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Item does not contain fulltext, PURPOSE: PARP-inhibitors have potent radiosensitizing properties in pre-clinical models. To identify the maximum tolerated dose (MTD) of the PARP-inhibitor Olaparib in combination with radiotherapy in patients with head and neck cancer, a single institutional phase-I dose escalation trial was initiated. PATIENTS AND METHODS: The starting dose of Olaparib was 25 mg BID, combined with radiotherapy (70 Gy in 35 fractions). The MTD was defined as the highest dose-level at which not more than 20 % of patients experience dose-limiting toxicities (DLT) or as the highest reached dose in the absence of DLT's. RESULTS: One week Olaparib-only treatment (25 mg QD) was administered to all patients prior to the start of radiotherapy. In dose-level I, Olaparib (25 mg BID) was combined with accelerated radiotherapy (70 Gy in 6 weeks). Because of DLT's in 3 of the 4 treated patients (acute tracheotomy 5 and 7 months and osteoradionecrosis 7 months after treatment), the Olaparib dose was de-escalated to 25 mg QD, and combined with conventional radiotherapy (70 Gy in 7 weeks) (dose-level II). There were no DLT's observed in 5 patients treated within dose-level II. After a median follow-up of 60 months, the 4-year LRC and OS rates were 77.8 % and 88.9 %, respectively. CONCLUSION: Olaparib 25 mg QD combined with conventionally fractionated radiotherapy was well tolerated and identified as the MTD while severe DLT's were observed when Olaparib 25 mg BID was combined with accelerated radiation. This combination might be further explored in future Olaparib dose escalation studies in patients with locally-advanced HNSCC unfit for cisplatin-based chemoradiotherapy., 01 januari 2024

Radboud University, 13 april 2022, Promotor : Verheij, M. Co-promotores : Triest, B. van, Vens, C., Contains fulltext : 247703.pdf (Publisher’s version ) (Closed access)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease where substantial heterogeneity in clinical presentation urgently requires a better stratification of patients for the development of drug trials and clinical care. In this study we explored stratification through a crowdsourcing approach, the DREAM Prize4Life ALS Stratification Challenge. Using data from >10,000 patients from ALS clinical trials and 1479 patients from community-based patient registers, more than 30 teams developed new approaches for machine learning and clustering, outperforming the best current predictions of disease outcome. We propose a new method to integrate and analyze patient clusters across methods, showing a clear pattern of consistent and clinically relevant sub-groups of patients that also enabled the reliable classification of new patients. Our analyses reveal novel insights in ALS and describe for the first time the potential of a crowdsourcing to uncover hidden patient sub-populations, and to accelerate disease understanding and therapeutic development.

Contains fulltext : 216693.pdf (publisher's version ) (Open Access)

Contains fulltext : 220434.pdf (Publisher’s version ) (Open Access), BACKGROUND: The prognosis of patients with HPV-negative advanced stage head and neck squamous cell carcinoma (HNSCC) remains poor. No prognostic markers other than TNM staging are routinely used in clinic. Epithelial-to-mesenchymal transition (EMT) has been shown to be a strong prognostic factor in other cancer types. The purpose of this study was to determine the role of EMT in HPV-negative HNSCC outcomes. METHODS: Pretreatment tumor material from patients of two cohorts, totalling 174 cisplatin-based chemoradiotherapy treated HPV-negative HNSCC patients, was RNA-sequenced. Seven different EMT gene expression signatures were used for EMT status classification and generation of HNSCC-specific EMT models using Random Forest machine learning. RESULTS: Mesenchymal classification by all EMT signatures consistently enriched for poor prognosis patients in both cohorts of 98 and 76 patients. Uni- and multivariate analyses show important HR of 1.6-5.8, thereby revealing EMT's role in HNSCC outcome. Discordant classification by these signatures prompted the generation of an HNSCC-specific EMT profile based on the concordantly classified samples in the first cohort (cross-validation AUC > 0.98). The independent validation cohort confirmed the association of mesenchymal classification by the HNSCC-EMT model with poor overall survival (HR = 3.39, p < 0.005) and progression free survival (HR = 3.01, p < 0.005) in multivariate analysis with TNM. Analysis of an additional HNSCC cohort from PET-positive patients with metastatic disease prior to treatment further supports this relationship and reveals a strong link of EMT to the propensity to metastasize. CONCLUSIONS: EMT in HPV-negative HNSCC co-defines patient outcome after chemoradiotherapy. The generated HNSCC-EMT prediction models can function as strong prognostic biomarkers.

Contains fulltext : 207152.pdf (publisher's version ) (Open Access), About half of advanced stage head and neck squamous cell carcinoma (HNSCC) patients can be cured by chemoradiotherapy. Patient outcome may be partially determined by the genetic alterations in HNSCC, rendering these alterations promising candidate prognostic factors and/or therapeutic targets. However, their relevance in patient outcome prognosis remains to be assessed in patients that receive standard-of-care chemoradiotherapy. We therefore tested whether frequent genetic alterations were associated with progression free survival (PFS) in advanced stage HNSCC patients who were uniformly treated with definitive platinum-based chemoradiotherapy. To this end, we performed targeted DNA sequencing on frozen pre-treatment tumor biopsy material from 77 patients with advanced stage oro- and hypopharyngeal carcinoma. This provided somatic point mutation and copy number aberration data of 556 genes. The most frequently mutated genes, TP53 (62%), CCND1 (51%), CDKN2A (30%) and PIK3CA (21%), were not associated with PFS. However, co-occurring CCND1 and CDKN2A mutations were associated with short PFS (HR 2.24, p = 0.028) in HPV-negative tumors. Furthermore, tumor mutational burden (sum of somatic point mutations) showed a trend towards decreased PFS (HR 1.9, p = 0.089), and chromosomal instability (CIN) was associated with shorter PFS (HR 2.3, p = 0.023), independent of HPV status. Our results show that tumor mutational burden, CIN markers, and co-occurring CCND1 and CDKN2A mutations are associated with chemoradiotherapy outcomes in advanced stage oro- and hypopharyngeal HNSCC patients, thereby highlighting their prognostic potential. Given their poor prognosis association and link to biological targets, they may also identify patients for novel targeted therapies and immunotherapies.

Item does not contain fulltext, BACKGROUND AND PURPOSE: Preclinical models are much needed to assess the effect of novel radio-sensitizers or mitigators on radiation dose limiting lung toxicity. Albeit showing radiation-induced lung pathologies, current mouse models lack the sensitivity to do so. Using micro image-guided radiotherapy (microIGRT) techniques, we aimed to establish murine models which enable the sensitive detection of lung damage aggravation and characterized functional, radiological and histological responses. MATERIALS AND METHODS: Right lungs of C57Bl/6J mice were irradiated using microIGRT with doses from 15 to 27Gy and with 21Gy and cisplatin as a radio-sensitizer in a second study. Mice were sacrificed for histological and pathological assessment at different time-points post-IR. Lung density was determined using the integrated micro cone-beam CT (microCBCT). Lung function was measured by double-chamber-plethysmography. RESULTS: microIGRT resulted in accurate deposition of the radiation dose in the right lung only as determined by H2AX staining. Lung fibrosis was confirmed by pathological assessments and increased significantly at 21Gy as determined by automated quantification of histochemical analyses. Lung function was affected in a dose-dependent manner. microCBCT-determined lung densities increased significantly over time in the irradiated lungs and showed a strong radiation dose-dependence. Importantly, the microCBCT analyses allowed the detection of additional lung damage caused by 3Gy dose increments or by the combination with cisplatin. CONCLUSION: microCBCT after right lung microIGRT enables the sensitive detection of effects inflicted by relative small dose increments or radio-sensitizers. Our preclinical model therefore facilitates the determination of lung damage exacerbation for the safety assessment of novel RT-drug combinations.

Contains fulltext : 208573.pdf (publisher's version ) (Open Access), BACKGROUND: Poly (ADP-ribose) Polymerase (PARP) inhibitors are promising novel radiosensitisers. Pre-clinical models have demonstrated potent and tumour-specific radiosensitisation by PARP inhibitors. Olaparib is a PARP inhibitor with a favourable safety profile in comparison to clinically used radiosensitisers including cisplatin when used as single agent. However, data on safety, tolerability and efficacy of olaparib in combination with radiotherapy are limited. METHODS: Olaparib is dose escalated in combination with radical (chemo-)radiotherapy regimens for non-small cell lung cancer (NSCLC), breast cancer and head and neck squamous cell carcinoma (HNSCC) in three parallel single institution phase 1 trials. All trials investigate a combination treatment of olaparib and radiotherapy, the NSCLC trial also investigates a triple combination of olaparib, radiotherapy and concurrent low dose cisplatin. The primary objective is to identify the maximum tolerated dose of olaparib in these combination treatments, defined as the dose closest to but not exceeding a 15% probability of dose limiting toxicity. Each trial has a separate dose limiting toxicity definition, taking into account incidence, duration and severity of expected toxicities without olaparib. Dose escalation is performed using a time-to-event continual reassessment method (TITE-CRM). TITE-CRM enables the incorporation of late onset toxicity until one year after treatment in the dose limiting toxicity definition while maintaining an acceptable trial duration. Olaparib treatment starts two days before radiotherapy and continues during weekends until two days after radiotherapy. Olaparib will also be given two weeks and one week before radiotherapy in the breast cancer trial and HNSCC trial respectively to allow for translational research. Toxicity is scored using common terminology criteria for adverse events (CTCAE) version 4.03. Blood samples, and tumour biopsies in the breast cancer trial, are collected for

Item does not contain fulltext, Head and neck squamous cell carcinoma (HNSCC) is characterized by the frequent manifestation of DNA crosslink repair defects. We established novel expression-based DNA repair defect markers to determine the clinical impact of such repair defects. Using hypersensitivity to the DNA crosslinking agents, mitomycin C and olaparib, as proxies for functional DNA repair defects in a panel of 25 HNSCC cell lines, we applied machine learning to define gene expression models that predict repair defects. The expression profiles established predicted hypersensitivity to DNA-damaging agents and were associated with mutations in crosslink repair genes, as well as downregulation of DNA damage response and repair genes, in two independent datasets. The prognostic value of the repair defect prediction profiles was assessed in two retrospective cohorts with a total of 180 patients with advanced HPV-negative HNSCC, who were treated with cisplatin-based chemoradiotherapy. DNA repair defects, as predicted by the profiles, were associated with poor outcome in both patient cohorts. The poor prognosis association was particularly strong in normoxic tumor samples and was linked to an increased risk of distant metastasis. In vitro, only crosslink repair-defective HNSCC cell lines are highly migratory and invasive. This phenotype could also be induced in cells by inhibiting rad51 in repair competent and reduced by DNA-PK inhibition. In conclusion, DNA crosslink repair prediction expression profiles reveal a poor prognosis association in HNSCC. SIGNIFICANCE: This study uses innovative machine learning-based approaches to derive models that predict the effect of DNA repair defects on treatment outcome in HNSCC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5597/F1.large.jpg.

Contains fulltext : 200373.pdf (publisher's version ) (Open Access), BACKGROUND: Large cancer genome studies continue to reveal new players in treatment response and tumorigenesis. The discrimination of functional alterations from the abundance of passenger genetic alterations still poses challenges and determines DNA sequence variant selection procedures. Here we evaluate variant selection strategies that select homozygous variants and rare SNPs and assess its value in detecting tumor cells with DNA repair defects. METHODS: To this end we employed a panel of 29 patient-derived head and neck squamous cell carcinoma (HNSCC) cell lines, of which a subset harbors DNA repair defects. Mitomycin C (MMC) sensitivity was used as functional endpoint of DNA crosslink repair deficiency. 556 genes including the Fanconi anemia (FA) and homologous recombination (HR) genes, whose products strongly determine MMC response, were capture-sequenced. RESULTS: We show a strong association between MMC sensitivity, thus loss of DNA repair function, and the presence of homozygous and rare SNPs in the relevant FA/HR genes. Excluding such selection criteria impedes the discrimination of crosslink repair status by mutation analysis. Applied to all KEGG pathways, we find that the association with MMC sensitivity is strongest in the KEGG FA pathway, therefore also demonstrating the value of such selection strategies for exploratory analyses. Variant analyses in 56 clinical samples demonstrate that homozygous variants occur more frequently in tumor suppressor genes than oncogenes further supporting the role of a homozygosity criterion to improve gene function association or tumor suppressor gene identification studies. CONCLUSION: Together our data show that the detection of relevant genes or of repair pathway defected tumor cells can be improved by the consideration of allele zygosity and SNP allele frequencies.

Item does not contain fulltext, BACKGROUND: PARP inhibitors are currently evaluated in combination with radiotherapy and/or chemotherapy. As sensitizers, PARP inhibitors are active at very low concentrations therefore requiring highly sensitive pharmacodynamic (PD) assays. Current clinical PD-assays partly fail to provide such sensitivities. The aim of our study was to enable sensitive PD evaluation of PARP inhibitors for clinical sensitizer development. MATERIAL AND METHODS: PBMCs of healthy individuals and of olaparib and radiotherapy treated lung cancer patients were collected for ELISA-based PD-assays. RESULTS: PAR-signal amplification by ex vivo irradiation enabled an extended quantification range for PARP inhibitory activities after ex vivo treatment with inhibitors. This "radiation-enhanced-PAR" (REP) assay provided accurate IC50 values thereby also revealing differences among healthy individuals. Implemented in clinical radiotherapy combination Phase I trials, the REP-assay showed sensitive detection of PARP inhibition in patients treated with olaparib and establishes strong PARP inhibitory activities at low daily doses. CONCLUSIONS: Combination trials of radiotherapy and novel targeted agent(s) often require different and more sensitive PD assessments than in the monotherapy setting. This study shows the benefit and relevance of sensitive and adapted PD-assays for such combination purposes and provides proof of clinically relevant cellular PARP inhibitory activities at low daily olaparib doses.

Contains fulltext : 193611.pdf (Publisher’s version ) (Open Access), Mutations in Fanconi Anemia or Homologous Recombination (FA/HR) genes can cause DNA repair defects and could therefore impact cancer treatment response and patient outcome. Their functional impact and clinical relevance in head and neck squamous cell carcinoma (HNSCC) is unknown. We therefore questioned whether functional FA/HR defects occurred in HNSCC and whether they are associated with FA/HR variants. We assayed a panel of 29 patient-derived HNSCC cell lines and found that a considerable fraction is hypersensitive to the crosslinker Mitomycin C and PARP inhibitors, a functional measure of FA/HR defects. DNA sequencing showed that these hypersensitivities are associated with the presence of bi-allelic rare germline and somatic FA/HR gene variants. We next questioned whether such variants are associated with prognosis and treatment response in HNSCC patients. DNA sequencing of 77 advanced stage HNSCC tumors revealed a 19% incidence of such variants. Importantly, these variants were associated with a poor prognosis (p = 0.027; HR = 2.6, 1.1-6.0) but favorable response to high cumulative cisplatin dose. We show how an integrated in vitro functional repair and genomic analysis can improve the prognostic value of genetic biomarkers. We conclude that repair defects are marked and frequent in HNSCC and are associated with clinical outcome.

Item does not contain fulltext, OBJECTIVE: Locally advanced oral squamous cell carcinoma (OSCC) shows lower locoregional control and disease specific survival rates than laryngeal and pharyngeal squamous cell carcinoma (L/P-SCC) after definitive chemoradiotherapy treatment. Despite clinical factors, this can point towards a different tumor biology that could impact chemoradiotherapy response rates. This prompted us to compare the mutational profiles of OSCC with L/P-SCC. METHODS: We performed target capture DNA sequencing on 111 HPV-negative HNSCC samples (NKI dataset), 55 oral and 56 laryngeal/pharyngeal, and identified somatic point mutations and copy number aberrations. We next expanded our analysis with 276 OSCC and 134 L/P-SCC sample data from The Cancer Genome Atlas (TCGA dataset). We focused our analyses on genes that are frequently mutated in HNSCC. RESULTS: The mutational profiles of OSCC and L/P-SCC showed many similarities. However, OSCC was significantly enriched for CASP8 (NKI: 15% vs 0%; TCGA: 17% vs 2%) and HRAS (TCGA: 10% vs 1%) mutations. LAMA2 (TCGA: 5% vs 19%) and NSD1 (TCGA: 7% vs 25%) mutations were enriched in L/P-SCC. Overall, we find that OSCC had fewer somatic point mutations and copy number aberrations than L/P-SCC. Interestingly, L/P-SCC scored higher in mutational and genomic scar signatures associated with homologous recombination DNA repair defects. CONCLUSION: Despite showing a similar mutational profile, our comparative genomic analysis revealed distinctive features in OSCC and L/P-SCC. Some of these genes and cellular processes are likely to affect the cellular response to radiation or cisplatin. Genomic characterizations may guide or enable personalized treatment in the future.

Data Cleaning represents a crucial and error prone activity in KDD that might have unpredictable effects on data analytics, affecting the believability of the whole KDD process. In this paper we describe how a bridge between AI Planning and Data Quality communities has been made, by expressing both the data quality and cleaning tasks in terms of AI planning. We also report a real-life application of our approach.

The rapid growth of Web usage for advertising job positions provides a great opportunity for real-time labour market monitoring. This is the aim of Labour Market Intelligence (LMI), a field that is becoming increasingly relevant to EU Labour Market policies design and evaluation. The analysis of Web job vacancies, indeed, represents a competitive advantage to labour market stakeholders with respect to classical survey-based analyses, as it allows for reducing the time-to-market of the analysis by moving towards a fact-based decision making model. In this paper, we present our approach for automatically classifying million Web job vacancies on a standard taxonomy of occupations. We show how this problem has been expressed in terms of text classification via machine learning. Then, we provide details about the classification pipelines we evaluated and implemented, along with the outcomes of the validation activities. Finally, we discuss how machine learning contributed to the LMI needs of the European Organisation that supported the project.

Item does not contain fulltext, BACKGROUND AND PURPOSE: The PARP inhibitor olaparib is currently tested in clinical phase 1 trials to define safe dose levels in combination with RT. However, certain clinically relevant insights are still lacking. Here we test, while comparing to single agent activity, the olaparib dose and genetic background dependence of olaparib-mediated radiosensitization. MATERIALS AND METHODS: Long-term growth inhibition and clonogenic assays were used to assess radiosensitization in BRCA2-deficient and BRCA2-complemented cells and in a panel of human head and neck squamous cell carcinoma cell lines. RESULTS: The extent of radiosensitization greatly depended on the olaparib dose, the radiation dose and the homologous recombination status of cells. Olaparib concentrations that resulted in radiosensitization prevented PAR induction by irradiation. Seven hours olaparib exposures were sufficient for radiosensitization. Importantly, the radiosensitizing effects can be observed at much lower olaparib doses than the single agent effects. CONCLUSION: Extrapolation of these data to the clinic suggests that low olaparib doses are sufficient to cause radiosensitization, underlining the potential of the treatment. Here we show that drug doses achieving radiosensitization can greatly differ from those achieving single agent activities, an important consideration when developing combined radiotherapy strategies with novel targeted agents.

Contains fulltext : 153904.pdf (publisher's version ) (Open Access), Breast cancers with BRCA1 germline mutation have a characteristic DNA copy number (CN) pattern. We developed a test that assigns CN profiles to be 'BRCA1-like' or 'non-BRCA1-like', which refers to resembling a BRCA1-mutated tumor or resembling a tumor without a BRCA1 mutation, respectively. Approximately one third of the BRCA1-like breast cancers have a BRCA1 mutation, one third has hypermethylation of the BRCA1 promoter and one third has an unknown reason for being BRCA1-like. This classification is indicative of patients' response to high dose alkylating and platinum containing chemotherapy regimens, which targets the inability of BRCA1 deficient cells to repair DNA double strand breaks. We investigated whether this classification can be reliably obtained with next generation sequencing and copy number platforms other than the bacterial artificial chromosome (BAC) array Comparative Genomic Hybridization (aCGH) on which it was originally developed. We investigated samples from 230 breast cancer patients for which a CN profile had been generated on two to five platforms, comprising low coverage CN sequencing, CN extraction from targeted sequencing panels (CopywriteR), Affymetrix SNP6.0, 135K/720K oligonucleotide aCGH, Affymetrix Oncoscan FFPE (MIP) technology, 3K BAC and 32K BAC aCGH. Pairwise comparison of genomic position-mapped profiles from the original aCGH platform and other platforms revealed concordance. For most cases, biological differences between samples exceeded the differences between platforms within one sample. We observed the same classification across different platforms in over 80% of the patients and kappa values of at least 0.36. Differential classification could be attributed to CN profiles that were not strongly associated to one class. In conclusion, we have shown that the genomic regions that define our BRCA1-like classifier are robustly measured by different CN profiling technologies, providing the possibility to retro- and prospectively in

Breast cancers with BRCA1 germline mutation have a characteristic DNA copy number (CN) pattern. We developed a test that assigns CN profiles to be ‘BRCA1-like’ or ‘non-BRCA1-like’, which refers to resembling a BRCA1-mutated tumor or resembling a tumor without a BRCA1 mutation, respectively. Approximately one third of the BRCA1-like breast cancers have a BRCA1 mutation, one third has hypermethylation of the BRCA1 promoter and one third has an unknown reason for being BRCA1-like. This classification is indicative of patients' response to high dose alkylating and platinum containing chemotherapy regimens, which targets the inability of BRCA1 deficient cells to repair DNA double strand breaks. We investigated whether this classification can be reliably obtained with next generation sequencing and copy number platforms other than the bacterial artificial chromosome (BAC) array Comparative Genomic Hybridization (aCGH) on which it was originally developed. We investigated samples from 230 breast cancer patients for which a CN profile had been generated on two to five platforms, comprising low coverage CN sequencing, CN extraction from targeted sequencing panels (CopywriteR), Affymetrix SNP6.0, 135K/720K oligonucleotide aCGH, Affymetrix Oncoscan FFPE (MIP) technology, 3K BAC and 32K BAC aCGH. Pairwise comparison of genomic position-mapped profiles from the original aCGH platform and other platforms revealed concordance. For most cases, biological differences between samples exceeded the differences between platforms within one sample. We observed the same classification across different platforms in over 80% of the patients and kappa values of at least 0.36. Differential classification could be attributed to CN profiles that were not strongly associated to one class. In conclusion, we have shown that the genomic regions that define our BRCA1-like classifier are robustly measured by different CN profiling technologies, providing the possibility to retro- and prospectively in, Intelligent Systems, Electrical Engineering, Mathematics and Computer Science

Item does not contain fulltext, Radiotherapy is used to treat approximately 50% of all cancer patients, with varying success. The dose of ionizing radiation that can be given to the tumour is determined by the sensitivity of the surrounding normal tissues. Strategies to improve radiotherapy therefore aim to increase the effect on the tumour or to decrease the effects on normal tissues. These aims must be achieved without sensitizing the normal tissues in the first approach and without protecting the tumour in the second approach. Two factors have made such approaches feasible: namely, an improved understanding of the molecular response of cells and tissues to ionizing radiation and a new appreciation of the exploitable genetic alterations in tumours. These have led to the development of treatments combining pharmacological interventions with ionizing radiation that more specifically target either tumour or normal tissue, leading to improvements in efficacy.

1 oktober 2010, Contains fulltext : 89072.pdf (publisher's version ) (Closed access), Cellular DNA repair determines survival after ionizing radiation. Human tumors commonly exhibit aberrant DNA repair since they drive mutagenesis and chromosomal instability. Recent reports have shown alterations in the base excision repair (BER) and single strand break repair (SSBR) pathways in human tumors. Here we review these reports with respect to radiation sensitivity and the attempts to target such tumor-specific BER/SSBR aberrations. These aberrations can alter cellular resistance to therapeutic agents, including radiation. Some strategies therefore aim to counteract the radioresistance mediated by such aberrant DNA repair. Other strategies aim to exploit the dependence of the tumor, but not the normal cells, on backup repair mechanisms after radiation, therefore increasing the therapeutic window. Such tumor-targeted radiosensitization holds promise for increasing the efficacy of radiotherapy.

Contains fulltext : 89068.pdf (publisher's version ) (Closed access), Ionizing radiation (IR) is an effective anticancer treatment, although failures still occur. To improve radiotherapy, tumor-targeted strategies are needed to increase radiosensitivity of tumor cells, without influencing normal tissue radiosensitivity. Base excision repair (BER) and single-strand break repair (SSBR) contribute to the determination of sensitivity to IR. A crucial protein in BER/SSBR is DNA polymerase beta (polbeta). Aberrant polbeta expression is commonly found in human tumors and leads to inhibition of BER. Here, we show that truncated polbeta variant (polbeta-Delta)-expressing cells depend on homologous recombination (HR) for survival after IR, indicating that a considerable fraction of polbeta-Delta-induced lesions are subject to repair by HR. Increased sensitization was found not to result from involvement in DNA-dependent protein kinase-dependent nonhomologous end joining, the other major double-strand break repair pathway. Caffeine and the ATM inhibitor Ku55933 cause polbeta-Delta-dependent radiosensitization. Consistent with the observed HR dependence and the known HR-modulating activity of ATM, polbeta-Delta-expressing cells showed increased radiosensitization after BRCA2 knockdown that is absent under ATM-inhibited conditions. Our data suggest that treatment with HR modulators is a promising therapeutic strategy for exploiting defects in the BER/SSBR pathway in human tumors.

Contains fulltext : 79524.pdf (publisher's version ) (Closed access), 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

RU Radboud Universiteit Nijmegen, 12 november 2009, Promotor : Begg, A.C. Co-promotor : Vens, C., Contains fulltext : 74587.pdf (publisher's version ) (Open Access)

RU Radboud Universiteit Nijmegen, 9 januari 2009, Promotor : Begg, A.C. Co-promotor : Vens, C., Contains fulltext : 80268.pdf (publisher's version ) (Open Access)

Contains fulltext : 69923.pdf (publisher's version ) (Closed access), 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.

Contains fulltext : 53393.pdf (publisher's version ) (Closed access), 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.

Contains fulltext : 51692.pdf (publisher's version ) (Open Access), 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.

Contains fulltext : 51693.pdf (publisher's version ) (Open Access), 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.

Contains fulltext : 50367.pdf (publisher's version ) (Closed access), 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.

Promotores : Begg, A.C., Balm, A.J.M., Haustermans, K., Co-promotor : Vens, C., RU Radboud Universiteit Nijmegen, 11 september 2006, Promotores : Begg, A.C., Balm, A.J.M., Haustermans, K. Co-promotor : Vens, C., Contains fulltext : mmubn000001_457951125.pdf (publisher's version ) (Open Access)

Contains fulltext : 47693.pdf (publisher's version ) (Closed access), 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.

Contains fulltext : 186706.pdf (publisher's version ) (Open Access), 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.

Item does not contain fulltext, To test modifications in sensitization to radiation or drugs in preclinical studies of cancer therapy, the colony-forming assay is regarded as the gold standard. Because this assay is time consuming, somewhat laborious, and unsuitable for rapid screening, development of other assays is desirable. We describe here an assay based on the detection of enhanced green fluorescence protein (EGFP) with flow cytometry that is particularly suitable for genetic manipulation studies in which the gene of interest is introduced together with EGFP as reporter. It is easily adaptable to other reporters, however, whether naturally fluorescent or requiring immunochemical staining. Cells are irradiated as mixed populations of a known standard cell line (nonfluorescent) together with the genetically manipulated cell line expressing EGFP. Ratios of fluorescent and nonfluorescent cells are measured before treatment and several days after treatment. If the cell populations have equal radiosensitivities, the ratio remains unchanged. Changes in the ratio indicate changes in radiosensitivity. The assay was validated for two situations in which dominant negative peptides inhibiting DNA repair were expressed in A549 human lung cells and affected radiosensitivity.