Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta.

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polbetaDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polbetaDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polbetaDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H(2)O(2) treatment, polbetaDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H(2)O(2), these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polbetaDN induced increase in residual gammaH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual gammaH2AX foci, indicating an escape from gammaH2AX-mediated DSB repair. In addition, we show that in the polbetaDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.

Molecular markers predict outcome in squamous cell carcinoma of the head and neck after concomitant cisplatin-based chemoradiation.

Not all patients with squamous cell carcinomas of the head and neck (HNSCC) benefit from concurrent cisplatin-based chemoradiation, but reliable predictive markers for outcome after chemoradiation are scarce. We have investigated potential prognostic biomarkers for outcome in a large group of patients. Ninety-one tumor biopsies taken from consecutive HNSCC patients were evaluated for protein expression on a tissue microarray. Using immunohistochemistry, 18 biomarkers, involved in various cellular pathways were investigated. Univariable and multivariable proportional hazard analyses were performed to investigate associations between each individual marker and outcome. In addition, the global test was used to test all variables simultaneously and selected combinations of markers for an overall association with local control. Univariable analysis showed statistically significant increased relative risks of RB, P16 and MRP2 for local control and MDR1 and HIF-1alpha for overall survival. MRP2, MDR1 and P16 levels were positively associated with outcome whereas RB and HIF-1alpha had a negative relationship. Using Goeman's global testing no combination of markers was identified that was associated with local control. Grouping the markers according to their function revealed an association between a combination of 3 markers (P16, P21 and P27) and outcome (p = 0.05) was found. In the multivariable analysis, MRP2 and RB remained significant independent predictive markers for local control. This study describes the prognostic value of biomarkers for the outcome in patients uniformly treated with concurrent chemoradiation. MRP2 and RB were found to be associated with outcome in patients treated with concurrent chemoradiation.

Role for DNA polymerase beta in response to ionizing radiation.

Evidence for a role of DNA polymerase beta in determining radiosensitivity is conflicting. In vitro assays show an involvement of DNA polymerase beta in single strand break repair and base excision repair of oxidative damages, both products of ionizing radiation. Nevertheless the lack of DNA polymerase beta has been shown to have no effect on radiosensitivity. Here we show that mouse embryonic fibroblasts deficient in DNA polymerase beta are considerably more sensitive to ionizing radiation than wild-type cells, but only when confluent. The inhibitor methoxyamine renders abasic sites refractory to the dRP lyase activity of DNA polymerase beta. Methoxyamine did not significantly change radiosensitivity of wild-type fibroblasts in log phase. However, DNA polymerase beta deficient cells in log phase were radiosensitized by methoxyamine. Alkaline comet assays confirmed repair inhibition of ionizing radiation induced damage by methoxyamine in these cells, indicating both the existence of a polymerase beta-dependent long patch pathway and the involvement of another methoxyamine sensitive process, implying the participation of a second short patch polymerase(s) other than DNA polymerase beta. This is the first evidence of a role for DNA polymerase beta in radiosensitivity in vivo.

Cell cycle phase dependent role of DNA polymerase beta in DNA repair and survival after ionizing radiation.

PURPOSE: The purpose of the present study was to determine the role of DNA polymerase beta in repair and response after ionizing radiation in different phases of the cell cycle. METHODS AND MATERIALS: Synchronized cells deficient and proficient in DNA polymerase beta were irradiated in different phases of the cell cycle as determined by BrdU/flow cytometry. Cell kill and DNA repair were assessed by colony formation and alkaline comet assays, respectively. RESULTS: We first demonstrated delayed repair of ionizing radiation induced DNA damage in confluent polymerase beta deficient cells. Cell synchronization experiments revealed a cell cycle phase dependence by demonstrating radiation hypersensitivity of polymerase beta-deficient cells in G1, but not in the S-phase. Complementing polymerase beta-deficient cells with polymerase beta reverted the hypersensitivity in G1. Ionizing radiation damage repair was found to be delayed in beta-deficient cells when irradiated in G1, but not in S. CONCLUSIONS: The data show a differential role of DNA polymerase beta driven base excision and single strand break repair throughout the cell cycle after ionizing radiation damage.

Phase III trial to evaluate the efficacy of maintaining hemoglobin levels above 12.0 g/dL with erythropoietin vs above 10.0 g/dL without erythropoietin in anemic patients receiving concurrent radiation and cisplatin for cervical cancer.

PURPOSE: To determine whether maintaining HGB levels > or = 12.0 g/dL with recombinant human erythropoietin (R-HUEPO) compared to "standard" treatment (transfusion for HGB < or = 10.0 g/dL) improves progression-free survival (PFS), overall survival (OS) and local control (LC) in women receiving concurrent weekly cisplatin and radiation (CT/RT) for carcinoma of the cervix. In addition, to determine whether platinum-DNA adducts were associated with clinical characteristics or outcome. METHODS: Patients with stage IIB-IVA cervical cancer and HGB < 14.0 g/dL were randomly assigned to CT/RT+/-R-HUEPO (40,000 units s.c. weekly). R-HUEPO was stopped if HGB > 14.0 g/dL. Endpoints were PFS, OS and LC. Platinum-DNA adducts were quantified using immunocytochemistry assay in buccal cells. RESULTS: Between 08/01 and 09/03, 109 of 114 patients accrued were eligible. Fifty-two received CT/RT and 57 CT/RT+R-HUEPO. The study closed prematurely, with less than 25% of the planned accrual, due to potential concerns for thromboembolic event (TE) with R-HUEPO. Median follow-up was 37 months (range 9.8-50.4 months). PFS and OS at 3 years should be 65% and 75% for CT/RT and 58% and 61% for CT/RT+R-HUEPO, respectively. TE occurred in 4/52 receiving CT/RT and 11/57 with CT/RT+R-HUEPO, not all considered treatment related. No deaths occurred from TE. High-platinum adducts were associated with inferior PFS and LC. CONCLUSION: TE is common in cervical cancer patients receiving CT/RT. Difference in TE rate between the two treatments was not statistically significant. The impact of maintaining HGB level > 12.0 g/dL on PFS, OS and LC remains undetermined.

Gene expression profiling to predict outcome after chemoradiation in head and neck cancer.

PURPOSE: The goal of the present study was to improve prediction of outcome after chemoradiation in advanced head and neck cancer using gene expression analysis. MATERIALS AND METHODS: We collected 92 biopsies from untreated head and neck cancer patients subsequently given cisplatin-based chemoradiation (RADPLAT) for advanced squamous cell carcinomas (HNSCC). After RNA extraction and labeling, we performed dye swap experiments using 35k oligo-microarrays. Supervised analyses were performed to create classifiers to predict locoregional control and disease recurrence. Published gene sets with prognostic value in other studies were also tested. RESULTS: Using supervised classification on the whole series, gene sets separating good and poor outcome could be found for all end points. However, when splitting tumors into training and validation groups, no robust classifiers could be found. Using Gene Set Enrichment analysis, several gene sets were found to be enriched in locoregional recurrences, although with high false-discovery rates. Previously published signatures for radiosensitivity, hypoxia, proliferation, "wound," stem cells, and chromosomal instability were not significantly correlated with outcome. However, a recently published signature for HNSCC defining a "high-risk" group was shown to be predictive for locoregional control in our dataset. CONCLUSION: Gene sets can be found with predictive potential for locoregional control after combined radiation and chemotherapy in HNSCC. How treatment-specific these gene sets are needs further study.

Impact of supervised gene signatures of early hypoxia on patient survival.

BACKGROUND AND PURPOSE: Hypoxia is a common feature of solid tumors associated with therapy resistance, increased malignancy and poor prognosis. Several approaches have been developed with the hope of identifying patients harboring hypoxic tumors including the use of microarray based gene signatures. However, studies to date have largely ignored the strong time dependency of hypoxia-regulated gene expression. We hypothesized that use of time-dependent patterns of gene expression during hypoxia would enable development of superior prognostic expression signatures. MATERIALS AND METHODS: Using published data from the microarray study of Chi et al., we extracted gene signatures correlating with induction during either early or late hypoxic exposure. Gene signatures were derived from in vitro exposed human mammary epithelial cell line (HMEC) under 0% or 2% oxygen. Gene signatures correlating with early and late up-regulation were tested by means of Kaplan-Meier survival, univariate, and multivariate analysis on a patient data set with primary breast cancer treated conventionally (surgery plus on indication radiotherapy and systemic therapy). RESULTS: We found that the two early hypoxia gene signatures extracted from 0% and 2% hypoxia showed significant prognostic power (log-rank test: p=0.004 at 0%, p=0.034 at 2%) in contrast to the late hypoxia signatures. Both early gene signatures were linked to the insulin pathway. From the multivariate Cox-regression analysis, the early hypoxia signature (p=0.254) was found to be the 4th best prognostic factor after lymph node status (p=0.002), tumor size (p=0.016) and Elston grade (p=0.111). On this data set it indeed provided more information than ER status or p53 status. CONCLUSIONS: The hypoxic stress elicits a wide panel of temporal responses corresponding to different biological pathways. Early hypoxia signatures were shown to have a significant prognostic power. These data suggest that gene signatures identified from in vitro experiments could contribute to individualized medicine.

Involvement of DNA polymerase beta in repair of ionizing radiation damage as measured by in vitro plasmid assays.

Characteristic of damage introduced in DNA by ionizing radiation is the induction of a wide range of lesions. Single-strand breaks (SSBs) and base damages outnumber double-strand breaks (DSBs). If unrepaired, these lesions can lead to DSBs and increased mutagenesis. XRCC1 and DNA polymerase beta (polbeta) are thought to be critical elements in the repair of these SSBs and base damages. XRCC1-deficient cells display a radiosensitive phenotype, while proliferating polbeta-deficient cells are not more radiosensitive. We have recently shown that cells deficient in polbeta display increased radiosensitivity when confluent. In addition, cells expressing a dominant negative to polbeta have been found to be radiosensitized. Here we show that repair of radiation-induced lesions is inhibited in extracts with altered polbeta or XRCC1 status, as measured by an in vitro repair assay employing irradiated plasmid DNA. Extracts from XRCC1-deficient cells showed a dramatically reduced capacity to repair ionizing radiation-induced DNA damage. Extracts deficient in polbeta or containing a dominant negative to polbeta also showed reduced repair of radiation-induced SSBs. Irradiated repaired plasmid DNA showed increased incorporation of radioactive nucleotides, indicating use of an alternative long-patch repair pathway. These data show a deficiency in repair of ionizing radiation damage in extracts from cells deficient or altered in polbeta activity, implying that increased radiosensitivity resulted from radiation damage repair deficiencies.

Ionizing radiation sensitivity of DNA polymerase lambda-deficient cells.

Ionizing radiation induces a diverse spectrum of DNA lesions, including strand breaks and oxidized bases. In mammalian cells, ionizing radiation-induced lesions are targets of non-homologous end joining, homologous recombination, and base excision repair. In vitro assays show a potential involvement of DNA polymerase lambda in non-homologous end joining and base excision repair. In this study, we investigated whether DNA polymerase lambda played a significant role in determining ionizing radiation sensitivity. Despite increased sensitivity to hydrogen peroxide, lambda-deficient mouse embryonic fibroblasts displayed equal survival after exposure to ionizing radiation compared to their wild-type counterparts. In addition, we found increased sensitivity to the topoisomerase inhibitors camptothecin and etoposide in the absence of polymerase lambda. These results do not reveal a major role for DNA polymerase lambda in determining radiosensitivity in vivo.

Radiosensitization of squamous cell carcinoma by the alkylphospholipid perifosine in cell culture and xenografts.

PURPOSE: Combined modality treatment has improved outcome in various solid tumors. Besides classic anticancer drugs, a new generation of biological response modifiers has emerged that increases the efficacy of radiation. Here, we have investigated whether perifosine, an orally applicable, membrane-targeted alkylphospholipid, enhances the antitumor effect of radiation in vitro and in vivo. EXPERIMENTAL DESIGN: Several long-term and short-term in vitro assays (clonogenic survival, sulforhodamine B cytotoxicity, apoptosis, and cell cycle analysis) were used to assess the cytotoxic effect of perifosine in combination with radiation. In vivo, the response of human KB squamous cell carcinoma xenografts was measured after treatment with perifosine, irradiation, and the combination. Radiolabeled perifosine was used to determine drug disposition in tumor and normal tissues. At various intervals after treatment, tumor specimens were collected to document histopathologic changes. RESULTS: In vitro, perifosine reduced clonogenic survival, enhanced apoptosis, and increased cell cycle arrest after radiation. In vivo, radiation and perifosine alone induced a dose-dependent tumor growth delay. When combining multiple perifosine administrations with single or split doses of radiation, complete and sustained tumor regression was observed. Histopathologic analysis of tumor specimens revealed a prominent apoptotic response after combined treatment with radiation and perifosine. Radiation-enhanced tumor response was observed at clinically relevant plasma perifosine concentrations and accumulating drug disposition of >100 microg/g in tumor tissue. CONCLUSIONS: Perifosine enhances radiation-induced cytotoxicity, as evidenced by reduced clonogenic survival and increased apoptosis induction in vitro and by complete tumor regression in vivo. These data provide strong support for further development of this combination in clinical studies.

Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status.

PURPOSE: To determine the role of DNA repair in hypoxic radioresistance. METHODS AND MATERIALS: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. RESULTS: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. CONCLUSION: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity.

Hypoxia in human colorectal adenocarcinoma: comparison between extrinsic and potential intrinsic hypoxia markers.

PURPOSE: To detect and quantify hypoxia in colorectal adenocarcinomas by use of pimonidazole and iododeoxyuridine (IdUrd) as extrinsic markers and carbonic anhydrase IX (CA IX), microvessel density (MVD), epidermal growth-factor receptor (EGFR), and vascular endothelial growth factor (VEGF) as intrinsic markers of hypoxia. METHODS AND MATERIAL: Twenty patients with an adenocarcinoma of the left colon and rectum treated by primary surgery were injected with pimonidazole and IdUrd. Serial sections of tumor biopsies were single stained for VEGF, EGFR, Ki67, and double stained for blood vessels in combination with either pimonidazole, IdUrd, or CA IX. Percentage of expression was scored as well as colocalization of pimonidazole with CA IX. RESULTS: The median percentage of hypoxia, as judged by pimonidazole staining, was 16.7% (range, 0-52.4%). The expression of pimonidazole correlated inversely with the total MVD and endothelial cord MVD (R = -0.55, p = 0.01; R = -0.47, p = 0.04). Good colocalization was found between pimonidazole and CA IX in only 30% of tumors, with no correlation overall between pimonidazole and CA IX, VEGF, or EGFR or between the different intrinsic markers. Cells around some vessels (0.08-11%) were negative for IdUrd but positive for Ki 67, which indicated their lack of perfusion at the time of injection. CONCLUSION: Chronic and acute hypoxic regions are present in colorectal tumors, as shown by pimonidazole and IdUrd staining. Only in a minority of tumors did an association exist between the areas stained by pimonidazole and those positive for CA IX. Pimonidazole also did not correlate with expression of other putative intrinsic hypoxia markers (VEGF, EGFR).

Microsatellite alterations in head and neck squamous cell carcinoma and relation to expression of pimonidazole, CA IX and GLUT-1.

BACKGROUND AND PURPOSE: Because the locoregional control for HNSCC is still disappointing, research efforts focus on the exploration of new molecular markers located in both tumour and microenvironment, which could help stratify patients. The aim of the present work was therefore first to assess microsatellite alterations and hypoxia in HNSCC as possible molecular markers. Second, a relation between both was investigated, as hypoxia is known to select for genetic alterations. MATERIAL AND METHODS: Forty-eight patients with advanced HNSCC treated by surgery+/-radiotherapy were included. MSI and LOH were investigated with microsatellite markers using automatic fragment analysis. The presence of hypoxia was assessed by immunohistochemistry for pimonidazole, CA IX and GLUT-1. The mutual relationship between MSI/LOH and hypoxia was evaluated. RESULTS: No MSI was detected in this patient group. LOH occurred mostly on chromosomal arms 3p, 5q, 9p, 17p and 17q. Patients with LOH at D17S799, located in the near environment of p53, showed a higher CA IX expression (p=0.01). CONCLUSIONS: LOH is a possible molecular marker in HNSCC. The positive correlation between LOH at D17S799 and CA IX is in full concordance with previous publications linking hypoxia to selective pressure on the p53 gene.

The role of DNA polymerase beta in determining sensitivity to ionizing radiation in human tumor cells.

Lethal lesions after ionizing radiation are thought to be mainly unrepaired or misrepaired DNA double-strand breaks, ultimately leading to lethal chromosome aberrations. However, studies with radioprotectors and repair inhibitors indicate that single-strand breaks, damaged nucleotides or abasic sites can also influence cell survival. This paper reports on studies to further define the role of base damage and base excision repair on the radiosensitivity of human cells. We retrovirally transduced human tumor cells with a dominant negative form of DNA polymerase beta, comprising the 14 kDa DNA-binding domain of DNA polymerase beta but lacking polymerase function. Radiosensitization of two human carcinoma cell lines, A549 and SQD9, was observed, achieving dose enhancement factors of 1.5-1.7. Sensitization was dependent on expression level of the dominant negative and was seen in both single cell clones and in unselected virally transduced populations. Sensitization was not due to changes in cell cycle distribution. Little or no sensitization was seen in G(1)-enriched populations, indicating cell cycle specificity for the observed sensitization. These results contrast with the lack of effect seen in DNA polymerase beta knockout cells, suggesting that polDN also inhibits the long patch, DNA polymerase beta-independent repair pathway. These data demonstrate an important role for BER in determining sensitivity to ionizing radiation and might help identify targets for radiosensitizing tumor cells.

Nbs1 promotes ATM dependent phosphorylation events including those required for G1/S arrest.

Cell lines from Nijmegen Breakage Syndrome (NBS) and ataxia telangiectasia (A-T) patients show defective S phase checkpoint arrest. In contrast, only A-T but not NBS cells are significantly defective in radiation-induced G1/S arrest. Phosphorylation of some ATM substrates has been shown to occur in NBS cells. It has, therefore, been concluded that Nbs1 checkpoint function is S phase specific. Here, we have compared NBS with A-T cell lines (AT-5762ins137) that express a low level of normal ATM protein to evaluate the impact of residual Nbs1 function in NBS cells. The radiation-induced cell cycle response of these NBS and 'leaky' A-T cells is almost identical; normal G2/M arrest after 2 Gy, intermediate G1/S arrest depending on the dose and an A-T-like S phase checkpoint defect. Thus, the checkpoint assays differ in their sensitivity to low ATM activity. Radiation-induced phosphorylation of the ATM-dependent substrates Chk2, RPAp34 and p53-Ser15 are similarly impaired in AT-5762ins137 and NBS cells in a dose dependent manner. In contrast, NBS cells show normal ability to activate ATM kinase following irradiation in vitro and in vivo. We propose that Nbs1 facilitates ATM-dependent phosphorylation of multiple downstream substrates, including those required for G1/S arrest.

Thymidine analogues to assess microperfusion in human tumors.

PURPOSE: To validate the use of the thymidine analogues as local perfusion markers in human tumors (no labeling indicates no perfusion) by comparison with the well-characterized perfusion marker Hoechst 33342. METHODS AND MATERIALS: Human tumor xenografts from gliomas and head-and-neck cancers were injected with iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) and the fluorescent dye Hoechst 33342. In frozen sections, each blood vessel was scored for the presence of IdUrd/BrdUrd labeling and Hoechst in surrounding cells. The percentage of analogue-negative vessels was compared with the fraction of Hoechst-negative vessels. Collocalization of the two markers was also scored. RESULTS: We found considerable intertumor variation in the fraction of perfused vessels, measured by analogue labeling, both in the human tumor xenografts and in a series of tumor biopsies from head-and-neck cancer patients. There was a significant correlation between the Hoechst-negative and IdUrd/BrdUrd-negative vessels in the xenografts (r = 85, p = 0.0004), despite some mismatches on a per-vessel basis. CONCLUSIONS: Thymidine analogues can be successfully used to rank tumors according to their fraction of perfused vessels. Whether this fraction correlates with the extent of acute hypoxia needs further confirmation.

Hypoxic cell turnover in different solid tumor lines.

PURPOSE: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. METHODS AND MATERIALS: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). RESULTS: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. CONCLUSIONS: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h.

Molecular markers predicting radiotherapy response: report and recommendations from an International Atomic Energy Agency technical meeting.

PURPOSE: There is increasing interest in radiogenomics and the characterization of molecular profiles that predict normal tissue and tumor radioresponse. A meeting in Amsterdam was organized by the International Atomic Energy Agency to discuss this topic on an international basis. METHODS AND MATERIALS: This report is not completely exhaustive, but highlights some of the ongoing studies and new initiatives being carried out worldwide in the banking of tumor and normal tissue samples underpinning the development of molecular marker profiles for predicting patient response to radiotherapy. It is generally considered that these profiles will more accurately define individual or group radiosensitivities compared with the nondefinitive findings from the previous era of cellular-based techniques. However, so far there are only a few robust reports of molecular markers predicting normal tissue or tumor response. RESULTS: Many centers in different countries have initiated tissue and tumor banks to store samples from clinical trials for future molecular profiling analysis, to identify profiles that predict for radiotherapy response. The European Society for Therapeutic Radiology and Oncology GENEtic pathways for the Prediction of the effects of Irradiation (GENEPI) project, to store, document, and analyze sample characteristics vs. response, is the most comprehensive in this regard. CONCLUSIONS: The next 5-10 years are likely to see the results of these and other correlative studies, and promising associations of profiles with response should be validated in larger definitive trials.

Radiosensitization by a dominant negative to DNA polymerase beta is DNA polymerase beta-independent and XRCC1-dependent.

BACKGROUND AND PURPOSE: DNA base damages and single strand breaks after ionizing radiation are repaired by base excision repair (BER) and single strand break repair (SSBR), with both DNA polymerase beta (polbeta) and XRCC1 playing key roles. We previously showed that a dominant negative to polbeta (polbetaDN) sensitized human tumor cells to ionizing radiation. However, polbeta-deficient cells, in contrast to XRCC1-deficient cells, are not more radiosensitive. The purpose of the present study was to further elucidate the mechanism of action of the polbetaDN to better understand the roles of BER and SSBR in determining radiosensitivity. MATERIALS AND METHODS: Mouse embryonic fibroblasts, both polbeta wildtype and knockout, and hamster XRCC1-deficient EM9 cells together with its parental line, were transfected with the polbetaDN. Clones with equal polbetaDN expression levels were selected and used in clonogenic assays to determine radiosensitivity. RESULTS: Radiosensitization of polbeta deficient cells by the polbetaDN is shown here, demonstrating inhibition of a polbeta-independent pathway. In addition, we observed radiosensitization of wildtype hamster cells but no radiosensitization of the XRCC1-deficient EM9 cells. CONCLUSIONS: The polbetaDN acts independently of polbeta status and inhibits a pathway, which is dependent on XRCC1, consistent with inhibition of BER and/or SSBR. The data further indicate involvement of other polymerases, which are inhibited by polbetaDN. Finally, they demonstrate that inhibition of BER and SSBR can increase radiosensitivity, with potential clinical relevance.

Pretreatment microRNA Expression Impacting on Epithelial-to-Mesenchymal Transition Predicts Intrinsic Radiosensitivity in Head and Neck Cancer Cell Lines and Patients.

PURPOSE: Predominant causes of head and neck cancer recurrence after radiotherapy are rapid repopulation, hypoxia, fraction of cancer stem cells, and intrinsic radioresistance. Currently, intrinsic radioresistance can only be assessed by ex vivo colony assays. Besides being time-consuming, colony assays do not identify causes of intrinsic resistance. We aimed to identify a biomarker for intrinsic radioresistance to be used before start of treatment and to reveal biologic processes that could be targeted to overcome intrinsic resistance. EXPERIMENTAL DESIGN: We analyzed both microRNA and mRNA expression in a large panel of head and neck squamous cell carcinoma (HNSCC) cell lines. Expression was measured on both irradiated and unirradiated samples. Results were validated using modified cell lines and a series of patients with laryngeal cancer. RESULTS: miRs, mRNAs, and gene sets that correlated with resistance could be identified from expression data of unirradiated cells. The presence of epithelial-to-mesenchymal transition (EMT) and low expression of miRs involved in the inhibition of EMT were important radioresistance determinants. This finding was validated in two independent cell line pairs, in which the induction of EMT reduced radiosensitivity. Moreover, low expression of the most important miR (miR-203) was shown to correlate with local disease recurrence after radiotherapy in a series of patients with laryngeal cancer. CONCLUSIONS: These findings indicate that EMT and low expression of EMT-inhibiting miRs, especially miR-203, measured in pretreatment material, causes intrinsic radioresistance of HNSCC, which could enable identification and treatment modification of radioresistant tumors. Clin Cancer Res; 21(24); 5630-8. ©2015 AACR.