of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) complex, into the nucleus. After 0.5 or 0.7 Gy of X-rays, the intracellular protein amounts of up (p38) and downstream molecules (Akt) of NF-κB were reduced in activated macrophages, as were the pro- and cleaved forms of IL-1β. Conclusions: Distinct low doses of X-rays induce an anti-inflammatory phenotype of activated macrophages by lowering the amount of secreted IL-1β in a NF-κB dependent manner. (authors)