[en] The use of molecular medicine is now merging into clinical practice with the advent of molecular targeting agents, molecular pathology and molecular imaging for both diagnosis and treatment response. Radiation oncologists must therefore gain expertise in utilizing this information to drive new treatment protocols. Recognizing the importance of this issue, the Canadian Association of Radiation Oncologists (CARO) charged a Task Force in Translational Radiobiology to: (1) critically assess training programs and research infrastructure in relation to current and future translational radiobiology requirements; and (2) make specific recommendations to accelerate the implementation of translational science into day-to-day practice. Selected Task Force recommendations included the principle that universities and departmental Chairs increase the opportunities for academic promotion, funding, and tenure track positions of radiobiologists and translational radiation oncologists. The dedication of 4 to 5 national centers as translational 'hubs', can serve as an interface between clinicians, clinical specimens and radiobiological sciences within the context of correlative clinical trials. The model of the clinician-scientist was encouraged as an important adjunct to good clinical care to be associated with strong enticement, training and mentoring programs and 75%-protected research time. Finally, an integrated model of radiobiological training programs and mutual continuing education between clinicians and basic scientists can be facilitated through a new national radiobiology meeting sponsored by CARO. These recommendations have been accepted by the national radiation oncology membership. Such a framework may serve useful for national programs wishing to develop rapid conduits from the lab to the clinic as a means of integrating molecular biology and the day-to-day practice of radiation oncology

[en] Background and purpose: Experimental studies have implicated the normal or 'wild type' p53 protein (i.e. WTp53) in the cellular response to ionizing radiation and other DNA damaging agents. Whether altered WTp53 protein function can lead to changes in cellular radiosensitivity and/or clinical radiocurability remains an area of ongoing study. In this review, we describe the potential implications of altered WTp53 protein function in normal and tumour cells as it relates to clinical radiotherapy, and describe novel treatment strategies designed to re-institute WTp53 protein function as a means of sensitizing cells to ionizing radiation. Methods and Materials: A number of experimental and clinical studies are critically reviewed with respect to the role of the p53 protein as a determinant of cellular oncogenesis, genomic stability, apoptosis, DNA repair and radioresponse in normal and transformed mammalian cells. Results: In normal fibroblasts, exposure to ionizing radiation leads to a G1 cell cycle delay (i.e. a 'G1 checkpoint') as a result of WTp53-mediated inhibition of G1-cyclin-kinase and retinoblastoma (pRb) protein function. The G1 checkpoint response is absent in tumour cells which express a mutant form of the p53 protein (i.e. MTp53), leading to acquired radioresistance in vitro. Depending on the cell type studied, this increase in cellular radiation survival can be mediated through decreased radiation-induced apoptosis, or altered kinetics of the radiation-induced G1 checkpoint. Recent biochemical studies support an indirect role for the p53 protein in both nucleotide excision and recombinational DNA repair pathways. However, based on clinicopathologic data, it remains unclear as to whether WTp53 protein function can predict for human tumour radiocurability and normal tissue radioresponse. Conclusions: Alterations in cell cycle control secondary to aberrant WTp53 protein function may be clinically significant if they lead to the acquisition of mutant cellular phenotypes, including the radioresistant phenotype. Pre-clinical studies suggest that these phenotypes may be reversed using adenovirus-mediated gene therapy or pharmacologic strategies designed to re-institute WTp53 protein function. Our analysis of the published data strongly argues for the use offunctional assays for the determination of WTp53 protein function in studies which attempt to correlate normal and tumour tissue radioresponse with p53 genotype, or p53 protein expression

[en] Purpose: Changes in wild-type p53 protein function occur in the majority of human tumors, and may alter genomic stability and the cellular response to ionizing radiation. Whether oncoproteins can render tumor cells both radioresistant and metastatic, may have implications for clinical strategies designed to improve local tumor control. In the studies reported here, we tested the hypothesis that acquired radioresistance correlates with metastatic potential within a large panel of transformed rat embryo cell (REF) lines following transfection with activated H-ras, mutant p53, and HPV16-E7 alleles. Methods and Materials: Rat embryo cells (REF cells) were transfected using the calcium-phosphate technique with an activated H-ras gene alone, or in combination with human papillomavirus HPV16-E7 and/or human or murine mutant p53 sequences. Other rat embryo cell clones expressing transfected HPV-E7 and activated ras sequences subsequently acquired endogenous p53 gene mutations during culture in vitro. The relative expression of p21ras and p53 protein for each REF transformant was determined by Western blot analysis following transfection. REF clones were phenotypically characterized at early passage (i.e., passages 5-7) and late passage (i.e., passages 10-20) for their: (a) relative tumor growth rate, and (b) their ability to undergo spontaneous metastasis following intramuscular injection into the hind legs of SCID mice. In vivo phenotypic end points were then compared to previously measured parameters of in vitro radiosensitivity for each cell line. Additionally, the expression of the cellular protease, plasminogen activator, was determined for a number of metastatic and nonmetastatic cell lines. Results: We found no evidence that selected oncogene-transfected REF transformants that were radioresistant in culture had a greater spontaneous metastatic potential than nonradioresistant REF transformants. Neither the level of expression of the p21ras protein nor that of the p53 protein was correlated with the spontaneous metastatic phenotype when tested at early passage. The metastatic phenotype appeared to be independent of p53 genotype. The majority of metastatic REF clones tested (7 out of 9 clones) expressed plasminogen activator following oncogene transfection, in contrast to nonmetastatic REF transformed cell lines. Conclusions: Our results suggest that (a) intrinsic radioresistance does not correlate with spontaneous metastatic potential in oncogene-expressing REF transformant cell lines, and (b), novel clinical strategies designed to overcome oncogene-mediated radioresistance could potentially impact on overall survival, as gains in local tumor control may not be offset by a greater risk of distant metastasis

[en] Purpose: Pretreatment hemoglobin (Hb) level has been reported to be an important prognostic factor for local control and survival in various malignancies. However, in many settings, the adverse effect of a low Hb may be related to more advanced disease. The purpose of this analysis was to assess the influence of pretreatment Hb on local control in a large series of patients with a localized cancer (T1/T2 glottic cancer, AJCC 1992) treated in a standard fashion. Materials and Methods: Between January 1981 and December 1989, 735 patients (median age 63; 657 males, 78 females) with T1/T2 glottic cancer were treated with radiation therapy (RT). The standard RT prescription was 50 Gy in 20 fractions over 4 weeks (97% of patients). Factors studied for prognostic importance for local failure included pretreatment Hb, age, sex, T category, anterior commissure involvement, subglottic extension, and tumor bulk (presence of visible tumor vs. subclinical disease). Results: With a median follow-up of 6.8 years (range 0.2-14.3), 131 patients have locally relapsed for an actuarial 5-year relapse-free rate of 81.7%. The 5-year actuarial survival was 75.8%. The mean pretreatment hemoglobin level was 14.8 g/dl and was similar in all prognostic categories. On multivariate analysis, using the Cox proportional hazards model, pretreatment Hb predicted for local failure after RT. The hazard ratio (HR) for relapse was calculated for various Hb levels. For example, the HR for a Hb of 12 g/dl vs. a Hb of 15 g/dl was 1.8 (95% confidence interval 1.2-2.5). Previously established factors, including gender, T category, subglottic extension, as well as tumor bulk, were also prognostically important for local control. Conclusions: This analysis, in a large number of similarly treated patients, indicates that pretreatment Hb is an independent prognostic factor for local control in patients with T1/T2 carcinoma of the glottis treated with RT. The underlying biology of this observation needs to be explored, and using this information, it may be possible to develop strategies to improve treatment outcome

[en] Background and purpose: RAD51 is a key protein involved in homologous recombination (HR) and a potential target for radiation- and chemotherapies. Amuvatinib (formerly known as MP470) is a novel receptor tyrosine kinase inhibitor that targets c-KIT and PDGFRα and can sensitize tumor cells to ionizing radiation (IR). Here, we studied amuvatinib mechanism on RAD51 and functional HR. Materials and methods: Protein and RNA analyses, direct repeat green fluorescent protein (DR-GFP) assay and polysomal fractioning were used to measure HR efficiency and global translation in amuvatinib-treated H1299 lung carcinoma cells. Synergy of amuvatinib with IR or mitomycin c (MMC) was assessed by clonogenic survival assay. Results: Amuvaninib inhibited RAD51 protein expression and HR. This was associated with reduced ribosomal protein S6 phosphorylation and inhibition of global translation. Amuvatinib sensitized cells to IR and MMC, agents that are selectively toxic to HR-deficient cells. Conclusions: Amuvatinib is a promising agent that may be used to decrease tumor cell resistance. Our work suggests that this is associated with decreased RAD51 expression and function and supports the further study of amuvatinib in combination with chemotherapy and radiotherapy.

[en] The ATM kinase is activated by chromatin modification following exogenous and endogenous DSBs or cell stress, including acute anoxia. The p53 binding protein 1 (53BP1) contains multiple ATM-consensus phosphorylation sites in its N- and C-termini and may therefore be a distal read-out of ATM function. We have examined the cellular activation of these phosphorylation sites for the first time in situ following anoxic/hypoxic stress and IR-induced exogenous DSBs. We show that multiple residues of 53BP1 are phosphorylated and that these phosphoforms form discrete nuclear foci following IR or during DNA replication as exogenous or endogenous DNA double strand breaks (DSBs), respectively. Novel data pertaining to the phosphorylation of 53BP1^Ser25in situ supports its dependency on the ATM kinase; but this occurs independently of p53 function. We show that 53BP1^Ser25 is activated specifically in S-phase cells during anoxia in an ATM-dependent manner. Exogenous DSBs form discrete IR-induced foci whereas oxygen stress induced non-localized 53BP1^Ser25 activation. Our in vitro data are supported by irradiated xenograft studies in vivo whereby 53BP1^Ser25 phosphorylation does not occur in sub-regions positive for the hypoxia marker EF5. We propose a model whereby DSBs induce chromatin modification at sites of DNA damage which are tracked by the ATM substrates γ H2AX and 53BP1^Ser25 in a mechanism distinct from p53-mediated cell cycle arrest. Together this work indicates 53BP1^Ser25, and possibly other 53BP1 phosphoforms, as a bona fide DSB-biomarkers for surveying ongoing DNA-damage related signaling in oxic and hypoxic cells during clinical radiotherapy.

[en] The novel agent PRIMA-1^met can reactivate WTp53 function in MTp53-expressing cells. We investigated PRIMA-1^met as a radiosensitizer of WTp53, p53Null or MTp53 prostate cancer cells. Radiosensitization was observed in PC3 (p53Null) cells, even under hypoxia. In certain circumstances, PRIMA-1^met may therefore act independently of MTp53 status and WTp53 transactivation

[en] Using elegant targeting techniques such as IMRT, radiation oncology has improved the therapeutic ratio of prostate cancer radiotherapy through increased physical precision (e.g. increased local control through dose-escalation without increased normal tissue toxicity). The therapeutic ratio might be further improved by the addition of 'biologic precision and escalation' pertaining to the use of molecular inhibitors of DNA damage sensing and repair. Indeed, proteins involved in the ATM-p53 damage signaling axis and the homologous (HR) and non-homologous end-joining (NHEJ) pathways of DNA double-strand break (DNA-dsb) rejoining pathways may be attractive candidates to elucidate cancer risk, prognosis, prediction of response and to develop sensitizers towards oxic and hypoxic prostate tumor cells. This review highlights DNA-dsb in prostate cancer research in terms of novel molecular inhibitors, the role of the microenvironment in DNA-dsb repair and potential DNA-dsb biomarkers for clinical trials

[en] ObjectivesKallikreins are serine proteases over expressed in many malignancies. In this study, we measure changes in serum kallikrein (KLKs) levels during intensity-modulated radiotherapy (IMRT) in prostate cancer patients, and find potential correlations between serum kallikrein level and normal tissues toxicity during radiation.

[en] Background and purpose: Radiation proctitis is a potential complication following pelvic radiation therapy. There are no standard treatments and treatment outcomes are unpredictable. We report our experience with the use of hyperbaric oxygen treatment (HBOT) for radiation proctitis cases refractory to standard medical or laser therapy. Patients and methods: During the period 2000-2004, 10 patients with radiation proctitis were treated with HBOT (three males and seven females; mean age of 65). The median follow-up period was 25 months (range 6-43 months). Patient symptoms were retrospectively scored prior to, and following HBOT, based on the LENT-SOMA scale. Results: Prior to treatment, three patients had Grade 3 toxicity (i.e. requiring blood transfusions) and seven had Grade 2 toxicity with dominant symptoms of rectal pain and/or diarrhoea. HBOT was well tolerated and 9 of the 10 patients completed a full HBOT treatment program. Rectal bleeding completely stopped in four of nine symptomatic patients and improved in three others. Rectal pain completely remitted in three of five symptomatic patients. Diarrhea remitted completely in one of five patients and improved in three others. Of the 10 patients treated, only two did not respond to HBOT. Conclusions: Significant improvement of rectal bleeding, diarrhea and rectal pain is possible using HBOT. HBOT should be offered to patients who fail conventional treatments for radiation proctitis